Subtype-selective nicotinic receptor antagonists: potential as tobacco use cessation agents

N-n-Alkylpicolinium and N,N'-alkyl-bis-picolinium analogues were assessed in nicotinic receptor (nAChR) assays. The most potent and subtype-selective analogue, N,N'-dodecyl-bis-picolinium bromide (bPiDDB), inhibited nAChRs mediating nicotine-evoked [(3)H]dopamine release (IC(50)=5 nM; I(max) of 60%), and did not interact with alpha4beta2* or alpha7* nAChRs. bPiDDB represents the current lead compound for development as a tobacco use cessation agent.     

Tris-azaaromatic quaternary ammonium salts: Novel templates as antagonists at nicotinic receptors mediating nicotine-evoked dopamine release

A series of tris-azaaromatic quaternary ammonium salts has been synthesized and evaluated for their ability to inhibit neuronal nicotinic acetylcholine receptors (nAChRs) mediating nicotine-evoked [³H]dopamine release from superfused rat striatal slices and for inhibition of [³H]nicotine and [³H]methyllycaconitine binding to whole rat brain membranes. The 3-picolinium compound 1,3,5-tri-{5-[1-(3-picolinium)]-pent-1-ynyl}benzene tribromide (tPy3PiB), 3b, exhibited high potency and selectivity for nAChR subtypes mediating nicotine-evoked [³H]dopamine release with an IC₅₀ of 0.2 nM and I_max of 67%.     

Identification and synthesis of novel alkaloids from the root system of Nicotiana tabacum: affinity for neuronal nicotinic acetylcholine receptors

A novel pyridine derivative, 3,5-bis-(1-methyl-pyrrolidin-2-yl)-pyridine, and a pair of diastereomers of 1,1'-dimethyl-[2,3']bipyrrolidinyl were isolated from the root of Nicotiana tabacum plants and identified as novel alkaloids by GC-MS analysis. The structures of these new alkaloids were confirmed by total synthesis. The affinities of these novel alkaloids, and other structurally related compounds for alpha4beta2*, alpha7* neuronal nicotinic acetylcholine receptors (nAChRs), and for nAChRs mediating nicotine-evoked dopamine release from rat striatum were also assessed. The results indicate that these compounds do not interact with alpha7* nAChRs, but inhibit [3H]nicotine binding to the alpha4beta2* nAChR subtype. The results also demonstrate that these compounds act as antagonists at nAChRs mediating nicotine-evoked dopamine release from rat striatum.     

Novel bis-2,2,6,6-tetramethylpiperidine (bis-TMP) and bis-mecamylamine antagonists at neuronal nicotinic receptors mediating nicotine-evoked dopamine release

By linking two or three mecamylamine or 2,2,6,6-tetramethylpiperidine (TMP) molecules together via a linear lipophilic bis-methylene linker or a specially designed conformationally restricted tris-linker, a series of bis- and tris-tertiary amine analogs has been synthesized and evaluated as potent antagonists at nAChRs mediating nicotine-evoked [³H]dopamine release from rat striatal slices. Compounds 7e, 14b and 16 demonstrated high potency in decreasing nicotine-evoked [³H]dopamine release (IC₅₀ = 2.2, 46, and 107 nM, respectively). The preliminary structure–activity data obtained with these new analogs suggest the importance of the length of the methylene linker in the bis-analog series. Such bis-tertiary amino analogs may provide a new strategy for the design of drugable ligands that have high inhibitory potency against nAChRs mediating nicotine-evoked dopamine release in striatum, which have been suggested to be target receptors of interest in the development of potential smoking cessation therapies.     

Nicotine-evoked transmitter release from synaptosomes: functional association of specific presynaptic acetylcholine receptors and voltage-gated calcium channels.

It has previously been shown that nicotine-evoked dopamine release from rat striatal synaptosomes and nicotine-evoked norepinephrine release from hippocampal synaptosomes are mediated by distinct nicotinic acetylcholine receptor (nAChR) subtypes. In the present study, the functional association of these nicotinic receptors with specific subtypes of voltage-gated calcium channels was examined. Cd(2+) (200 microM), as well as omega-conotoxin MVIIC (5 microM), blocks approximately 85% of nicotine-evoked dopamine release from striatal synaptosomes, indicating a major involvement of calcium channels. Furthermore, the toxin-susceptibility suggests that these calcium channels contain alpha(1A) and/or alpha(1B) subunits. Inhibition of nicotine-evoked dopamine release by conotoxins alpha-MII and omega-GVIA is additive and indicates that presynaptic alpha3beta2 nAChRs are functionally coupled to alpha(1A), but not alpha(1B), calcium channel subtypes. Conversely, insensitivity to alpha-AuIB and sensitivity to omega-MVIIC indicate that non-alpha3beta2/alpha3beta4-containing nAChRs are functionally coupled to alpha(1B)-containing calcium channels. In contrast, Cd(2+) blocks only 65% of nicotine-evoked norepinephrine release from hippocampal synaptosomes, indicating that a substantial fraction of this release occurs through mechanisms not involving calcium channels. This Cd(2+)-insensitive component of release is blocked by alpha-AuIB and therefore appears to be triggered by Ca(2+) flowing directly through the channels of presynaptic alpha3beta4 nAChRs. Thus, these data indicate that different presynaptic termini can have distinctive functional associations of specific nAChRs and voltage-gated calcium channels.     

Novel bis-, tris-, and tetrakis-tertiary amino analogs as antagonists at neuronal nicotinic receptors that mediate nicotine-evoked dopamine release

A series of tertiary amine analogs derived from lead azaaromatic quaternary ammonium salts has been designed and synthesized. The preliminary structure-activity relationships of these new analogs suggest that such tertiary amine analogs, which potently inhibit nicotine-evoked dopamine release from rat striatum, represent drug-like inhibitors of α6-containing nicotinic acetylcholine receptors. The bis-tertiary amine analog 7 exhibited an IC₅₀ of 0.95 nM, while the tris-tertiary amine analog 19 had an IC₅₀ of 0.35 nM at nAChRs mediating nicotine-evoked dopamine release.     

The neurotoxin histrionicotoxin interacts with the putative ion channel of the nicotinic acetylcholine receptors in the central nervous system

Perhydrohistrionicotoxin at micromolar concentrations blocked the nicotine-evoked transmitter release from perfused striatal (dopaminergic) and hippocampal (cholinergic) nerve terminals. Perhydrohistrionicotoxin failed to compete with [3H]nicotine for its high-affinity binding site in rat brain, suggesting that the action of this toxin on central nicotinic receptors is noncompetitive. From the dose-response curve, 50% inhibition of nicotine-evoked striatal dopamine release occurred at 5 microM perhydrohistrionicotoxin, a value similar to that obtained in frog sartorius muscle and Electrophorus electroplax. This close agreement may suggest that the ionic channel of the presynaptic nicotinic acetylcholine receptor of brain neurons has similar properties to those of the peripheral receptor.     

Regulation of nicotine-evoked dopamine release from PC12 cells.

The nature of second messengers involved in the nicotine-evoked release of dopamine from PC12 cells was examined. Calmidazolium, a calmodulin inhibitor, abolished the nicotine-evoked release. A23187, a Ca2+ ionophore, enhanced dopamine release, and this was inhibited by calmidazolium. Further, 2', 5'-dideoxyadenosine abolished both the nicotine- and A23187-evoked release. Forskolin, dibutyryl-cyclic AMP, and rolipram (a cyclic AMP phosphodiesterase inhibitor) all enhanced dopamine release. 1, 9-Dideoxyforskolin, a forskolin analog which does not activate adenylate cyclase, did not alter dopamine release. These results suggest an obligatory role for Ca2+ and calmodulin-sensitive adenylate cyclase in the nicotine-evoked release process.     

Characterization of Nicotinic Receptor-Mediated [3H]Dopamine Release from Synaptosomes Prepared from Mouse Striatum

This study establishes that presynaptic nicotinic receptors modulate dopamine release in the mouse striatum. Nicotinic agonists elicit a dose-dependent increase in the release of [3H]dopamine from synaptosomes prepared from mouse striatum. At low concentrations, this release is Ca2+ dependent, whereas at higher concentrations Ca(2+)-independent, mecamylamine-insensitive release was also observed. The Ca(2+)-dependent nicotine-evoked release was not blocked by alpha-bungarotoxin but was effectively blocked by neuronal bungarotoxin as well as several other nicotinic receptor antagonists. The relationship between potency for stimulation of release for agonists and potency for inhibition of release for antagonists was compared to the affinity of these compounds for the [3H]nicotine binding site. The overall correlation between release and binding potency was not high, but the drugs may be classified into separate groups, each of which has a high correlation with binding. This finding suggests either that more than one nicotinic receptor regulates dopamine release or that not all agonists interact with the same receptor in an identical fashion.     

The pharmacological activity of nicotine and nornicotine on nAChRs subtypes: relevance to nicotine dependence and drug discovery

Cigarette smoking and other forms of tobacco use deliver an array of pharmacologically active alkaloids, including nicotine and ultimately various metabolites of these substances. While nornicotine is a significant component in tobacco as well as a minor systemic metabolite of nicotine, nornicotine appears to be N-demethylated locally in the brain where it accumulates at relatively high levels after chronic nicotine administration. We have now examined the effects of nornicotine on specific combinations of neuronal nicotinic acetylcholine receptor (nAChR) subunits expressed in Xenopus oocytes and compared these responses to those evoked by acetylcholine and nicotine. Of the nAChR subtypes studied, we have found that alpha7 receptors are very responsive to nornicotine (EC50 approximately 17 micromol/L I(max) 50%, compared with acetylcholine (ACh)). nAChRs containing the ligand-binding domain of the alpha6 subunits (in the form of an alpha6/alpha3 chimera) are also strongly responsive to nornicotine (EC50 approximately 4 micromol/L I(max) 50%, compared with ACh). Alpha7-type nAChRs have been suggested to be potential therapeutic targets for Alzheimer's disease, schizophrenia and possibly other pathologies. nAChRs containing alpha6 subunits have been suggested to have a role in nicotine-evoked dopamine release. Thus, understanding the actions of nornicotine in the brain may have significance for both emerging therapeutics and the management of nicotine dependence.     

Development of subtype-selective ligands as antagonists at nicotinic receptors mediating nicotine-evoked dopamine release

N-n-Alkylation of nicotine converts it from an agonist into an antagonist at neuronal nicotinic acetylcholine receptor subtypes mediating nicotine-evoked dopamine release. Conformationally restricted analogues exhibit both high affinity and selectivity at this site, and are able to access the brain due to their ability to act as substrates for the blood-brain barrier choline transporter.     

bis-Azaaromatic quaternary ammonium analogues: ligands for alpha4beta2* and alpha7* subtypes of neuronal nicotinic receptors

A series of bis-nicotinium, bis-pyridinium, bis-picolinium, bis-quinolinium and bis-isoquinolinium compounds was evaluated for their binding affinity at nicotinic acetylcholine receptors (nAChRs) using rat brain membranes. N,N'-Decane-1,12-diyl-bis-nicotinium diiodide (bNDI) exhibited the highest affinity for [(3)H]nicotine binding sites (K(i)=330 nM), but did not inhibit [(3)H]methyllycaconitine binding (K(i) >100 microM), indicative of an interaction with alpha4beta2*, but not alpha7* receptor subtypes, respectively. Also, bNDI inhibited (IC(50)=3.76 microM) nicotine-evoked (86)Rb(+) efflux from rat thalamic synaptosomes, indicating antagonist activity at alpha4beta2* nAChRs. N,N'-Dodecane-1,12-diyl-bis-quinolinium dibromide (bQDDB) exhibited highest affinity for [(3)H]methyllycaconitine binding sites (K(i)=1.61 microM), but did not inhibit [(3)H]nicotine binding (K(i)>100 microM), demonstrating an interaction with alpha7*, but not alpha4beta2* nAChRs. Thus, variation of N-n-alkyl chain length together with structural modification of the azaaromatic quaternary ammonium moiety afforded selective antagonists for the alpha4beta2* nAChR subtype, as well as ligands with selectivity at alpha7* nAChRs.     

Presynaptic Nicotinic Modulation of Dopamine Release in the Three Ascending Pathways Studied by In Vivo Microdialysis: Comparison of Naive and Chronic Nicotine-Treated Rats

Abstract: The modulation of dopamine release by presynaptic nicotinic receptors in vitro is well established, but the significance of this effect in vivo is unclear. We have characterised the effect of nicotine, locally applied via a microdialysis probe, on dopamine release from the terminal regions of three ascending dopaminergic pathways in conscious, freely moving rats. Nicotine caused a dose-dependent increase in dopamine release in the striatum, the nucleus accumbens, and, to a lesser extent, the frontal cortex. Metabolite levels were unaltered by any concentration of nicotine. Prior administration of mecamylamine via the probe abolished the nicotine-evoked increase in dopamine release, confirming the mediation of nicotinic receptors. The dose dependence of mecamylamine-sensitive, nicotine-evoked dopamine release was similar in all three brain regions. However, 10⁻⁵ M tetrodotoxin totally blocked nicotine-stimulated dopamine release in the striatum and the accumbens but not the cortex. Daily subcutaneous injections of nicotine (0.4 mg kg⁻¹ for 7 days) increased the response to a subsequent local application of nicotine in the striatum, and a similar trend was found in the other brain areas. The same daily dose of nicotine given as a continuous infusion had no effect, whereas infusion of 4 mg kg⁻¹ day⁻¹ increased the response to a subsequent nicotine challenge. The localisation and regulation of nicotinic receptors in the terminal fields of dopaminergic pathways are discussed.     

nAChR-mediated calcium responses and plasticity in Drosophila Kenyon cells

In Drosophila, nicotinic acetylcholine receptors (nAChRs) mediate fast excitatory synaptic transmission in mushroom body Kenyon cells, a neuronal population involved in generation of complex behaviors, including responses to drugs of abuse. To determine whether activation of nAChRs can induce cellular changes that contribute to functional plasticity in these neurons, we examined nicotine-evoked responses in cells cultured from brains of late stage OK107-GAL4 pupae. Kenyon cells can be identified by expression of green fluorescent protein (GFP+). Nicotine activates alpha-bungarotoxin-sensitive nAChRs, causing a rapid increase in intracellular calcium levels in over 95% of the Kenyon cells. The nicotine-evoked calcium increase has a voltage-gated calcium channel (VGCC) dependent component and a VGCC-independent component that involves calcium influx directly through nAChRs. Thapsigargin treatment reduces the nicotine response consistent with amplification by calcium release from intracellular stores. The response to nicotine is experience-dependent: a short conditioning pulse of nicotine causes a transient 50% reduction in the magnitude of the response to a test pulse of nicotine when the interpulse interval is 4 h. This cellular plasticity is dependent on activation of the VGCC-component of the nicotine response and on cAMP-signaling, but not on protein synthesis. These data demonstrate that activation of nAChRs induces a calcium-dependent plasticity in Kenyon cells that could contribute to adult behaviors involving information processing in the mushroom bodies including responses to nicotine.     

Tetrakis-azaaromatic quaternary ammonium salts: novel subtype-selective antagonists at neuronal nicotinic receptors that mediate nicotine-evoked dopamine release

A series of tetrakis-azaaromatic quaternary ammonium salts was synthesized to identify compounds with higher affinity and selectivity as antagonists at neuronal nicotinic receptor subtypes (nAChR) that mediate nicotine-evoked DA release. A high hit rate was achieved in identifying potent analogs that inhibit these nAChRs. Three tetrakis analogs, 11j, 11f, and 11g, were identified as potent (IC(50)=3, 28 and 56nM, respectively) antagonists at these receptors. These compounds represent a novel structural class of nicotinic receptor antagonists.     

Evidence for Functional Activity of Up-Regulated Nicotine Binding Sites in Rat Striatal Synaptosomes

A number of studies have found that the chronic administration of nicotine causes an increase in the density of nicotinic binding sites in the brain, but it is not known whether these additional binding sites are functionally active receptors. In this study, the effects of 1-week administration of the potent nicotinic agonist, (+)-anatoxin-a (96 nmol/day via osmotic minipumps), was assessed on [3H]nicotine binding and [3H]dopamine uptake and release in rat striatal synaptosomes. Chronic (+)-anatoxin-a treatment resulted in a 32% increase in the Bmax of [3H]nicotine binding in anatoxin-treated animals compared to control. There was a 43% increase in the activity of 3 microM nicotine to release [3H]dopamine from synaptosomes of anatoxin-treated animals, but the release induced by 20 mM K+ depolarization was unaffected. There was no effect of chronic (+)-anatoxin-a treatment on the uptake of [3H]dopamine. A strong positive correlation (r = 0.64) was found between the density of [3H]nicotine binding sites and the nicotine-induced stimulation of [3H]dopamine release in individual animals. These results indicate that (+)-anatoxin-a, like nicotine, produces an up-regulation of nicotine binding sites following chronic administration, and that these additional sites are functional receptors capable of mediating the release of dopamine from striatal synaptosomes.     

Release-enhancing pre-synaptic muscarinic and nicotinic receptors co-exist and interact on dopaminergic nerve endings of rat nucleus accumbens

Dopaminergic nerve endings in the corpus striatum possess nicotinic (nAChRs) and muscarinic cholinergic receptors (mAChRs) mediating release of dopamine (DA). Whether nAChRs and mAChRs co-exist and interact on the same nerve endings is unknown. We here investigate on these possibilities using rat nucleus accumbens synaptosomes pre-labeled with [³H]DA and exposed in superfusion to cholinergic receptor ligands. The mixed nAChR–mAChR agonists acetylcholine (ACh) and carbachol provoked [³H]DA release partially sensitive to the mAChR antagonist atropine but totally blocked by the nAChR antagonist mecamylamine. Addition of the mAChR agonist oxotremorine at the minimally effective concentration of 30 μmol/L, together with 3, 10, or 100 μmol/L (−)nicotine provoked synergistic effect on [³H]DA overflow. The [³H]DA overflow elicited by 100 μmol/L (−)nicotine plus 30 μmol/L oxotremorine was reduced by atropine down to the release produced by (−)nicotine alone and it was abolished by mecamylamine. The ryanodine receptor blockers dantrolene or 8-bromo-cADP-ribose, but not the inositol 1,4,5-trisphosphate receptor blocker xestospongin C inhibited the (−)nicotine/oxotremorine evoked [³H]DA overflow similarly to atropine. This overflow was partly sensitive to 100 nmol/L methyllycaconitine which did not prevent the synergistic effect of (−)nicotine/oxotremorine. Similarly to (−)nicotine, the selective α4β2 nAChR agonist RJR2403 exhibited synergism when added together with oxotremorine. To conclude, in rat nucleus accumbens, α4β2 nAChRs exert a permissive role on the releasing function of reportedly M₅ mAChRs co-existing on the same dopaminergic nerve endings.     

Pre-synaptic nicotinic and D2 receptors functionally interact on dopaminergic nerve endings of rat and mouse nucleus accumbens

The existence of pre-synaptic auto- and hetero receptors which modulate neurotransmitter release is well documented. Emerging evidence show that in some cases these pre-synaptic receptors may also cross-talk with each other. The aim of the present work was to investigate whether acetylcholine receptors (nAChRs) and dopamine (DA) autoreceptors, which are both able to modulate DA release, functionally interact on the same nerve endings. We used rat and mouse nucleus accumbens synaptosomes pre-labeled with [³H]DA and exposed to nicotinic and dopaminergic receptor ligands. Both nicotinic agonists and 4-aminopyridine (4-AP) provoked [³H]DA release which was inhibited by quinpirole and blocked by sulpiride and raclopride. Both the inhibitory effect of quinpirole and the stimulatory effect of (−)nicotine did not change when the nAChRs or the DA receptors were desensitized. (−)Nicotine and 4-AP were able to stimulate [³H]DA overflow also in mouse synaptosomes and this overflow was partially inhibited by quinpirole. In the β₂ knockout mice quinpirole was still able to inhibit the [³H]DA overflow elicited by 4-AP. To conclude: in rat and mouse the (−)nicotine evoked-release can be modulated by D₂/D₃ autoreceptors present on the DA terminals and nAChRs function is independent from D₂/D₃ autoreceptors which themselves may function independently from the activation of nAChRs.     

Differential Inhibition by α-Conotoxin-MII of the Nicotinic Stimulation of [3H]Dopamine Release from Rat Striatal Synaptosomes and Slices

Abstract: The presynaptic nicotinic modulation of dopamine release from striatal nerve terminals is well established, but the subtype(s) of neuronal nicotinic acetylcholine receptor (nAChR) underlying this response has not been identified. Recently, α-conotoxin-MII has been reported to inhibit potently and selectively the rat α3/β2 combination of nAChR subunits. Here we have synthesised the peptide, confirmed its specificity, and examined its effect on the (±)-anatoxin-a-evoked release of [³H]dopamine from rat striatal synaptosomes and slices. α-Conotoxin-MII (112 nM) completely blocked acetylcholine-evoked currents of α3β2 nAChRs expressed in Xenopus oocytes (IC₅₀ = 8.0 ± 1.1 nM). Pairwise combinations of other nicotinic subunits were not blocked by 112 nMα-conotoxin-MII. On perfused striatal synaptosomes and slices, α-conotoxin-MII dose-dependently inhibited [³H]dopamine release evoked by 1 µM (±)-anatoxin-a with IC₅₀ values of 24.3 ± 2.9 and 17.3 ± 0.1 nM, respectively. The dose-response curve was shifted to the right with increasing agonist concentrations. However, the maximal inhibition of responses achieved by α-conotoxin-MII (112 nM) was 44.9 ± 5.4% for synaptosomes and 25.0 ± 4.1% for slices, compared with an inhibition by 10 µM mecamylamine of 77.9 ± 3.7 and 88.0 ± 2.1%, respectively. These results suggest the presence of presynaptic α3β2-like nAChRs on striatal dopaminergic terminals, but the incomplete block of (±)-anatoxin-a-evoked [³H]dopamine release by α-conotoxin-MII also supports the participation of nAChRs composed of other subunits. The lower inhibition found in slices is consistent with an additional indirect nicotinic stimulation of dopamine release via an α-conotoxin-MII-insensitive nAChR.