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.
acetylcholine; nicotinic; epibatidine; α-bungarotoxin; suberyldicholine
The diversity of nicotinic acetylcholine receptor (nAChR) subtypes was explored by measuring the effects of gene deletion and pharmacological diversity of epibatidine binding sites in mouse brain. All epibatidine binding sites require expression of either the α7, β2, or β4 subunit. In agreement with general belief, the α4β2*-nAChR and α7-nAChR subtypes are major components of the epibatidine binding sites. α4β2*-nAChR sites account for approximately 70% of total high- and low-affinity epibatidine binding sites, while α7-nAChR accounts for 16% of the total sites all of which have lower affinity for epibatidine. The other subtypes are structurally diverse. Although these minor subtypes account for only 14% of total binding in whole brain, they are expressed at relatively high concentrations in specific brain areas indicating unique functional roles.
Neuronal nicotinic acetylcholine receptors; Epibatidine; Null mutant mice; α-Bungarotoxin; Cytisine; α-Conotoxin MII
In 1992, John Daly et al. reported the isolation and structure determination of epibatidine. Epibatidine’s unique structure and its potent nicotinic agonist activity have had a tremendous impact on nicotine receptor research. This research has led to a better understanding of the nicotinic acetylcholine receptor (nAChR) pharmacophore and to epibatidine analogues with potential as pharmacotherapies for treating various CNS disorders. In this study, we report the synthesis, receptor binding ([3H]epibatidine and [125I]iodoMLA), and in vivo pharmacological properties (mouse tail flick, hot plate, hypothermia, and spontaneous activity) of a series of 3′-(substituted phenyl)epibatidine analogues (5a–m). Results from these studies have added to the understanding of the nAChR pharmacophore and led to nicotinic partial agonists that may have potential for smoking cessation. All the analogues had affinities for the α4β2 nAChR similar to epibatidine (1). 3′-(3-Dimethylaminophenyl)epibatidine (5m) has a nicotinic partial agonist pharmacological profile similar to the smoking cessation drug varenicline. Other analogues are partial agonists with varying degrees of nicotinic functional agonist and antagonist activity. 3′-(3-Aminophenyl)epibatidine (5j) is a more potent functional agonist and antagonist in all tests than varenicline. 3′-(3-Fluorophenyl)epibatidine and 3′-(3-chlorophenyl)epibatidine (5c and 5e) are more potent than varenicline when tested as agonists in four pharmacological tests and antagonists when evaluated against nicotine in the analgesia hot-plate test.
Cellular membranes obtained from the 1321N1 and A172 astrocytoma cell lines were immobilized on a chromatographic phase to create cellular membrane affinity chromatography (CMAC) columns, CMAC(1321N1) and CMAC(A172). The columns were characterized using frontal affinity chromatography with [3H]-epibatidine as the marker ligand and epibatidine, nicotine, and methyllycaconitine as the displacers. The results indicated that the columns contained homomeric α7 nicotinic acetylcholine receptors (α7 nAChR) and heteromeric nicotinic acetylcholine receptors (αxβy nAChRs), which was confirmed by the addition of subtype-specific inhibitors, κ-bungarotoxin (α7 nAChR) and K-bungarotoxin (αxβy nAChR) to the mobile phase. The presence of two additional ligand-gated ion channels (LGICs), γ-aminobutyric acid (GABAA) and N-methyl-d-aspartic acid (NMDA), was established using frontal affinity chromatography with flunitrazepam and diazepam (GABAA receptor) and MK-801 and NMDA (NMDA receptor). The presence of the four LGICs was confirmed using confocal microscopy and flow cytometry. The results indicate that the CMAC(1321N1) and CMAC(A172) columns contain four independently functioning LGICs, that the columns can be used to characterize binding affinities of small molecules to each of the receptors, and that the CMAC approach can be used to probe the expression of endogenous membrane receptors.
Neuronal nicotinic acetylcholine receptors (nAChR) composed of α4 + β2 subunits, the high affinity nicotine-binding site in the mammalian brain, up-regulate in response to chronic nicotine exposure. The identities of endogenous mediators of this process are unknown. We find that choline also up-regulates α4 + β2 nAChRs stably expressed by HEK293 cells as measured by increased [3H]epibatidine density. Choline-mediated up-regulation is dose-dependent and corresponds with an increase in β2 subunit protein expression. The choline kinase inhibitor hemicholinium-3 inhibits ∼60% of choline-mediated up-regulation revealing both an HC3-dependent and -independent pathway. Furthermore, choline-mediated up-regulation is not additive with up-regulation agents such as nicotine, but it is additive with weaker promoters of the up-regulation process. When co-applied with the pro-inflammatory cytokine tumor necrosis factor α, choline-mediated up-regulation is increased further through a mechanism that includes an increase in both α4 and β2 protein expression, and this is inhibited by the p38 MAPK inhibitor SB202190. These findings extend the view that up-regulation of α4 + β2 nAChRs is a normal physiological response to altered metabolic and inflammatory conditions.
Inflammation; Nicotinic Acetylcholine Receptors; p38 MAPK; Receptor Regulation; Tumor Necrosis Factor (TNF); Choline
In the enteric nervous system (ENS) excitatory nicotinic cholinergic transmission is mediated by neuronal nicotinic acetylcholine receptors (nAChR) and is critical for the regulation of gastric motility. nAChRs are ligand-gated pentameric ion channels found in the central and peripheral nervous systems. The expression of heteromeric nAChR and receptor subunit mRNAs was investigated in the neonatal rat ENS using receptor autoradiography with the radiolabeled ligand 125I-Epibatidine, and in situ hybridization with subtype specific probes for ligand binding alpha (α2, α3, α4, α5, α6) and structural beta (β2, β3, β4) subunits. The results showed strong nicotine sensitive binding of 125I-Epibatidine around the stomach, and small and large intestines. The binding was partially displaced by A85380, a nicotinic ligand which differentiates between different heteromeric nAChR subtypes, suggesting a mixed receptor population. Radioactive in situ hybridization detected expression of α3, α5, α7, β2 and β4 mRNA in the myenteric plexus of the stomach, and small and large intestines. In the submucosal plexus of the small and large intestines expression of α3, α5 and β4 was found in some ganglia. There was no signal for α4, α6 and β3 in the ENS but positive hybridization signal for α2 transcripts was seen in some areas of the small intestines. However, the signal was not associated with any ganglion cells. The results confirm the presence of heteromeric nAChRs in the ENS similar to those found in the peripheral nervous system, with the majority being composed of α3(α5)β4, and a few α3β2 nAChRs. In addition, homomeric α7 nAChRs could be present.
gastrointestinal; nicotine; myenteric plexus; submucosal; in situ hybridization; epibatidine
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.
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-evoked neurotransmitter release.
Antagonist activity at the alpha3beta4 nicotinic acetylcholine receptor (nAChR) is thought to contribute to the anti-addictive properties of several compounds. However, truly selective ligands for the α3β4 nAChR have not been available. We report the discovery and SAR of a novel class of compounds that bind to the α3β4 nAChR and have no measurable affinity for the α4β2 or α7 subtypes. In functional assays the lead compound antagonized epibatidine-induced Ca2+ flux in α3β4-transfected cells in a noncompetitive manner.
A series of 3’-(substituted phenyl)deschloroepibatidine analogs (5a–j) were synthesized. The α4β2* and α7 nicotinic acetylcholine receptor (nAChR) binding properties and functional activity in the tail-flick, hot-plate, locomotor, and body temperature tests in mice of 5a–j were compared to those of the nAChR agonist, nicotine (1), epibatidine (4), and deschloroepibatidine (13) the partial agonist, varenicline (3) and the antagonist 2’-fluoro-3’-(substituted phenyl)deschloroepibatidine analogs (7a–j). Unlike epibatidine and deschloroepibatidine, which are potent agonists in the tail-flick test, 5a–k show no or very low antinociceptive activity in the tail-flick or hot-plate test. However, they are potent antagonists in nicotine-induced antinociception in the tail-flick test, but weaker than the corresponding 2’-fluoro-3’-(substituted phenyl)deschloroepibatidines.
nicotinic antagonist; varenicline; nAChR bindng; epibatidine analogs
Agonists activating nicotinic acetylcholine receptors (nAChR) include potential therapeutic agents and also toxicants such as epibatidine and neonicotinoid insecticides with a chloropyridinyl substituent. Nicotinic agonist interactions with mollusk (Aplysia californica) acetylcholine binding protein, a soluble surrogate of the nAChR extracellular domain, are precisely defined by scanning with 17 methionine and tyrosine mutants within the binding site by photoaffinity labeling with 5-azido-6-chloropyridin-3-yl probes that have similar affinities to their nonazido counterparts. Methionine and tryrosine are the only residues found derivatized, and their reactivity exquisitely depends on the direction of the azido moiety and its apposition to the reactive amino acid side chains.
Nine nicotinic receptor subunits are expressed in the central nervous system indicating that a variety of nicotinic acetylcholine receptors (nAChR) may be assembled. A useful method with which to identify putative nAChR is radioligand binding. In the current study the binding of [125I]α-bungarotoxin, [125I]α-conotoxinMII, 5[125I]-3-((2S)-azetidinylmethoxy)pyridine (A-85380), and [125I]epibatidine has been measured autoradiographically to provide data on many nAChR binding sites. Each binding sites was evaluated semiquantitatively for samples prepared from wild-type and α2, α4, α6, α7, β2, β4, α5 and β3 null mutant mice. Deletion of the α7 subunit completely and selectively eliminated [125I]α-bungarotoxin binding. The binding of [125I]αConotoxinMII was eliminated in most brain regions by deletion of either the α6 or β2 subunit and is reduced by deletion of either the α4 or β3 subunit. The binding of 5[125I]A-85380 was completely eliminated by deletion of the β2 subunit and significantly reduced by deletion of the α4 subunit. Most, but not all, α4-independent sites require expression of the α6 subunit. The effect of gene deletion on total [125I]epibatidine binding was very similar to that on [125I]A-85380 binding. [125I]Epibatidine also labels β4* nAChR, which was readily apparent for incubations conducted in the presence of 100 nM cytisine. The effects of α3 gene deletion could not be evaluated, but persistence of residual sites implies the expression of α3* nAChR. Taken together these results confirm and extend previously published evaluations of the effect of nAChR gene deletion and help to define the nAChR subtypes measurable by ligand binding.
nicotinic acetylcholine receptor; null mutant mice; epibatidine; A-85380; α-conotoxin MII; α-bungarotoxin
The spontaneously hypertensive rat (SHR) is widely used as a model of attention-deficit/hyperactivity disorder (ADHD). Deficits in central nicotinic receptors (nAChRs) have previously been observed in SHRs, which is interesting since epidemiological studies have identified an association between smoking and ADHD symptoms in humans. Here we examine whether nAChR deficits in SHRs compared to Wistar Kyoto rat (WKY) controls are nAChR subtype-specific and whether these deficits correlate with changes at the level of mRNA transcription in specific brain regions. Levels of binding sites (Bmax) and dissociation constants (Kd) for nAChRs were determined from saturation curves of high-affinity [3H]epibatidine- and [3H]MLA binding to membranes from cortex, striatum, hippocampus and cerebellum. In additional brain regions, nAChRs were examined by autoradiography with [125I]A-85380 and [125I]α-bungarotoxin. Levels of mRNA encoding nAChR subunits were measured using quantitative real-time PCR (qPCR). We show that the number of α4β2 nAChR binding sites is lower globally in the SHR brain compared to WKY in the absence of significant differences in mRNA levels, with the exception of lower α4 mRNA in cerebellum of SHR compared to WKY. Further, nAChR deficits were subtype- specific because no strain difference was found in α7 nAChR binding or α7 mRNA levels. Our results suggest that the lower α4β2 nAChR number in SHR compared to WKY may be a consequence of dysfunctional post-transcriptional regulation of nAChRs.
Attention-deficit/hyperactivity disorder; mRNA; nicotinic receptor; post translational; spontaneous hypertensive rat; Wistar Kyoto rat
The development of nicotinic acetylcholine receptor (nAChR) agonists, particularly those that discriminate between neuronal nAChR subtypes, hold promise as potential therapeutic agents for many neurological diseases and disorders. To this end, we photoaffinity labeled human α4β2 and rat α4β4 nAChRs affinity-purified from stably transfected HEK-293 cells, with the agonists [125I]epibatidine and 5[125I]A-85380. Our results show that both agonists photoincorporated into the β4 subunit with little or no labeling of the β2 and α4 subunits respectively. [125I]epibatidine labeling in the β4 subunit was mapped to two overlapping proteolytic fragments that begin at β4V102 and contain Loop E (β4I109-P120) of the agonist binding site. We were unable to identify labeled amino acid(s) in Loop E by protein sequencing, but we were able to demonstrate that β4Q117 in Loop E is the principal site of [125I]epibatidine labeling. This was accomplished by substituting residues in the β2 subunit with the β4 homologs and finding [125I]epibatidine labeling in β4 and β2F119Q subunits with little, if any, labeling in α4, β2, or β2S113R subunits. Finally, functional studies established that the β2F119/β4Q117 position is an important determinant of the receptor subtype-selectivity of the agonist 5I-A-85380, affecting both binding affinity and channel activation.
Cys-loop receptor; neuronal nicotinic receptor; HEK-293 cell; affinity-purification; photoaffinity labeling; protein sequencing
Nicotinic acetylcholine receptors (nAChRs) of the cerebral cortex and cerebellum of rats were evaluated by a radioligand binding assay, employing tissue segments, or homogenates as materials. [3H]-epibatidine specifically bound to nAChRs in rat cortex or cerebellum, but the dissociation constants for [3H]-epibatidine differed between segments and homogenates (187 pM for segments and 42 pM for homogenates in the cortex and 160 pM for segments and 84 pM for homogenates in the cerebellum). The abundance of total nAChRs was approximately 310 fmol/mg protein in the segments of cortex and 170 fmol/mg protein in the segments of cerebellum, which were significantly higher than those estimated in the homogenates (115 fmol/mg protein in the homogenates of the cortex and 76 fmol/mg protein in the homogenates of the cerebellum). Most of the [3H]-epibatidine binding sites in the cortex segments (approximately 70% of the population) showed high affinity for nicotine (pKi = 7.9), dihydro-β-erythroidine, and cytisine, but the binding sites in the cerebellum segments had slightly lower affinity for nicotine (pKi = 7.1). An upregulation of nAChRs by chronic administration of nicotine was observed in the cortex segments but not in the cerebellum segments with [3H]-epibatidine as a ligand. The upregulation in the cortex was caused by a specific increase in the high-affinity sites for nicotine (probably α4β2). The present study shows that the native environment of nAChRs is important for a precise quantitative as well as qualitative estimation of nAChRs in rat brain.
nicotinic receptor; tissue-segment binding; upregulation
The analog of epibatidine having a fluoro- substituent at the 3’ position of the pyridine ring has been recently developed and shown to possess binding affinity in the pM range to α4β2 nAChRs and in the nM range to α7 nAChRs and to exhibit potent agonist activity in nicotine-induced analgesia tests. Here we used patch-clamp technique in a whole-cell configuration to compare functional activity of 3’-fluoroepibatidine to that of epibatidine by itself on recombinant α4β2, α7 and α3β4 neuronal nAChRs. The agonist effect of (±)-epibatidine was partial and yielded comparable EC50s of 0.012 µM (72% efficacy) and 0.027 µM (81% efficacy) at α4β2 and α3β4 nAChRs, respectively, but was full at α7 nAChRs with an EC50 of 4.8 µM. Testing of the analog at different concentrations revealed that it acts as a full agonist with an EC50 of 0.36 µM at α4β2 nAChRs and induces partial agonist effect (66% efficacy) at α7 nAChRs with an EC50 of 9.8 µM and an IC50 corresponding to 225 µM. In contrast, the analog caused only 24% maximal activation at the range of concentrations from 0.1–100 µM and, in addition, induced an inhibition of α3β4 nAChR function with an IC50 of 8.3 µM. Our functional data, which are in agreement with previous binding and behavioral findings, demonstrate that 3’-fluoro substitution in the pyridine ring of epibatidine results in an improved pharmacological profile as observed by an increased efficacy and selectivity for α4β2 versus α3β4 nAChRs.
recombinant neuronal nicotinic receptors; epibatidine; epibatidine analogs; whole-cell; nicotine-induced analgesia; binding
Clinical and preclinical studies suggest that regulation of nicotinic acetylcholine receptors (nAChR) maybe involved in the etiology of withdrawal symptoms.
We evaluated heteromeric nAChR regulation via [3H]epibatidine binding following cessation of chronic nicotine or varenicline treatment. Animals were concurrently tested in the marble-burying test to evaluate treatment-related effects.
We found that both nicotine (18 mg/kg/day, free base) and varenicline (1.8 mg/kg/day) chronically administered for 14 days upregulated nAChRs significantly in the cortex, hippocampus, striatum, and thalamus. The duration of upregulation (up to 72 hr) was both drug and region specific. In addition to nAChR upregulation, chronic administration of both nicotine and varenicline had anxiolytic-like effects in the marble-burying test. This effect was maintained for 48 hr following cessation of varenicline but was absent 24 hr following cessation from nicotine. Additionally, marble-burying behavior positively correlated to the regulation of cortical nAChRs following cessation of either treatment.
Varenicline has been shown to be an efficacious smoking cessation aid, with a proposed mechanism of action that includes modulation of dopamine release in reward areas of the brain. Our studies show that varenicline elicits both anxiolytic effects in the marble-burying test as well as region- and time-specific receptor upregulation. These findings suggest receptor upregulation as a mechanism for its efficacy as a smoking cessation therapy.
Nitrosamines are carcinogens formed in the mammalian organism from amine precursors contained in food, beverages, cosmetics and drugs. The potent carcinogen, NNK, and the weaker carcinogen, NNN, are nitrosamines formed from nicotine. Metabolites of the nitrosamines react with DNA to form adducts responsible for genotoxic effects. We have identified NNK as a high affinity agonist for the alpha7 nicotinic acetylcholine receptor (α7nAChR) whereas NNN bound with high affinity to epibatidine-sensitive nAChRs. Diethylnitrosamine (DEN) bound to both receptors but with lower affinity. High levels of the α7nAChR were expressed in human small cell lung cancer (SCLC) cell lines and in hamster pulmonary neuroendocrine cells (PNECs), which serve as a model for the cell of origin of human SCLC. Exposure of SCLC or PNECs to NNK or nicotine increased expression of the a7nAChR and caused influx of Ca2+, activation of PKC, Raf-1, ERK1/2, and c-myc, resulting in the stimulation of cell proliferation. Signaling via the α7nAChR was enhanced when cells were maintained in an environment of 10–15% CO2 similar to that in the diseased lung. Hamsters with hyperoxia-induced pulmonary fibrosis developed neuroendocrine lung carcinomas similar to human SCLC when treated with NNK, DEN, or nicotine. The development of the NNK-induced tumors was prevented by green tea or theophylline. The beta-adrenergic receptor agonist, isoproterenol or theophylline blocked NNK-induced cell proliferation in vitro. NNK and nicotine-induced hyperactivity of the α7nAChR/RAF/ERK1/2 pathway thus appears to play a crucial role in the development of SCLC in smokers and could be targeted for cancer prevention.
Tobacco nitrosamines; nicotinic acetylcholine receptor; small cell lung cancer; pulmonary neuroendocrine cell
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.
Background and purpose
Nicotinic agonists increase sympathetic field-stimulus-evoked contraction of the rodent vas deferens, presumably by increasing evoked neurotransmitter release. This presumption was tested in two species.
The effect of the nicotinic acetylcholine receptor (nAChR) agonist epibatidine on neurotransmitter release in mouse and guinea pig isolated vas deferens was investigated using contraction studies and conventional intracellular recording techniques.
In 12 of 14 mouse vasa deferentia, slow bath application of epibatidine (100 nM) had no significant effect on excitatory junction potential (EJP) amplitude and spontaneous EJP (SEJP) frequency. However, rapid application of epibatidine to the mouse vas deferens caused an increase in SEJP frequency (by 530%), with no effect on EJP amplitude. Despite the absence of an effect on EJPs, electrically-evoked contractions of the mouse vas deferens were significantly increased in the presence of epibatidine (by 50%). A transient contraction was reliably induced by a higher epibatidine concentration (1 μM). This contraction was significantly reduced in the presence of prazosin, tetrodotoxin, or α,β-methyleneATP. Epibatidine did not induce a contraction in the presence of a combination of prazosin, α,β-methyleneATP and cyclopentolate.
In the guinea pig, bath-applied epibatidine potentiated EJP amplitude in a biphasic pattern, lasting for at least 30 minutes.
Conclusion and Implications
The nAChR-mediated augmentation of neurogenic contraction is indeed prejunctional, but in the mouse arises from an increase in spontaneous neurotransmitter release that primes smooth muscle for subsequent contraction, while in the guinea pig there is a direct augmentation of evoked neurotransmitter (ATP) release.
Prejunctional; nicotinic; epibatidine; intracellular recording; mouse; guinea pig; vas deferens; sympathetic; electrophysiology; neurotransmission
The electron diffraction structure of nicotinic acetylcholine receptor (nAChR) from Torpedo marmorata and the X-ray crystallographic structure of acetylcholine binding protein (AChBP) are providing new answers to persistent questions about how nAChRs function as biophysical machines and as participants in cellular and systems physiology. New high-resolution information about nAChR structures might come from advances in crystallography and NMR, from extracellular domain nAChRs as high fidelity models, and from prokaryotic nicotinoid proteins. At the level of biophysics, structures of different nAChRs with different pharmacological profiles and kinetics will help describe how agonists and antagonists bind to orthosteric binding sites, how allosteric modulators affect function by binding outside these sites, how nAChRs control ion flow, and how large cytoplasmic domains affect function. At the level of cellular and systems physiology, structures of nAChRs will help characterize interactions with other cellular components, including lipids and trafficking and signaling proteins, and contribute to understanding the roles of nAChRs in addiction, neurodegeneration, and mental illness. Understanding nAChRs at an atomic level will be important for designing interventions for these pathologies.
Acetylcholine; Addiction; Neurodegeneration; Nicotine; Protein Design; Protein Folding; Protein Structure; Cys-Loop Receptors; Review
The nicotinic acetylcholine receptors (nAChR) assembled from α4 and β2 subunits are the most densely expressed subtype in the brain. Concentration-effect curves for agonist activation of α4β2*-nAChR are biphasic. This biphasic agonist sensitivity is ascribed to differences in subunit stoichiometry. The studies described here evaluated desensitization elicited by low concentrations of epibatidine, nicotine, cytisine or methylcarbachol of brain α4β2-nAChR function measured with acetylcholine stimulated 86Rb+ efflux from mouse thalamic synaptosomes. Each agonist elicited concentration-dependent desensitization. The agonists differed in potency. However, IC50 values for each agonist for desensitization of 86Rb+ efflux both with high (EC50≈3 μM) and low (EC50≈ 150 μM) acetylcholine sensitivity were not significantly different. Concentrations required to elicit desensitization were higher that their respective KD values for receptor binding. Even though the two components of α4β2*-nAChR mediated 86Rb+ efflux from mouse brain differ markedly in EC50 values for agonist activation, they are equally sensitive to desensitization by exposure to low agonist concentrations. Mice were also chronically treated with nicotine by continuous infusion of 0, 0.5 or 4.0 mg/kg/hr and desensitization induced by nicotine was evaluated. Consistent with previous results, chronic nicotine treatment increased the density of epibatidine binding sites. Acute exposure to nicotine also elicited concentration-dependent desensitization of both high sensitivity and low sensitivity acetylcholine-stimulated 86Rb+ efflux from cortical and thalamic synaptosomes. Although chronic nicotine treatment reduced maximal 86Rb+ efflux from thalamus, IC50 values in both brain regions were unaffected by chronic nicotine treatment.
nicotinic acetylcholine receptor; desensitization; nicotine; epibatidine; cytisine; methylcarbachol
A series of methyllycaconitine (1a, MLA) analogs was synthesized where the (S)-2-methylsuccinimidobenzoyl group in MLA was replaced with a (R)-2-methyl, 2,2-dimethyl-, 2,3-dimethyl, 2-phenyl- and 2-cyclohexylsuccinimidobenzoyl (1b–f) group. The analogs 1b–f were evaluated for their inhibition of [125I]iodo MLA binding at rat brain α7 nicotinic acetylcholine receptors (nAChR). In order to determine selectivity, MLA and the analogs 1b–f were evaluated for inhibition of binding to rat brain α, β nAChR using [3H]epibatidine. At the α7 nAChR, MLA showed a Ki value of 0.87 nM, analogs 1b–e possessed Ki values of 1.68–2.16 nM, and 1f showed a Ki value of 26.8 nM. Surprisingly, the analog 1e containing the large phenyl substituent (Ki = 1.68 nM) possessed the highest affinity. None of the compounds possessed appreciable affinity for α, β nAChRs. MLA antagonized nicotine-induced seizures with an AD50 = 2mg/kg. None of the MLA analogs were as potent as MLA in this assay. MLA and all of the MLA analogs, with the exception of 1b, antagonized nicotine’s antinociceptive effects in the tail-flick assay. Compound 1c (Ki = 1.78 nM at α7 nAChR) with an AD50 value of 1.8 mg/kg was 6.7 times more potent than MLA (AD50 = 12 mg/kg) in antagonizing nicotine’s antinociceptive effects but was 5-fold less potent than MLA in blocking nicotine-induced seizures. Since MLA has been reported to show neuroprotection against β-amyloid1–42, these new analogs which have high α7 nAChR affinity and good selectivity relative to α, β nAChRs will be useful biological tools for studying the effects of α7 nAChR antagonist and neuroprotection.
methyllycaconitine; α7 nAChR; antagonist; and MLA analogs
[3H]Epibatidine binds to nAChR subtypes in mouse brain with higher (KD≈0.02 nM) and lower affinity (KD≈7 nM), which can be further subdivided through inhibition by selected agonists and antagonists. These subsets are differentially affected by targeted deletion of α7, β2 or β4 subunits. Most, but not all, higher and lower affinity binding sites require β2 (Marks et al., 2006). Effects of functional α4 gene deletion are reported here. Deletion of α4 virtually eliminated cytisine-sensitive, higher-affinity [3H]epibatidine binding as did β2 deletion, confirming that these sites are α4β2*-nAChR. Cytisine-resistant, higher-affinity [3H]epibatidine binding sites are diverse and some of these sites require α4 expression. Lower affinity [3H]epibatidine binding sites are also heterogeneous and can be subdivided into α-bungarotoxin-sensitive and - resistant components. Deleting α4 did not affect the α-bungarotoxin-sensitive component, but markedly reduced the α-bungarotoxin–resistant component. This effect was similar, but not quite identical, to the effect of β2 deletion. This provides the first evidence that lower-affinity epibatidine binding sites in the brain require expression of α4 subunits. The effects of α4 gene targeting on receptor function were measured using a 86Rb+ efflux assay. Concentration-effect curves for ACh-stimulated 86Rb+ efflux are biphasic (EC50 values = 3.3 µM and 300 µM). Targeting α4 produced substantial gene-dose dependent reductions in both phases in whole brain and in most of the 14 brain regions assayed. These effects are very similar to those following deletion of β2. Thus, α4β2*–nAChRs mediate a significant fraction of both phases of ACh stimulated 86Rb+ efflux.
Null mutant mice; Nicotinic acetylcholine receptor; Epibatidine; 86Rb+ efflux; Cytisine; Dihydro-β-erythroidine
The most abundant subtype of cerebral nicotinic acetylcholine receptors (nAChR), α4β2, plays a critical role in various brain functions and pathological states. Imaging agents suitable for visualization and quantification of α4β2 nAChRs by positron emission tomography (PET) would present unique opportunities to define the function and pharmacology of the nAChRs in the living human brain. In this study, we report the synthesis, nAChR binding affinity, and pharmacological properties of several novel 3-pyridyl ether compounds. Most of these derivatives displayed a high affinity to the nAChR and a high subtype selectivity for α4β2-nAChR. Three of these novel nAChR ligands were radiolabeled with the positron-emitting isotope 11C and evaluated in animal studies as potential PET radiotracers for imaging of cerebral nAChRs with improved brain kinetics.