Abstract
The aim of this study was to investigate the ability of sazetidine-A, a novel partial agonist at α4β2 nicotinic acetylcholine receptors (nAChRs), to affect the function of native α7 nAChRs in SH-SY5Y cells and primary cortical cultures. The α7-selective positive allosteric modulator PNU-120596 was used to reveal receptor activation, measured as an increase in intracellular calcium using fluorescent indicators. In the absence of PNU-120596, sazetidine-A elicited mecamylamine-sensitive increases in fluorescence in SH-SY5Y cells (EC50 4.2 µM) but no responses from primary cortical neurons. In the presence on PNU-120596, an additional response to sazetidine-A was observed in SH-SY5Y cells (EC50 0.4 µM) and robust responses were recorded in 14 % of cortical neurons. These PNU-120596-dependent responses were blocked by methyllycaconitine, consistent with the activation of α7 nAChRs. Preincubtion with sazetidine-A concentration-dependently attenuated subsequent responses to the α7-selective agonist PNU-282987 in SH-SY5Y cells (IC50 476 nM) and cortical cultures. These findings support the ability of sazetidine-A to interact with α7 nAChRs, which may contribute to sazetidine-A’s actions in complex physiological systems.
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Sazetidine-A (6-[5-[(2S)-2-azetidinylmethoxy]-3-pyridinyl]-5-hexyn-1-ol) has the profile of a potent partial agonist at α4β2 nicotinic acetylcholine receptors (nAChRs) and has attracted interest as a lead compound for several therapeutic targets, making detailed knowledge of its wider activity an important consideration. Sazetidine-A is a derivative of the nicotinic agonist A-85380, developed to bear a long side chain for potential attachment of fluorescent or photoaffinity probes [1]. In binding assays it showed improved selectivity for α4β2 nAChRs, compared with A-85380, with Ki values of 0.4 and 0.6 nM for rat and human α4β2 nAChRs respectively.
In contrast to the parent molecule, sazetidine-A appeared to be devoid of agonist activity at recombinant human α4β2 nAChRs and was described as a ‘silent desensitizer’ [1]. Subsequent studies revealed sazetidine-A to be a stoichiometry-dependent agonist capable of fully activating high sensitivity human α42β23 (HS-α42β23) nAChRs, whereas it had negligible efficacy, relative to acetylcholine, at lower sensitivity α43β22 (LS-α43β22) nAChRs [2, 3]. Presumably the stable cell lines employed in the original study predominantly expressed the low sensitivity form. The ‘accessory’ subunit occupying the fifth position in the pentameric nAChR, a position that does not directly contribute to either of the two high affinity, orthosteric agonist binding sites, nevertheless can influence receptor properties [4]. In this case the subunit occupying this position appears to determine the ability of sazetidine-A to affect the opening of the α4β2 nAChR channel. This notion is reinforced by the opposing effects of α5 and β3 subunits in the fifth position: inclusion of an α5 subunit to form α42β22α5 nAChRs abolished agonist efficacy [5], whereas sazetidine-A is a potent and efficacious agonist at α4α6β22β3 nAChRs (EC50 19 nM) [6].
The sobriquet ‘silent desensitizer’ reflected the observation that a brief preincubation with 1 µM sazetidine-A induced a long lasting inhibition of recombinant human α4β2 nAChRs expressed in SH-EP1 cells (IC50 value ~ 30 nM), compared with the rapid recovery following preincubation with nicotine [1]. More recent studies that have taken into account its differential interaction with the two stoichiometries of α4β2 nAChRs have shown that sazetidine-A selectively desensitizes HS-α42β23 nAChRs over LS-α43β22 [7, 8].
Its partial agonist profile has raised interest in sazetidine-A as a therapeutic lead for numerous indications, including drug dependence [9, 10], cognitive and attentional deficits [11], pain [12] and depression [13]. Given the complex contributions of multiple nAChRs in the CNS to brain function and behaviours, it is important to understand the specificty of agents such as sazetidine-A. It binds with much lower affinity but acts as a full or substantial agonist at recombinant α4β4, α3β4, and α6β2*nAChRs [1, 6, 7]. Sazetidine-A’s activation of α7 nAChRs has only recently been documented, with very different EC50 values and efficacies at human (1.2 µM; 100 %) and rat (60 µM; 6 %) α7 nAChRs [7, 14]. The contribution of α7 nAChRs to the clinical targets mentioned above [15, 16], as well as α7 nAChRs credited with functions in peripheral systems that could mediate side-efffects [17] suggest that this nAChR subtype, in particular, merits further attention. Hitherto, sazetidine-A’s activation of native α7 nAChRs has not been reported. In this study we examined the ability of sazetidine-A to activate and desensitize α7 nAChRs in SH-SY5Y cells and primary cortical neurons.
Materials and Methods
Materials
Triton X-100, (-)-nicotine hydrogen tartrate and mecamylamine hydrochloride, were purchased from Sigma-Aldrich (Poole, Dorset, UK); B27, l-glutamine, antibiotics, fluo-3 AM, fura-2 AM, and pluronic f127 were obtained from Life Technologies (Paisley, UK); sazetidine-A dihydrochloride, tetrodotoxin citrate, methyllycaconitine citrate and 5-iodo-A85380 dihydrochloride were purchased from Tocris Bioscience (Avonmouth, UK); PNU-120596 and PNU-282987 were provided by Pfizer Inc. USA; general reagents were purchased from Fisher Scientific (Loughborough, UK).
Methods
SH-SY5Y Cell Culture
Human neuroblastoma SH-SY5Y cells (ECACC, Salisbury, UK; passages 16–27) were cultured as previously described [18]. In brief, cultures were maintained in Advanced Dulbecco’s modified Eagle’s media (DMEM/F12), supplemented with 2 % fetal bovine serum (FBS), 2 mM l-glutamine, 190 U/ml penicillin and 0.2 mg/ml of streptomycin in 94 × 16 mm tissue culture dishes in a humidified chamber at 37 °C with 5 % CO2. Cells were seeded 1:2 into 96-well plates, experiments were performed 72 h later with confluent cultures.
Mouse Primary Cortical Cultures
Primary cultures were prepared from embryonic mouse cortices as previously described [19]. Briefly, time-mated pregnant female CD1 mice were killed by cervical dislocation and E18 embryos were harvested. Cortices were dissected in PBS with 30 % glucose (Ca2+- and Mg2+-free) and dissociated with a fire polished glass Pasteur pipette. Tissue was centrifuged at 500 g for 5 min, resuspended in neurobasal medium supplemented with B27, 2 mM l-glutamine and 60 μg/ml penicillin and 100 μg/ml streptomycin (12 ml medium per brain). For live imaging experiments, cells were plated on 25 mm round glass coverslips (thickness no. 1) coated with 20 μg/ml poly-d-lysine, in 6-well tissue culture plates (Corning, USA). Cells were allowed to grow for 10–14 days in vitro (DIV) at 37 °C in a humidified atmosphere of 95 % air and 5 % CO2.
Ca2+ Fluorimetry
SH-SY5Y Cells
Increases in [Ca2+]ic were measured as described previously [20]. Briefly, cells were washed twice with Tyrode’s salt solution (TSS: 137 mM NaCl, 2.7 mM KCl, 1.0 mM MgCl2, 1.8 mM CaCl2, 0.2 mM NaH2PO4, 12 mM NaHCO3, 5.5 mM glucose; pH 7.4) and incubated with the membrane-permeable, Ca2+ sensitive dye fluo-3 AM (10 μM) and 0.02 % pluronic F127 for 1 h at room temperature in darkness. Cells were then washed twice with TSS before pre-incubation (10 min) with 80 μl antagonists, modulators or TSS. Changes in fluorescence (excitation 485 nm, emission 538 mm) were monitored using a Fluoroskan Ascent fluorescent plate reader (Thermo Scientific, UK). Basal fluorescence was measured for 5 s before agonist (20 µl) was added and fluorescence was monitored for a further 20 s. Calibration of responses was achieved by determining the maximum and minimum fluorescence values of each fluo-3 AM signal, by application of 0.2 % Triton X-100 (Fmax) followed by 40 mM MnCl2 (Fmin). Data were calculated as a percentage of Fmax − Fmin. Concentration response data were fitted to the Hill equation and half maximal effective concentrations determined.
Cortical Cultures
Changes in [Ca2+]ic in individual cells of mouse E18 cortical cultures grown on glass coverslips were monitored using live cell imaging (Concord System, Perkin Elmer, UK). Cortical cultures (10–14 DIV) were washed twice with calcium buffer (140 mM NaCl, 5.0 mM KCl, 1.0 mM MgCl2, 1.8 mM CaCl2, 10 mM glucose, 5.0 mM HEPES; pH 7.4) and incubated with the ratiometric Ca2+-sensitive dye fura-2 AM (5 μM) and 0.02 % pluronic F127 for 1.5 h at room temperature in darkness. After another two washes with buffer, coverslips were assembled into a temperature controlled (37 °C) perfusion chamber (Series 20 PH2 platform with a RC-21BR chamber, Harvard Apparatus, MA, USA) and mounted on an inverted fluorescence microscope. Buffer and drug solutions were pre-heated to 37 °C and perfused at a rate of 5 ml/min. Fura-2 AM was excited at 340 and 380 nm using a SpectroMaster I and emissions at 510 nm were detected with an intensified Ultrapix PDCI low light level CCD camera. All experiments were carried out in the presence of 1 μM tetrodotoxin (TTX) pre-incubated for at least 1 min prior to recording. During long drug pre-incubations perfusion was switched off to reduce drug use, and recording was turned off to prevent photobleaching.
Data were analysed with Ultraview software (Perkin Elmer, UK) and expressed as a ratio of F340:F380 following subtraction of background fluorescence taken from a region in which no cells could be seen. For successive drug treatments on the same cells, initial peak F340:F380 ratio for each individual responding region of interest (ROI) was normalized to 100 % following subtraction of mean basal F340:F380 ratio recorded immediately before drug application. Subsequent responses in the presence of antagonists/modulators or after washout were calculated as a percentage of the original response from the same ROI. These values were then averaged within experiments, such that n values reflect the number of independent cultures examined.
Statistical Analysis
Statistical significance was evaluated by ANOVA with post-hoc test, or t-test as appropriate, with details given in figure legends.
Results
Effects of Sazetidine-A on Ca2+ Responses Initiated by Native Human nAChRs Expressed in SH-SY5Y Cells
SH-SY5Y cells express α3, α5, α7, β2, and β4 nAChR subunits [21–23] consistent with the formation of functional α3* and α7 nAChRs [20]. Based on the sensitivities of recombinant non-α4β2 nAChRs, sazetidine-A was examined at 10 and 100 µM in SH-SY5Y cells loaded with the Ca2+ indicator fluo-3 AM. Both concentrations of sazetidine-A produced a similar increase in fluorescence and this response was abolished in the presence of 30 µM mecamylamine (Fig. 1). The α7-selective antagonist methyllycaconitine (MLA; 100 nM) was without effect. This suggests that under the conditions of the assay, sazetidine-A activates α3-containing nAChRs but not α7 nAChRs, consistent with previous findings for other agonists [24]. However, in the presence of the α7-selective positive allosteric modulator (PAM) PNU-120596 (10 µM) [25], sazetidine-A evoked significantly larger increases in fluorescence that were partially blocked by both mecamylamine and by MLA (Fig. 1). This suggests that PNU-120596 reveals an α7 nAChR-mediated increase in intracellular Ca2+.
The response elicited by 10 µM sazetidine-A in the presence of PNU-120596 (2.3 ± 0.7 fold increase in fluorescence, Fig. 1) is comparable to that observed with the structurally related agonist 5-iodo-A85380 (1 µM; 2.3 ± 0.2 fold increase) and with nicotine (30 µM; 4.0 ± 0.2 fold increase), both tested in the presence of the PAM (data not shown). Increases in fluorescence in response to sazetidine-A were concentration dependent (Fig. 2). The concentration response curve was shifted to the left in the presence of PNU-120596. EC50 values of 4.2 and 0.4 µM were derived for sazetidine-A in the absence and presence of PNU-120596, respectively.
The propensity of sazetidine-A to antagonize nAChRs in SH-SY5Y cells was assessed by preincubating cultures with increasing concentrations of sazetidine-A for 10 min, followed by stimulation with 100 µM nicotine (to activate α3-containing nAChRs) or the α7-selective agonist PNU-282987 (10 µM), in the presence of the PAM PNU-120596. Maximally effective agonist concentrations were deployed to elicit the optimum signal for quantitating inhibition. In both cases sazetidine-A produced a concentration-dependent inhibition of agonist-evoked responses, with IC50 values of 522 and 476 nM respectively (Fig. 3).
Effects of Sazetidine-A on α7 nAChR-Mediated Ca2+ Signals in Mouse Cortical Neurons
Experiments were carried out on mouse E18 primary cortical cultures to assess the effects of sazetidine-A on native α7 nAChRs in cells with a more neuronal phenotype. Cortical cultures were loaded with fura-2 AM and changes in fluorescence indicative of changes in intracellular Ca2+ were monitored by live cell imaging. Sazetidine-A alone (10 nM–10 µM; 20 s application) failed to evoke any change in fluorescence, except for occasional, inconsistent increases at the highest concentration tested. In contrast, 40 mM KCl consistently produced robust responses from a majority of cells (data not shown). Following preincubation with PNU-120596, co-application of 10 µM sazetidine-A with the PAM evoked sustained responses from 14 % of cells (average from 6 experiments from 3 independent cultures). Responses were completely blocked by 100 nM MLA, with partial recovery (32.4 ± 9.4 % of initial response) following 10 min washout (Fig. 4).
Sazetidine-A was examined for its ability to attenuate responses from α7 nAChRs in cortical neurons by sequential application of the α7 nAChR agonist PNU-282987 alone (in the presence of PNU-120596) and following exposure to sazetidine-A for 10 min (Fig. 5). Sazetidine-A applied at 500 nM, a concentration approximating the IC50 value derived from SH-SY5Y cells (Fig. 3), decreased the response to PNU-282987 by 59 %, whereas preincubation with 10 µM sazetidine-A resulted in a stronger block of 86 %. This effect was not due to run-down of responses or exhaustion of the Ca2+ indicator as responses recovered to 57 and 60 % of control, respectively, after 10 min washout of sazetidine-A (Fig. 5).
Discussion
In this study we have exploited the PAM PNU-120596 to reveal activity of native α7 nAChRs [27], in order to examine the actions of sazetidine-A on α7 nAChRs expressed in SH-SY5Y cells and mouse cortical cultures. In the absence of the PAM, sazetidine-A evoked mecamylamine-sensitive increases in fluorescence in SH-SY5Y cells that were insensitive to MLA. The EC50 value of 4 µM is consistent with the activation of human α3β4* nAChRs in SH-SY5Y cells; at heterologously expressed α3β4 nAChRs sazetidine-A is a relatively weak agonist, with efficacy ranging from ~0 to 100 % in different studies, presumably reflecting differences in stoichiometry, species and methodology [1, 7, 9].
The lack of α7 nAChR responses in the absence of the PAM is likely to reflect the rapid kinetics of the receptor, as other agonists were previously found to be without effect in this assay [24]. However, sazatidine-A was recently reported to activate rat α7 nAChRs with very low efficacy [14] although another study using a different assay and overexpressed human α7 nAChRs, reported 100 % efficacy [7]. The MLA-sensitive enhancement of responses to sazetidine-A in the presence of the PAM PNU-120596 is indicative of the recruitment of α7 nAChRs. The lower EC50 determined in the presence of PNU-120596 is likely to underestimate the true EC50 for sazetidine-A at α7 nAChRs as this PAM shifts the agonist concentration–response relationship to the left by approximately 0.8 of a log unit [25]. This suggests that the EC50 value for activation of α7 nAChRs in SH-SY5Y cells by sazetidine-A would be in the low µM range, comparable with the recent report that sazetidine-A activated recombinant α7 nAChRs in the absence of a PAM with an EC50 value of 1.2 µM, using a sensitive fluorescence assay to measure changes in membrane potential [7]. A higher EC50 value of 60 µM was found using two-electrode voltage clamp recording from Xenopus oocytes expressing rat α7 nAChRs [14].The ability of a brief (10 min) incubation with sazetidine-A to ameliorate responses to subsequent stimulation of α3* or α7 nAChRs is consistent with its propensity to desensitize nAChRs. There was a concern that this experiment would be compromised by the requirement for preincubation with the PAM, alongside sazetidine-A, in order to reveal α7 nAChR-evoked responses. Although PNU-120596 prolongs the activation of α7 nAChRs [25], the duration of this effect is relatively short-lived with return to baseline within 5 min [26]. The very similar inhibition curves for nicotine-evoked responses in the absence of the PAM, attributed to α3β4* nAChRs, and for responses evoked by the α7-selective agonist PNU-282987 in the presence of PNU-120596 argues that an inhibitory effect of sazetidine-A is measured in both cases and that α3β4* and α7 nAChRs are similarly sensitive to inhibition by sazetidine-A.
The IC50 values for this effect were ~0.5 µM (Fig. 3). This could be relevant to clinical applications of sazetidine-A when therapeutic concentrations may approach these levels [28] (see below). Moreover, Campling et al. [7] recently highlighted ‘smouldering activation’ of nAChRs resulting from the balance within a population of receptors of sustained desensitization versus activation, such that the impact of chronic agonist concentrations will be complex.
The sensitivity of α7 nAChRs to sazetidine-A was reinforced by studies in primary cortical neurons. Interestingly, no changes in fluorescence were detected in response to sazetidine-A in the absence of the PAM. This was surprising as functional α4β2 nAChRs might have been anticipated to be present in cortical neurons. Possible explanations are that they are only present in the LS-α43β22 stoichiometry, or that they are absent at this developmental stage. Although α4β2 nAChRs have been documented on thalamocortical afferents [29], projection neurons would not be present in the cortical cultures. However α4β2 nAChRs may also occur on intrinsic cortical neurons [30, 31]. Alternatively, α4β2 nAChRs might not initiate detectable changes in intracellular Ca2+, due to the presence of TTX in the perfusing buffer.
In contrast, in the presence of PNU-120596 sazetidine-A elicited robust responses from a minority of cells, estimated as 14 % of the total population. This proportion is consistent with measurements using a selective α7 nAChR agonist together with the PAM (Brown and Wonnacott, unpublished observation). The almost total blockade of these responses by 100 nM MLA confirmed that they arise from activation of α7 nAChRs. Recovery following 3 min washout was partial and somewhat variable, possibly reflecting sazetidine-A’s propensity to desensitize nAChRs. This was supported by the ability of sazetidine-A to produce a concentration-dependent-decrease in responses to PNU-282987, with sensitivity similar to that observed in SH-SY5Y cells.
Together these data provide evidence for the ability of low micromolar concentrations of sazetidine-A to activate native human and mouse α7 nAChRs, whereas an inhibitory effect, likely reflecting desensitization of α7 nAChRs, was observed at sub-micromolar concentrations of sazetidine-A. Brain concentrations of sazetidine-A administered chronically to rodents via osmotic minipump (4.7 mg/kg/day) have been estimated to reach 32 nM, but repeated injection achieved transient levels that were 10 times higher [28]. This would be sufficient to elicit a degree of desensitization and/or ‘smouldering activation’ of α7 nAChRs [7], which could either compromise or contribute to the beneficial effects of a selective agonist such as sazetidine-A.
References
Xiao Y, Fan H, Musachio JL, Wei Z-L, Chellappan SK, Kozikowski AP, Kellar KJ (2006) Sazetidine-A, a novel ligand that desensitizes alpha4beta2 nicotinic acetylcholine receptors without activating them. Mol Pharmacol 70:1454–1460
Zwart R, Carbone AL, Moroni M, Bermudez I, Mogg AJ, Folly EA, Broad LM, Williams AC, Zhang D, Ding C, Heinz BA, Sher E (2008) Sazetidine-A is a potent and selective agonist at native and recombinant alpha 4 beta 2 nicotinic acetylcholine receptors. Mol Phamacol 73:1838–1843. doi:10.1124/mol.108.045104
Carbone AL, Moroni M, Groot-Kormelink PJ, Bermudez I (2009) Pentameric concatenated (alpha4)(2)(beta2)(3) and (alpha4)(3)(beta2)(2) nicotinic acetylcholine receptors: subunit arrangement determines functional expression. Br J Pharmacol 156:970–981. doi:10.1111/j.1476-5381.2008.00104.x
Kuryatov A, Onksen J, Lindstrom J (2008) Roles of accessory subunits in alpha4beta2(*) nicotinic receptors. Mol Pharmacol 74:132–143. doi:10.1124/mol.108.046789
Zhang J, Xiao YD, Jordan KG, Hammond PS, Van Dyke KM, Mazurov AA, Speake JD, Lippiello PM, James JW, Letchworth SR, Bencherif M, Hauser TA (2012) Analgesic effects mediated by neuronal nicotinic acetylcholine receptor agonists: correlation with desensitization of α4β2* receptors. Eur J Pharm Sci 47:813–823. doi:10.1016/j.ejps.2012.09.014
Kuryatov A, Lindstrom J (2011) Expression of functional human α6β2β3* acetylcholine receptors in Xenopus laevis oocytes achieved through subunit chimeras and concatamers. Mol Pharmacol 79:126–140. doi:10.1124/mol.110.066159
Campling BG, Kuryatov A, Lindstrom J (2013) Acute activation, desensitization and smoldering activation of human acetylcholine receptors. PLoS ONE 14(8):e79653. doi:10.1371/journal.pone.0079653
Eaton JB, Lucero LM, Stratton H, Chang Y, Cooper JF, Lindstrom JM, Lukas RJ, Whiteaker P (2014) The unique α4 ± α4 agonist binding site in (α4)3(β2)2 subtype nicotinic acetylcholine receptors permits differential agonist desensitization pharmacology versus the (α4)2(β2)3 subtype. J Pharmacol Exp Ther 348:46–58. doi:10.1124/jpet.113.208389
Liu Y, Richardson J, Tran T, Al-Muhtasib N, Xie T, Yenugonda VM, Sexton HG, Rezvani AH, Levin ED, Sahibzada N, Kellar KJ, Brown ML, Xiao Y, Paige M (2013) Chemistry and pharmacological studies of 3-alkoxy-2,5-disubstituted-pyridinyl compounds as novel selective α4β2 nicotinic acetylcholine receptor ligands that reduce alcohol intake in rats. J Med Chem 11(56):3000–3011. doi:10.1021/jm4000374
Johnson JE, Slade S, Wells C, Petro A, Sexton H, Rezvani AH, Brown ML, Paige MA, McDowell BE, Xiao Y, Kellar KJ, Levin ED (2012) Assessing the effects of chronic sazetidine-A delivery on nicotine self-administration in both male and female rats. Psychopharmacology 222:269–276. doi:10.1007/s00213-012-2642-z
Rezvani AH, Cauley M, Sexton H, Xiao Y, Brown ML, Paige MA, McDowell BE, Kellar KJ, Levin ED (2012) Sazetidine-A, a selective α4β2 nicotinic acetylcholine receptor ligand: effects on dizocilpine and scopolamine-induced attentional impairments in female Sprague-Dawley rats. Psychopharmacology 215:621–630. doi:10.1007/s00213-010-2161-8
Alsharari SD, Carroll FI, McIntosh JM, Damaj MI (2012) The antinociceptive effects of nicotinic partial agonists varenicline and sazetidine-A in murine acute and tonic pain models. J Pharmacol Exp Ther 342:742–749. doi:10.1124/jpet.112.194506
Liu J, Yu LF, Eaton JB, Caldarone B, Cavino K, Ruiz C, Terry M, Fedolak A, Wang D, Ghavami A, Lowe DA, Brunner D, Lukas RJ, Kozikowski AP (2011) Discovery of isoxazole analogues of sazetidine-A as selective α4β2-nicotinic acetylcholine receptor partial agonists for the treatment of depression. J Med Chem 54:7280–7288. doi:10.1021/jm200855b
Zwart R, Strotton M, Ching J, Astles PC, Sher E (2014) Unique pharmacology of heteromeric α7β2 nicotinic acetylcholine receptors expressed in Xenopus laevis oocytes. Eur J Pharmacol 726C:77–86. doi:10.1016/j.ejphar.2014.01.031
Lendvai B, Kassai F, Szájli A, Némethy Z (2013) α7 nicotinic acetylcholine receptors and their role in cognition. Brain Res Bull 93:86–96. doi:10.1016/j.brainresbull.2012.11.003
Wallace TL, Bertrand D (2013) Importance of the nicotinic acetylcholine receptor system in the prefrontal cortex. Biochem Pharmacol 85:1713–1720. doi:10.1016/j.bcp.2013.04.001
Filippini P, Cesario A, Fini M, Locatelli F, Rutella S (2012) The Yin and Yang of non-neuronal α7-nicotinic receptors in inflammation and autoimmunity. Curr Drug Targets 13:644–655
Ridley DL, Pakkanen J, Wonnacott S (2002) Effects of chronic drug treatments on increases in intracellular calcium mediated by nicotinic acetylcholine receptors in SH-SY5Y cells. Br J Pharmacol 135:1051–1059
Hoey SE, Williams RJ, Perkinton MS (2009) Synaptic NMDA receptor activation stimulates alpha-secretase amyloid precursor protein processing and inhibits amyloid-beta production. J Neurosci 29:4442–4460. doi:10.1523/JNEUROSCI.6017-08.2009
Dajas-Bailador FA, Mogg AJ, Wonnacott S (2002) Intracellular Ca2+ signals evoked by stimulation of nicotinic acetylcholine receptors in SH-SY5Y cells: contribution of voltage-operated Ca2+ channels and Ca2+ stores. J Neurochem 81:606–614
Lukas RJ, Norman SA, Lucero L (1993) Characterization of Nnicotinic acetylcholine receptors expressed by cells of the SH-SY5Y human neuroblastoma clonal line. Mol Cell Neurosci 4:1–12. doi:10.1006/mcne.1993.1001
Peng X, Katz M, Gerzanich V, Anand R, Lindstrom J (1994) Human alpha 7 acetylcholine receptor: cloning of the alpha 7 subunit from the SH-SY5Y cell line and determination of pharmacological properties of native receptors and functional alpha 7 homomers expressed in Xenopus oocytes. Mol Pharmacol 45:546–554
Riganti L, Matteoni C, Di Angelantonio S, Nistri A, Gaimarri A, Sparatore F, Canu-Boido C, Clementi F, Gotti C (2005) Long-term exposure to the new nicotinic antagonist 1,2-bisN-cytisinylethane upregulates nicotinic receptor subtypes of SH-SY5Y human neuroblastoma cells. Br J Pharmacol 146:1096–1109
Innocent N, Livingstone PD, Hone A, Kimura A, Young T, Whiteaker P, McIntosh JM, Wonnacott S (2008) Alpha-conotoxin Arenatus IB[V11L, V16D] [corrected] is a potent and selective antagonist at rat and human native alpha7 nicotinic acetylcholine receptors. J Pharmacol Exp Ther 327:529–537. doi:10.1124/jpet.108.142943
Grønlien JH, Håkerud M, Ween H, Thorin-Hagene K, Briggs CA, Gopalakrishnan M, Malysz J (2007) Distinct profiles of alpha7 nAChR positive allosteric modulation revealed by structurally diverse chemotypes. Mol Pharmacol 72:715–724
Malysz J, Anderson DJ, Grønlien JH, Ji J, Bunnelle WH, Håkerud M, Thorin-Hagene K, Ween H, Helfrich R, Hu M, Gubbins E, Gopalakrishnan S, Puttfarcken PS, Briggs CA, Li J, Meyer MD, Dyhring T, Ahring PK, Nielsen EØ, Peters D, Timmermann DB, Gopalakrishnan M (2010) In vitro pharmacological characterization of a novel selective alpha7 neuronal nicotinic acetylcholine receptor agonist ABT-107. J Pharmacol Exp Ther 334:863–874. doi:10.1124/jpet.110.167072
Kalappa BI, Gusev AG, Uteshev VV (2010) Activation of functional α7-containing nAChRs in hippocampal CA1 pyramidal neurons by physiological levels of choline in the presence of PNU-120596. PLoS ONE 12(5):e13964. doi:10.1371/journal.pone.0013964
Hussmann GP, Turner JR, Lomazzo E, Venkatesh R, Cousins V, Xiao Y, Yasuda RP, Wolfe BB, Perry DC, Rezvani AH, Levin ED, Blendy JA, Kellar KJ (2012) Chronic sazetidine-A at behaviorally active doses does not increase nicotinic cholinergic receptors in rodent brain. J Pharmacol Exp Ther 343:441–450. doi:10.1124/jpet.112.198085
Lambe EK, Picciotto MR, Aghajanian GK (2003) Nicotine induces glutamate release from thalamocortical terminals in prefrontal cortex. Neuropsychopharmacology 28:216–225
Aracri P, Amadeo A, Pasini ME, Fascio U, Becchetti A (2013) Regulation of glutamate release by heteromeric nicotinic receptors in layer V of the secondary motor region (Fr2) in the dorsomedial shoulder of prefrontal cortex in mouse. Synapse 67:338–357. doi:10.1002/syn.21655
Poorthuis RB, Bloem B, Schak B, Wester J, de Kock CP, Mansvelder HD (2013) Layer-specific modulation of the prefrontal cortex by nicotinic acetylcholine receptors. Cereb Cortex 23:148–161. doi:10.1093/cercor/bhr390
Acknowledgments
This study was supported by a studentship to JLB from the Biological and Biotechnological Sciences Research Council (BBSRC). We are grateful to Pfizer for providing the PNU-120596 and PNU-282987.
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The manuscript does not contain clinical studies or patient data. Standards of animal care were in accordance with the ARRIVE guidelines and UK law.
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The authors declare that they have no conflict of interest.
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Special Issue: In honor of Lynn Wecker.
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Brown, J.L., Wonnacott, S. Sazetidine-A Activates and Desensitizes Native α7 Nicotinic Acetylcholine Receptors. Neurochem Res 40, 2047–2054 (2015). https://doi.org/10.1007/s11064-014-1302-6
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DOI: https://doi.org/10.1007/s11064-014-1302-6