Expression of nicotinic acetylcholine receptors in human and rat adrenal medulla.

Neuronal nicotinic receptors (nAChRs) are expressed in the brain but also in the peripheral tissues including the adrenal medulla. However, it is unclear which nAChRs are present in the human adrenal medulla. In the study, receptor binding assay, Western blot and RT-PCR have been performed to investigate the expression of nAChRs in adrenal medulla from human, rat and mouse. The results showed that in human adult adrenal medulla, mRNAs for nAChR alpha3, alpha4, alpha5, alpha7, beta2, beta3, and beta4 subunits but not beta2 in the fetal human adrenal medulla were expressed. Saturation binding of [3H]epibatidine showed two binding sites in human aged adrenal medulla. The specific binding of [3H]epibatidine (0.1 nM) was significantly higher in human fetal compared to human aged adrenal medulla. mRNAs for the alpha3, alpha4, alpha5, alpha7, beta2, and beta4 subunits but not the beta3 were detectable in adult rat and mouse adrenal medulla. No differences in gene-expression of the nAChRs were observed between new born, adult and aged rat adrenal medulla. Saturation binding of [3H]epibatidine showed only one binding site in rat adrenal medulla. Lower protein levels for the nAChR subunits were observed in the rat adrenal medulla compared to rat brain. There was lower protein levels of the nAChRs in aged rat adrenal medulla compared to the young rats. Sub-chronic treatment of nicotine to rats did not influence level of the nAChRs in the adrenal medulla. In conclusion, the expression of nAChRs in adrenal medulla is age- related and species dependent.     

Neuronal nicotinic receptor deficits in Alzheimer patients with the Swedish amyloid precursor protein 670/671 mutation

The influence of beta-amyloid on cholinergic neurotransmission was studied by measuring alterations in nicotinic acetylcholine receptors (nAChRs) in autopsy brain tissue from subjects carrying the Swedish amyloid precursor protein (APP) 670/671 mutation. Significant reductions in numbers of nAChRs were observed in various cortical regions of the Swedish 670/671 APP mutation family subjects (-73 to -87%) as well as in sporadic Alzheimer's disease (AD) cases (-37 to -57%) using the nicotinic agonists [3H]epibatidine and [3H]nicotine, which bind with high affinity to both alpha3 and alpha4 and to alpha4 nAChR subtypes, respectively. Saturation binding studies with [3H]epibatidine revealed two binding sites in the parietal cortex of AD subjects and controls. A significant decrease in Bmax (-82%) for the high-affinity site was observed in APP 670/671 subjects with no change in K(D) compared with controls (0.018 nM APP 670/671; 0.036 nM control). The highest load of neuronal plaques (NPs) was observed in the parietal cortex of APP 670/671 brains, whereas the number of [3H]nicotine binding sites was less impaired compared with other cortical brain regions. Except for a positive significant correlation between the number of [3H]nicotine binding sites and number of NPs in the parietal cortex, no strict correlation was observed between nAChR deficits and the presence of NPs and neurofibrillary tangles, suggesting that these different processes may be closely related but not strictly dependent on each other.     

Extracellular domain nicotinic acetylcholine receptors formed by alpha4 and beta2 subunits

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

Interaction of the nicotinic agonist (R,S)-3-pyridyl-1-methyl-2-(3-pyridyl)-azetidine (MPA) with nicotinic acetylcholine receptor subtypes expressed in cell lines and rat cortex

The interaction of the nicotinic agonist (R,S)-3-pyridyl-1-methyl-2-(3-pyridyl)-azetidine (MPA) with different nicotinic acetylcholine receptor (nAChR) subtypes was studied in cell lines and rat cortex. MPA showed an affinity (Ki = 1.21 nM) which was higher than anatoxin-a > (−)-nicotine > (+)-[R]nornicotine > (−)-[S]nornicotine > and (+)-nicotine, but lower than cytisine (Ki = 0.46 nM) in competing for (−)-[³H]nicotine binding in M10 cells, which stably express the recombinant ₄β₂ nAChR subtype. A one-binding site model was observed in all competing experiments between (−)-[³H]nicotine binding and each of the agonists studied in M10 cells. MPA showed a 13-fold higher affinity for (−)-[³H]nicotine binding sites compared to the [³H]epibatidine binding sites in rat cortical membranes. In human neuroblastoma SH-SY5Y cells, which predominantly express the ₃ nAChR subunit mRNA, MPA displaced [³H]epibatidine binding from a single population of the binding sites with an affinity in the same nM range as that observed MPA in displacing [³H]epibatidine binding in rat cortical membranes. Chronic treatment of M10 cells with MPA significantly up-regulated the number of (−)-[³H]nicotine binding sites in a concentration dependent manner. Thus MPA appears to have higher affinity to 4-subunit containing receptor subtype than ₃-subunit containing receptor subtype of nAChRs. Furthermore MPA binds to ₄β₂ receptor subtype with higher affinity than (−)-nicotine and behaves, opposite to cytisine, as a full agonist in up-regulating the number of nAChRs. © 1998 Elsevier Science Ltd. All rights reserved.     

Measuring nicotinic receptors with characteristics of alpha4beta2, alpha3beta2 and alpha3beta4 subtypes in rat tissues by autoradiography

Comparison of [125I]epibatidine and 5-[125I]iodo-3-(2-azetidinylmethoxy)pyridine ([125I]A-85380) autoradiography showed evidence for nicotinic receptor heterogeneity. To identify the receptor subtypes, we performed [125I]epibatidine autoradiography in the presence of cytisine or A-85380. By comparing these results with binding data from human embryonic kidney (HEK) 293 cells stably transfected with different combinations of rat nicotinic receptor subunits, we were able to quantify three distinct populations of [125I]epibatidine binding sites with characteristics of alpha4beta2, alpha3beta2 and alpha3beta4 receptors. Although the predominant subtype in rat brain was alpha4beta2, non-alpha4beta2 binding sites were prominent in many regions. In the habenulo-peduncular system, cerebellum, substantia gelatinosa, and many medullary nuclei, alpha3beta4-like binding accounted for more than 40% of [125I]epibatidine binding, and nearly all binding in superior cervical ganglion and pineal gland. Other regions enriched in alpha3beta4-like binding included locus ceruleus, dorsal tegmentum, subiculum and anteroventral thalamic nucleus. Regions enriched in alpha3beta2-like binding included the habenulo-peduncular system, many visual system structures, certain geniculate nuclei, and dopaminergic regions. The combination of autoradiography using a broad spectrum radioligand in the presence of selective competitors, and data from binding to defined receptor subtypes in expression systems, allowed us to quantify the relative populations of these three subtypes.     

Methadone increases intracellular calcium in SH-SY5Y and SH-EP1-halpha7 cells by activating neuronal nicotinic acetylcholine receptors

(-)-Methadone acts as an agonist at opioid receptors. Both (+)- and (-)-enantiomers of methadone have been suggested to be potent non-competitive antagonists of alpha3beta4 neuronal nicotinic acetylcholine receptors (nAChRs). In the present study, we have examined interactions of methadone with nAChRs by using receptor binding assays, patch-clamp recording and calcium fluorometry imaging with SH-SY5Y cells naturally expressing alpha7 and alpha3* nAChR subtypes and SH-EP1-halpha7 cells heterologously expressing human alpha7 nAChRs. Methadone potently inhibited binding of [3H]methyllycaconitine to alpha7 nAChRs and that of [3H]epibatidine to alpha3* nAChRs. Methadone pretreatment induced up-regulation of epibatidine binding sites in SH-SY5Y cells. Using whole-cell patch-clamp recording, both isomers of methadone activated cation currents via mecamylamine-sensitive nAChRs in SH-SY5Y cells. Nicotine and both (+)- and (-)-methadone evoked increases in [Ca2+]i in both fluo-3AM loaded cell lines, and these effects were blocked by mecamylamine and by the alpha7 selective antagonist methyllycaconitine, suggesting effects of methadone as alpha7-nAChR agonist. Sensitivity of sustained nicotine and methadone effects to blockade by CdCl2, ryanodine and xestospongin-c implicates voltage-operated Ca2+ channels and intracellular Ca2+ stores as downstream modulators of elevated [Ca2+]i. Collectively, our results suggest that methadone engages in complex and potentially pharmacologically significant interactions with nAChRs.     

Binding and functional activity of nicotinic cholinergic receptors in selected rat brain regions are increased following long-term but not short-term nicotine treatment

Chronic nicotine exposure up-regulates neuronal nicotinic receptors, but the functional consequences for these receptors is less well understood. Following 2 weeks of nicotine or saline treatment by osmotic minipump, the functional activity of nicotinic receptors was measured by concentration-response curves for epibatidine-stimulated (86)Rb efflux. Nicotine-treated animals had a significantly higher maximal efflux in cerebral cortex and superior colliculus, but not in thalamus or interpeduncular nucleus plus medial habenula. This increase was confirmed in a separate experiment with stimulation by single concentrations of epibatidine (cortex, superior colliculus) or nicotine (cortex only). Chronic nicotine did not alter (86)Rb efflux stimulated by cytisine, an alpha3beta4-selective agonist, or by potassium chloride, in any region. Short-term (16 h) nicotine exposure caused no changes in either (86)Rb efflux or receptor binding measured with [(3)H]epibatidine. Binding was significantly increased after 2 weeks nicotine exposure in cortex, superior colliculus and thalamus, but not in interpeduncular nucleus plus medial habenula. The increases in epibatidine-stimulated (86)Rb efflux in the four regions tested was linearly correlated with the increases in [(3)H]epibatidine binding in these regions (R(2) = 0.91), suggesting that rat brain receptors up-regulated by chronic nicotine are active. These results have important consequences for understanding nicotinic receptor neurobiology in smokers and users of nicotine replacement therapy.     

Neuronal Nicotinic Receptor Deficits in Alzheimer Patients with the Swedish Amyloid Precursor Protein 670/671 Mutation

Abstract : The influence of β‐amyloid on cholinergic neurotransmission was studied by measuring alterations in nicotinic acetylcholine receptors (nAChRs) in autopsy brain tissue from subjects carrying the Swedish amyloid precursor protein (APP) 670/671 mutation. Significant reductions in numbers of nAChRs were observed in various cortical regions of the Swedish 670/671 APP mutation family subjects (‐73 to ‐87%) as well as in sporadic Alzheimer's disease (AD) cases (‐37 to ‐57%) using the nicotinic agonists [³H]epibatidine and [³H]nicotine, which bind with high affinity to both α3 and α4 and to α4 nAChR subtypes, respectively. Saturation binding studies with [³epibatidine revealed two binding sites in the parietal cortex of AD subjects and controls. A significant decrease in B_max (‐82%) for the high‐affinity site was observed in APP 670/671 subjects with no change in K_D compared with controls (0.018 nM APP 670/671 ; 0.036 nM control). The highest load of neuronal plaques (NPs) was observed in the parietal cortex of APP 670/671 brains, whereas the number of [³H]nicotine binding sites was less impaired compared with other cortical brain regions. Except for a positive significant correlation between the number of [³H]nicotine binding sites and number of NPs in the parietal cortex, no strict correlation was observed between nAChR deficits and the presence of NPs and neurofibrillary tangles, suggesting that these different processes may be closely related but not strictly dependent on each other.     

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.     

Pharmacological profiles of recombinant and native insect nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors (nAChRs) are targets for insect-selective neonicotinoid insecticides exemplified by imidacloprid (IMI) and mammalian-selective nicotinoids including nicotine and epibatidine (EPI). Despite their importance, insect nAChRs are poorly understood compared with their vertebrate counterparts. This study characterizes the [(3)H]IMI, [(3)H]EPI, and [(3)H]alpha-bungarotoxin (alpha-BGT) binding sites in hybrid nAChRs consisting of Drosophila melanogaster (fruit fly) or Myzus persicae (peach-potato aphid) alpha2 coassembled with rat beta2 subunits (Dalpha2/Rbeta2 and Mpalpha2/Rbeta2) and compares them with native insect and vertebrate alpha4beta2nAChRs. [(3)H]IMI and [(3)H]EPI bind to Dalpha2/Rbeta2 and Mpalpha2/Rbeta2 hybrids but [(3)H]alpha-BGT does not. In native Drosophila receptors, [(3)H]EPI has a single high-affinity binding site that is independent from that for [(3)H]IMI and, interestingly, overlaps the [(3)H]alpha-BGT site. In the Mpalpha2/Rbeta2 hybrid, [(3)H]IMI and [(3)H]EPI bind to the same site and have similar pharmacological profiles. On considering both neonicotinoids and nicotinoids, the Dalpha2/Rbeta2 and Mpalpha2/Rbeta2 receptors display intermediate pharmacological profiles between those of native insect and vertebrate alpha4beta2 receptors, limiting the use of these hybrid receptors for predictive toxicology. These findings are consistent with the agonist binding site being located at the nAChR subunit interface and indicate that both alpha and beta subunits influence the pharmacological properties of insect nAChRs.     

5-Iodo-A-85380 binds to alpha-conotoxin MII-sensitive nicotinic acetylcholine receptors (nAChRs) as well as alpha4beta2* subtypes

Recent work suggests that 5-iodo-A-85380, a radioiodinated analog of the 3-pyridyl ether A-85380, represents a promising imaging agent for non-invasive, in vivo studies of alphaAbeta2* nicotinic acetylcholine receptors (nAChRs; *denotes receptors containing the indicated subunits), because of its low non-specific binding, low in vivo toxicity and high selectivity for alpha4beta2* nAChRs. As an approach to elucidate nAChR subtypes expressed in striatum, we carried out competitive autoradiography in monkey and rat brain using 5-[125I]iodo-A-85380 ([125I]A-85380) and [125I]alpha-conotoxin MII, a ligand that binds with high affinity to alpha6* and alpha3* nAChRs, but not to alpha4beta2* nAChRs. Although A-85380 is reported to be selective for alpha4beta2* nAChRs, we observed that A-85380 completely inhibited [125I]alpha-conotoxin MII binding in rat striatum and that A-85380 blocked >90% of [125I] alpha-conotoxin MII sites in monkey caudate and putamen. These results suggest that A-85380 binds to non-alpha4beta2* nAChRs, including putative alpha6* nAChRs. Experiments to determine the percentage of [125I]A-85380 sites that contain alpha-conotoxin MII-sensitive (alpha6beta2*) nAChRs indicate that they represent about 10% of [125I]A-85380 sites in rodent striatum and about 30% of sites in monkey caudate and putamen. These data are important for identifying alterations in nicotinic receptor subtypes in Parkinson's disease and other basal ganglia disorders both in in vitro and in in vivo imaging studies.     

Differential Effect of Nicotinic Agonists on the [3H]Norepinephrine Release from Rat Hippocampal Slices

The aim of this study was to investigate the mechanisms involved in the effect of nicotinic agonists on the [³H]norepinephrine ([³H]NE) release from rat hippocampal slices. The stimulatory effect of nicotine, cytisine, epibatidine and anatoxin-A was completely blocked by the nicotinic antagonist mecamylamine (10 M). In contrast, the effect of dimethylphenylpiperazinium (DMPP) was only partially inhibited by mecamylamine but was completely blocked by the NE uptake inhibitor desipramine (DMI, 10 M). Finally, the effect of lobeline was not affected by mecamylamine and was only partially blocked by DMI. Our data indicate that the majority of nicotinic agonists increase the release of [³H]NE exclusively via stimulation of nicotinic acetylcholine receptors (nAChRs). DMPP, in addition to the stimulation of nAChRs, also evokes a carrier-mediated release. Lobeline has no stimulatory effect on nAChRs, induces a carrier-mediated release and has a further action of unidentified mechanism. Our results suggest that special caution is required for the interpretation of data, when DMPP or lobeline are used as nicotinic agonists.     

Up-regulation of cell-surface alpha4beta2 neuronal nicotinic receptors by lower temperature and expression of chimeric subunits.

The predominant nicotinic acetylcholine receptor (nAChR) expressed in vertebrate brain is a pentamer containing alpha4 and beta2 subunits. In this study we have examined how temperature and the expression of subunit chimeras can influence the efficiency of cell-surface expression of the rat alpha4beta2 nAChR. Functional recombinant alpha4beta2 nAChRs, showing high affinity binding of nicotinic radioligands (K(d) = 41 +/- 22 pM for [(3)H]epibatidine), are expressed in both stably and transiently transfected mammalian cell lines. Despite this, only very low levels of alpha4beta2 nAChRs can be detected on the cell surface of transfected mammalian cells maintained at 37 degrees C. At 30 degrees C, however, cells expressing alpha4beta2 nAChRs show a 12-fold increase in radioligand binding (with no change in affinity), and a 5-fold up-regulation in cell-surface receptors with no increase in total subunit protein. In contrast to "wild-type" alpha4 and beta2 subunits, chimeric nicotinic/serotonergic subunits ("alpha4chi" and "beta2chi") are expressed very efficiently on the cell surface (at 30 degrees C or 37 degrees C), either as hetero-oligomeric complexes (e.g. alpha4chi+beta2 or alpha4chi+beta2chi) or when expressed alone. Compared with alpha4beta2 nAChRs, expression of complexes containing chimeric subunits typically results in up to 20-fold increase in nicotinic radioligand binding sites (with no change in affinity) and a similar increase in cell-surface receptor, despite a similar level of total chimeric and wild-type protein.     

Up-regulation of nicotinic receptors by nicotine varies with receptor subtype

Recent evidence suggests that in addition to alpha4beta2 and alpha3-containing nicotinic receptors, alpha6-containing receptors are present in midbrain dopaminergic neurons and involved in the nicotine reward pathway. Using heterologous expression, we found that alpha6beta2, like alpha3beta2 and alpha4beta2 receptors, formed high affinity epibatidine binding complexes that are pentameric, trafficked to the cell surface, and produced acetylcholine-evoked currents. Chronic nicotine exposure up-regulated alpha6beta2 receptors with differences in up-regulation time course and concentration dependence compared with alpha4beta2 receptors, the predominant high affinity nicotine binding site in brain. The alpha6beta2 receptor up-regulation required higher nicotine concentrations than for alpha4beta2 but lower than for alpha3beta2 receptors. The alpha6beta2 up-regulation occurred 10-fold faster than for alpha4beta2 and slightly faster than for alpha3beta2. Our data suggest that nicotinic receptor up-regulation is subtype-specific such that alpha6-containing receptors up-regulate in response to transient, high nicotine exposures, whereas sustained, low nicotine exposures up-regulate alpha4beta2 receptors.     

Synthesis and nicotinic acetylcholine receptor binding affinities of 2- and 3-isoxazolyl-8-azabicyclo[3.2.1]octanes

A series of epiboxidine homologues, 2- and 3-isoxazole substituted 8-azabicyclo[3.2.1]octane derivatives was synthesized and evaluated as potential ligands for neuronal nicotinic acetylcholine receptors in [(3)H]cytisine labeled rat brain. The 2beta-isoxazolyl-8-azabicyclo[3.2.1]octane 9b (K(i)=3 nM) was the most potent compound of the series with a binding affinity twice that of nicotine. The 3beta-isoxazolyl-8-azabicyclo[3.2.1]octane 15b (K(i)=148 nM) exhibited moderate affinity while the corresponding 2alpha- and 3alpha-isomers exhibited micromolar binding affinity.     

Pharmacological and immunological identification of native alpha7 nicotinic receptors: evidence for homomeric and heteromeric alpha7 receptors

Controversy surrounds the expression of alpha7 nicotinic acetylcholine receptors (nAChRs) in adrenal chromaffin cells. In these studies, alpha7 nAChRs expressed in bovine adrenal chromaffin cells are investigated. Using radiolabeled ligand binding techniques, [(125)I]alpha-bungarotoxin (alphaBGT) binding reaches equilibrium within 4 h and is saturable with a K(d) value of 4.2 nM. Using homologous competition experiments, the K(i) for binding of alphaBGT was 1.9 nM. These data are consistent with the expression of homomeric alpha7 nAChRs. Methyllycaconatine (MLA), which binds alpha7 nAChRs with high affinity, inhibits [(125)I]alphaBGT binding in a concentration-dependent manner with a K(i) of 30.6 nM; this value is approximately 10 fold higher than the reported affinity of MLA for alpha7 nAChRs. We also document the ability of bromoacetylcholine (brACh) to alkylate alpha7 nAChRs, as has been previous demonstrated for bovine adrenal alpha3beta4 nAChRs. When adrenal nAChRs are immunoprecipated with mAb319, an antibody which recognizes alpha7 nAChR protein, and then probed with mAb319 using Western blot analysis, a single band of approximately 53 kDa is identified. When adrenal nAChRs are immunoprecipated with mAb35, an antibody which recognizes alpha3 and alpha5 nAChR proteins, and then probed with mAb319 using Western blot analysis, a single band of approximately 53 kDa is identified. Together, these results support the expression of alpha7 nAChRs in bovine adrenal chromaffin cells. However, these data suggest that the subunit composition of some of these receptors may include heteromeric alpha7 nAChRs.     

Increased levels of tau protein in SH-SY5Y cells after treatment with cholinesterase inhibitors and nicotinic agonists

Several cholinesterase inhibitors used in the treatment of Alzheimer's disease (AD) have been shown to interact with an allosteric site on the nicotinic acetylcholine receptor (nAChR). A possible linkage between the phosphorylation state of tau, the major component of paired helical filaments found in AD brain, and stimulation of nAChRs by cholinesterase inhibitors and nicotinic agonists was investigated. Western blot analysis showed that treatment of SH-SY5Y cells for 72 h with the cholinesterase inhibitors tacrine (10(-5) M), donepezil (10(-5) M), and galanthamine (10(-5) M), nicotine (10(-5) M), and epibatidine (10(-7) M) increased tau levels as detected with Tau-1, AT 8, and AT 270 monoclonal antibodies and binding of [3H]epibatidine. The increase in tau immunoreactivity induced by nicotine, epibatidine, and tacrine, but not the up-regulation of nAChRs, was prevented by the antagonists d-tubocurarine and mecamylamine. Both antagonists were synergistic with the nicotinic agonists in causing up-regulation, but only d-tubocurarine showed a synergistic effect with tacrine. The increased tau immunoreactivity induced by tacrine was not prevented by atropine, indicating that in terms of cholinergic receptors, tacrine modulates tau levels mainly through interactions with nAChRs and not with muscarinic receptors. Additional work is needed to determine the exact mechanism by which cholinesterase inhibitors and nicotinic agonists modulate phosphorylation and levels of tau protein.     

Effects of Redox Reagents and Arsenical Compounds on [3H]-Cytisine Binding to Immunoisolated Nicotinic Acetylcholine Receptors from Chick Brain Containing α4 β2 Subunits

All known nicotinic receptor α subunits include a conserved disulfide bond that is essential for function and is a site for labeling via biochemical modification. In an effort to develop a universal ligand for all subtypes of nicotinic receptors, we previously studied the effects of arsenylation with two compounds, ρ-aminophenyldichloroarsine (APA) and bromoacetyl-ρ-aminophenylarsenòxide (BAPA) on nicotinic receptors from Torpedo electroplax. Here we apply these reagents to immunoisolated receptors containing α4, β2, and possibly other subunits from chick brain that bind [³H]cytisine with high affinity (K_D∼5 nM). These are distinct from another receptor subtype that also binds [³H]cytisine and [³H]nicotine and can be arsenylated with APA, but instead contains α5,β2, and probably other subunits. Reduction of α4 β2 receptors with dithiothreitol blocked [³H]cytisine binding and this effect was reversed upon reoxidation by dithiobisnitrobenzoic acid. APA or BAPA prevented the dithiobisnitrobenzoic acid reactivation of dithiothreitol-treated receptors with IC₅₀ values of 15 and 70 n M , respectively. However, the antiarsenical dimercaptopropanesulfonic acid restored function to APA- or BAPA- "arsenylated" receptors (EC₅₀∼100 μ M ). APA-treated receptors remained blocked for up to 24 h, but treatment with dimercaptopropanesulfonic acid at any time restored [³H]cytisine binding. APA treatment of reduced receptors protected against irreversible alkylation by Bromoacetyl choline, indicating that arsenylation occurs at least in part in the agonist binding site. Thus, these reagents have similar effects on different nicotinic receptor subtypes from both muscle and nerves.     

Computational evidence for the ligand selectivity to the alpha4beta2 and alpha3beta4 nicotinic acetylcholine receptors

The homology models of the alpha4beta2 and alpha3beta4 nicotinic acetylcholine receptors (nAChRs) suggest that the two nAChR subtypes are different in their ligand-binding pockets due to the non-conserved residues in the beta-subunits. The docking of nicotine, epibatidine, A-84543, and the two analogs of A-84543 ligands 1 and 2 to the homology models of alpha4beta2 and alpha3beta4 is presented. It is found that the protonated amino groups of these ligands bind to the alpha-subunits, whereas the remaining parts of the ligands bind to the beta-subunits. The two non-conserved amino acids Lys77 and Phe117 in the beta2-subunit corresponding to Ile77 and Gln117 in the beta4-subunit are identified to be the key players determining the binding modes of the ligands. We demonstrate how the increase in the number of the atoms connecting the pyrrolidine and pyridine rings in A-84543, 1, and 2, and an introduction of the alkynyl substituent in the pyridine ring affect the binding and shift the selectivity of these ligands toward the beta2-containing receptors. Further improvement in affinity and selectivity in this and other series of the ligands may be achieved by designing molecules that would specifically target the non-conserved regions in nAChRs.     

An extracellular protein microdomain controls up-regulation of neuronal nicotinic acetylcholine receptors by nicotine

In smoker's brain, rodent brain, and in cultured cells expressing nicotinic receptors, chronic nicotine treatment induces an increase in the total number of high affinity receptors for acetylcholine and nicotine, a process referred to as up-regulation. Up-regulation induced by 1 mm nicotine reaches 6-fold for alpha3beta2 nicotinic receptors transiently expressed in HEK 293 cells, whereas it is much smaller for alpha3beta4 receptors, offering a rationale to investigate the molecular mechanism underlying up-regulation. In this expression system binding sites are mainly intracellular, as shown by [(3)H]epibatidine binding experiments and competition with the impermeant ligand carbamylcholine. Systematic analysis of beta2/beta4 chimeras demonstrates the following. (i) The extracellular domain critically contributes to up-regulation. (ii) Only residues belonging to two beta2 segments, 74-89 and 106-115, confer up-regulation to beta4, mainly by decreasing the amount of binding sites in the absence of nicotine; on an atomic three-dimensional model of the alpha3beta2 receptor these amino acids form a compact microdomain that mainly contributes to the subunit interface and also faces the acetylcholine binding site. (iii) The beta4 microdomain is sufficient to confer to beta2 a beta4-like up-regulation. (iv) This microdomain makes an equivalent contribution to the up-regulation differences between alpha4beta2 and alpha4beta4. We propose that nicotine, by binding to immature oligomers, elicits a conformational reorganization of the microdomain, strengthening the interaction between adjacent subunits and, thus, facilitating maturation processes toward high affinity receptors. This mechanism may be central to nicotine addiction, since alpha4beta2 is the subtype exhibiting the highest degree of up-regulation in the brain.