Basic & Clinical Pharmacology & Toxicology, 2015, 116, 321–328
Doi: 10.1111/bcpt.12317
Methadone is a Non-Competitive Antagonist at the a4b2 and a3* Nicotinic Acetylcholine Receptors and an Agonist at the a7 Nicotinic Acetylcholine Receptor Reeta Talka, Outi Salminen and Raimo K. Tuominen Division of Pharmacology and Pharmacotherapy, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland (Received 21 March 2014; Accepted 26 August 2014) Abstract: Nicotine–methadone interactions have been studied in human beings and in various experimental settings regarding addiction, reward and pain. Most methadone maintenance treatment patients are smokers, and methadone administration has been shown to increase cigarette smoking. Previous in vitro studies have shown that methadone is a non-competitive antagonist at rat a3b4 nicotinic acetylcholine receptors (nAChR) and an agonist at human a7 nAChRs. In this study, we used cell lines expressing human a4b2, a7 and a3* nAChRs to compare the interactions of methadone at the various human nAChRs under the same experimental conditions. A [3H]epibatidine displacement assay was used to determine whether methadone binds to the nicotinic receptors, and 86Rb+ efflux and changes in intracellular calcium [Ca2+]i were used to assess changes in the functional activity of the receptors. Methadone displaced [3H]epibatidine from nicotinic agonist-binding sites in SH-EP1-ha7 and SH-SY5Y cells, but not in SH-EP1-ha4b2 cells. The Ki values for methadone were 6.3 lM in SH-EP1-ha7 cells and 19.4 lM and 1008 lM in SHSY5Y cells. Methadone increased [Ca2+]i in all cell lines in a concentration-dependent manner, and in SH-EP1-ha7 cells, the effect was more pronounced than the effect of nicotine treatment. In SH-EP1-ha4b2 cells, the effect of methadone was negligible compared to that of nicotine. Methadone pre-treatment abolished the nicotine-induced response in [Ca2+]i in all cell lines expressing nAChRs. In SH-EP1-ha4b2 and SH-SY5Y cells, methadone had no effect on the 86Rb+ efflux, but it antagonized the nicotine-induced 86Rb+ ion efflux in a non-competitive manner. These results suggest that methadone is an agonist at human a7 nAChRs and a non-competitive antagonist at human a4b2 and a3* nAChRs. This study adds further support to the previous findings that opioids interact with nAChRs, which may underlie their frequent co-administration in human beings and might be of interest to the field of drug discovery.
The cholinergic system in the brain is important in memory and learning as well as in the control of movement. Cholinergic neurons release acetylcholine (ACh) that binds to muscarinic and neuronal nicotinic acetylcholine receptors (nAChRs). The nAChRs are ligand-gated ion channels that are permeable to Na+, K+ and Ca2+ ions [1], and in addition to ACh, nicotine also binds with high affinity to these receptors. The channel is formed in the middle of five transmembrane subunits, which contain either a (a2–a10) or a and b (b2–b4) subunits [2]. The nAChRs modulate the function of several neurotransmitter systems, including dopaminergic transmission and endogenous opioids [3]. The opioid system modulates pain behaviour, antinociception, reward and addiction. To date, four different G-protein-coupled opioid receptors have been characterized, the l, d, j and opioid receptor like-1 receptors [4]. Nicotine–methadone interactions have been studied in human beings and in vitro settings regarding addiction, reward and pain. Methadone is a l-opioid receptor agonist that also has a low affinity for d and j receptors [5] and is in clinical use for treating moderate to severe pain and for the maintenance replacement treatment of opioid addiction. Almost all methadone maintenance treatment patients are smokers [6,7]. Methadone administration increases cigarette smoking in a Author for correspondence: Reeta Talka, Division of Pharmacology and Pharmacotherapy, Faculty of Pharmacy, University of Helsinki, Viikinkaari 5 E, P.O. Box 56, FI-00014 Helsinki, Finland (fax +358 2941 59471, e-mail [email protected]).
dose-dependent manner, and the maintenance patients have higher nicotine craving and withdrawal symptoms than smokers not receiving methadone [8,9]. Furthermore, smoking cessation is considered to be more demanding for maintenance patients, whereas quitting smoking has been shown to improve opioid abstinence [10]. In in vitro studies, methadone has been suggested to be an agonist at human a7 nAChRs [11] and a non-competitive antagonist at rat a3b4 nAChRs [12]. Furthermore, prolonged exposure to methadone up-regulates nAChRs expressed in SH-SY5Y cells [11]. We have previously shown that the opioid receptor agonist morphine has interactions with nAChRs that are independent of its agonist properties at opioid receptors [13]. The cell lines used in this study are of human origin and either have a native expression of nAChRs (SH-SY5Y cells) [14] or have been transfected with human cDNAs coding nAChR subunits (SH-EP1-ha4b2 and SH-EP1-ha7 cells) [15,16]. SH-SY5Y cells express the a3, a5, a7, b2 and b4 nAChR subunits and have native expression of functional opioid receptors [17]. The b2 subunit-containing receptors from SH-SY5Y cells have a higher affinity for [3H]epibatidine than the b4 or a7 subunit-containing receptors [18,19]. SH-EP1ha4b2 and SH-EP1-ha7 cells have no native expression of nAChRs [20], but have been transfected with human cDNAs coding the a4 and b2 subunits or a7 subunit, respectively [15,16]. SH-EP1-ha4b2 and SH-EP1-ha7 cells also have native expression of the l- and d-opioid receptors [21]. The
© 2014 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society)
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a4b2 nAChRs expressed in SH-EP1-ha4b2 cells have two distinct stoichiometric arrangements, (a4)2(b2)3 (high-affinity subtype) and (a4)3(b2)2 (low-affinity subtype) [22]. Untransfected SH-EP1-cells were used as a control cell line in the intracellular calcium assay. The purpose of this study was to investigate the interactions of nicotine and methadone at the receptor level using SH-EP1ha4b2, SH-SY5Y, SH-EP1-ha7 and untransfected SH-EP1 cell lines. Binding studies were performed with [3H]epibatidine, a nicotinic acetylcholine receptor agonist that binds to several nicotinic acetylcholine receptor subtypes [23], to determine whether methadone binds competitively to the orthosteric agonist-binding site in nicotinic acetylcholine receptors. Additionally, calcium fluorometry and 86Rb+ efflux assays were used to study the functional interactions. Our results show that methadone acts as a non-competitive antagonist at human a4b2 and a3* nAChRs expressed in SH-EP1-ha4b2 and SHSY5Y cells, respectively, and as an agonist at human a7 nAChRs. Materials and Methods Drugs and reagents. The ()-methadone hydrochloride was purchased from Star, Tampere, Finland. The bovine serum albumin, ( )-nicotine hydrogen tartrate, mecamylamine hydrochloride, carbamylcholine chloride, methyllycaconitine citrate salt hydrate, naltrexone hydrochloride, Bradford reagent, poly-D-lysine and poly (ethyleneimine) solution were purchased from Sigma-Aldrich (St. Louis, MO, USA). The bovine serum albumin standards were from Thermo Fisher Scientific Inc. (Rockford, IL, USA). The rubidium-86 radionuclide and [3H]epibatidine were purchased from PerkinElmer (Waltham, MA, USA). All cell culture plasticware was from Nunc (Nunc A/S, Roskilde, Denmark). The media, serum, non-essential amino acids, selection antibiotics and Fluo-3AM calcium indicator were from Invitrogen (Invitrogen, Carlsbad, CA, USA). All buffers were prepared as described in the study by Talka et al. [13]. Cell culture. All cell lines were grown as described in the study by Talka et al. [13]. SH-EP1-ha4b2 and SH-EP1-ha7 cells (kindly provided by Dr. Ronald J. Lukas, Barrow Neurological Institute, St Joseph’s Hospital and Medical Center, Phoenix, AZ, USA) were grown in Dulbecco’s modified Eagle’s medium containing foetal bovine serum, horse serum, penicillin–streptomycin, hygromycin, amphotericin B (Fungizoneâ (Invitrogen, Carlsbad, CA, USA)) and zeocin (only for SH-EP1-ha4b2 cells). Untransfected SH-EP1-cells were grown in Dulbecco’s modified Eagle’s medium containing foetal bovine serum, horse serum and penicillin–streptomycin. SH-SY5Y cells were grown in Dulbecco’s modified Eagle’s medium; Ham’s F12 was supplemented with non-essential amino acids, foetal bovine serum, penicillin and streptomycin. Cell cultures were maintained at 37°C in 5% CO2/humidified air, and passage numbers 10–30 were used in the assays. Cells were grown to confluency in 75- and 175cm2 flasks and split twice weekly. [3H]epibatidine binding assay. The [3H]epibatidine binding assays were carried out as described in the study by Talka et al. [13]. Briefly, the cells were grown to confluency, the medium was removed, and the cells were washed with phosphate-buffered saline. The cells were mechanically harvested and homogenized using an ultrasonic homogenizer (Ringo Ultrasonics Bio 70, Romanshorn, Switzerland) (75% amplitude, 6 9 5 sec.). The homogenates were centrifuged, and the pellet was resuspended in potassium phosphate
buffer and frozen. The binding studies were performed in 96-well glass-filter plates (Millipore MultiScreen HTS FC (Merck Millipore, Billerica, MA, USA)) with samples containing 50 lg of membrane protein (SH-EP1-ha4b2 cells, ligand depletion was minimized by reducing the protein concentration to 5 lg/well). The day before the binding assay, 200 ll of 0.1% poly(ethyleneimine) solution was added to the wells and left overnight at +2 to +8°C. The protein concentrations were measured using the Bradford method. A MultiScreen Vacuum Manifold (Merck Millipore, Billerica, MA, USA) was used to aspirate the assay mixtures. Cell membranes were incubated with [3H]epibatidine for 2 hr at room temperature. Nonspecific binding was defined using 1 mM nicotine. Optiphase HiSafe 3â (Perkin Elmer, Turku, Finland) scintillant was added to the wells, and radioactivity was quantified using a scintillation counter (Wallac 1450 MicroBeta TriLux Liquid Scintillation Counter & Luminometer, Turku, Finland). The binding of [3H]epibatidine to the cell membranes of cell lines expressing nAChRs was saturable [13]. Calcium fluorometry. The calcium fluorometry studies were carried out as described in the study by Talka et al. [13]. In brief, cells were seeded in 96-well microplates (in a 100 ll volume) coated with polyD-lysine the day before the experiment. On the following day, the culture medium was removed from the wells, and the cells were washed twice with Tyrode’s salt solution (TSS). The loading medium consisted of Fluo-3 AM and probenecid acid in TSS. After incubation with loading medium, the cells were washed twice with TSS. When pre-treatments were used, the cells were pre-incubated with drugs for 10 min. Fluorescence was measured with a FlexStation microplate reader (Molecular Devices, Sunnyvale, CA, USA). The test drug was injected, and the fluorescence was monitored for 80 sec. at the 485/ 525 nM wavelength. The drug-induced signals were calculated by subtracting the background signal (measured for 10 sec. before injection) from the signal (measured during the time interval from 20 to 50 sec.). The cells were also stimulated by injecting vehicle (TSS) as a control in each experiment. The changes in [Ca2+]i were expressed as the percentage of fluorescence increased by nicotine. 86
Rb+ efflux. The 86Rb+ efflux assays were carried out as described in the study by Talka et al. [13]. Briefly, cells were seeded in 24-well plates and allowed to adhere for 4 hr in a 37°C incubator, after which the medium was replaced with medium supplemented with 86Rb+. The 86 Rb+ efflux was measured 1 day after seeding the cells with the ‘flipplate’ method [24]. Each well was rinsed with fresh 86Rb+ efflux buffer for 1 min. to remove extracellular 86Rb+. The cells were exposed to efflux buffer containing the ligands being studied for 5 min. The radioactivity was counted using Cerenkov counting (Wallac 1450 MicroBeta TriLux Liquid Scintillation Counter & Luminometer) after placing cross-talk minimizing inserts (Perkin Elmer 1450-109) into each well. The specific efflux for each drug concentration was calculated by subtracting the non-specific efflux (buffer, three wells in each plate) from the total efflux (1 mM carbamylcholine, three wells in each plate). The remaining intracellular 86Rb+ was counted periodically [13]. The maximum 86 Rb+ efflux values depended on cell type, density and the concentration of 86Rb+ in the loading medium and were typically 30,000–50,000 cpm over non-specific backgrounds of 6000– 10,000 cpm. The 86Rb+ efflux assays could not be done with the SHEP1-ha7 cell line because a7 nAChRs are rapidly inactivating channels that are not open long enough to give a significant ion flux signal [24]. Data analysis. All data were analysed using GraphPad Prismâ (version 6.02; GraphPad Software, Inc., La Jolla, CA, USA). In the competition binding assay, the equilibrium dissociation constant (Ki) values were calculated using one-site and two-site curve fitting models
© 2014 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society)
THE ACTION OF METHADONE AT a4b2, a3 AND a7 nAChRs â
with Prism , and the Ki values were determined using best fit (pvalue < 0.05, extra sum-of-squares F test). The Ki values for one-site competition binding were derived from the equation logEC50 = log (10logKi(1 + L*/Kdhot)); y = min + (max min)/(1 + 10X logEC50), where L* is the concentration of [3H]epibatidine, Kdhot is the equilibrium dissociation constant of [3H]epibatidine, max and min are the plateaus of the Y axis and logKi is the log of the molar equilibrium dissociation constant of unlabelled ligand. The Ki values for two-site competition binding were derived from the Prismâ build-in model: L = 10X + 9; KiHi = 10LogKiHi + 9; KiLow = 10LogKiLow + 9; Site 1 = L*(max min)/(L* + KdL*_Ki* (1 + L/KiHi)); Site 2 = L*(max min)/(L* +KDL*_Low(1 + L/KiLow)); Y = Site 1 9 FractionHi) + min, where L* is the labelled FractionHi + Site 2 (1 ligand, L is unlabelled ligand, max and min are the plateaus in the units of the Y axis, FractionHi is the fraction of all the sites that have a high affinity for the competitor and logKiHi and logKiLow are the logarithms of the two molar Ki values. The KiHi and KiLow values are the equilibrium dissociation constant values for the high- and lowaffinity binding sites, respectively. The calcium fluorometry responses were calculated as the percentage of the increase in [Ca2+]i produced by a close to maximally effective concentration of nicotine (10 lM for SH-EP1-ha4b2 and SH-EP1-ha7 cells and 30 lM for SH-SY5Y cells). The intracellular calcium data were analysed statistically using one-way analysis of variance (ANOVA) and Dunnett’s multiplecomparison post hoc test. Values of p < 0.05 were taken to be statistically significant. Specific 86Rb+ efflux was defined as total 86 Rb+ efflux (1 mM carbamylcholine) minus non-specific 86Rb+ efflux (buffer). The specific 86Rb+ efflux was fit to the variable slope model or the biphasic dose–response model after comparing these two different fits (p-value 3000
8.30 0.30 7.09 0.13 NA
KiHi, KiLow (lM), (%) 0.005, 4.5, 10.6%, 89.4% 19.4, 1008, 48.6%, 51.4%
SH-EP1-ha7 LogKiHi S.D. logKiLow S.D. 8.31 5.35 4.71 3.00
0.60 0.07 0.28 0.50
Ki (lM)
LogKi S.D.
0.15
6.82 0.11
6.3
5.20 0.06
KiHi/KiLow, the equilibrium dissociation constant value for the high-/low-affinity binding sites; logHi/logKiLow, the equilibrium dissociation constant value for the high-/low-affinity binding sites in logarithmic scale; S.D., standard deviation; NA, not applicable.
A
B
C
Fig. 1. [3H]Epibatidine competition binding profiles in SH-EP1-ha4b2 (A), SH-SY5Y (B) and SH-EP1-ha7 (C) cell membranes for nicotine, mecamylamine and methadone. The assay mixtures contained 150 pM [3H]epibatidine, 5–50 lg cell membrane protein and nicotine, mecamylamine or methadone in increasing concentrations. To determine the specific binding, assays were performed in the absence (total binding) or presence of 1 mM nicotine (non-specific binding). The specific binding was calculated by subtracting the non-specific binding from the total binding. The data were fit to a two-sites or one-site competition binding model, and the Ki values were determined from the better fit after comparing the two fits (pvalue < 0.05, extra sum-of-squares F test). The statistical programme determined that the best fit was the two-sites model for nicotine (p < 0.001) in SH-EP1-ha4b2 cells and for nicotine (p < 0.05) and methadone (p < 0.05) in SH-SY5Y cells. The values are the mean S.E.M. from three to five assays, with each point assayed at least in triplicate. Ki and logKi values are provided in Table 1.
A
C
E
G
B
D
F
H
Fig. 2. Effects of mecamylamine and methadone on nicotine-evoked increases in [Ca2+]i in SH-EP1-ha4b2 (A), SH-SY5Y (C), SH-EP1-ha7 (E) and SH-EP1 (G) cells and methadone-induced increases in [Ca2+]i in SH-EP1-ha4b2 (B), SH-SY5Y (D), SH-EP1-ha7 (F) and SH-EP1 (H) cells. In figs A, C, E and G, Fluo-3 AM-loaded cells were incubated for 10 min. with mecamylamine or methadone and then stimulated with 10 or 30 lM nicotine. In figs B, D, F and H, Fluo-3 AM-loaded cells were incubated for 10 min. with vehicle (TSS) and then stimulated with nicotine (10 lM or 30 lM), methadone (10 lM, 50 lM or 100 lM) or vehicle (TSS). The responses are expressed as the percentage of the nicotine response measured in parallel in the absence of other agonists. The values are the mean S.E.M. from three to six separate assays. In each experiment, there were at least six replicates for each condition. Significantly different from nicotine response *p < 0.05, **p < 0.01 and ***p < 0.001; significantly different from vehicle response # p < 0.05, ##p < 0.01 and ###p < 0.001 using one-way ANOVA. Nic, nicotine; TSS, Tyrode’s salt solution.
Discussion and Conclusions Our study shows for the first time that methadone acts as a noncompetitive antagonist at a4b2 nAChRs. Additionally, we were
able to confirm previous results that postulated methadone as an antagonist at a3* nAChRs and an agonist at a7 nAChRs. Thus, methadone’s effects seem to vary by nAChR subtype, which might be of interest to the field of drug discovery.
© 2014 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society)
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C
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Fig. 3. Effects of nAChR and opioid antagonists on methadone-evoked increases in [Ca2+]i in SH-EP1-ha4b2 (A), SH-SY5Y (B), SH-EP1-ha7 (C) and SH-EP1 (D) cells. The Fluo-3 AM-loaded cells were incubated for 10 min. with vehicle, mecamylamine (Meca), methyllycaconitine (MLA), naloxone (Nalo) or naltrexone (Nalt) and then stimulated with 100 lM methadone. The responses are expressed as the percentage of the methadone response. The values are the mean S.E.M. from three to six separate assays. In each experiment, there were at least six replicates for each condition. Significantly different from methadone response *p < 0.05, **p < 0.01, and ***p < 0.001 using one-way ANOVA.
Our conclusion that methadone is a non-competitive antagonist at a4b2 nAChRs is based on several findings. Firstly, methadone does not possess binding affinity for the epibatidine-binding site of the receptor. Secondly, it has no effect on the 86Rb+ efflux when administered in buffer alone. Thirdly, methadone inhibits the effects of nicotine on a4b2 nAChRs in the functional assays used. The methadone-induced antagonism is non-competitive in nature, as it does not shift the EC50 value for nicotine, and the inhibition curve is not surmountable in the 86Rb+ efflux assay. Furthermore, the methadone-induced inhibition of a4b2 receptor function is comparable to that of the non-selective nicotinic antagonist mecamylamine, as both of them produce functional inhibition of a4b2 nAChRs without binding to the epibatidine-binding site. We have previously shown that morphine has affinity for a4b2 nAChRs and acts as a partial agonist at this subtype [13]. Thus, different opioids seem to have somewhat different effects on nAChRs. A high concentration of methadone (100 lM) increases [Ca2+]i in SH-EP1-ha4b2 cells, although to a considerably lesser extent than nicotine. This effect can be antagonized by mecamylamine, but not by opioid antagonists. In SH-EP1ha4b2 cells, the a4b2 nAChRs exist in two stoichiometries, that is the low-affinity (a4)3(b2)2 and the high-affinity (a4)2(b2)3 nAChRs [25]. The effect of methadone on [Ca2+]i may be due to the initial activation of the low-affinity (a4)3(b2)2 nAChRs because these receptors mediate agonistinduced responses when agonists are used at high concentrations [26]. Furthermore, racemic mecamylamine has been shown to be more selective in antagonizing the low-affinity a4b2 nAChRs, the (S)-(+) enantiomer being primarily respon-
IC50 and logIC50 values for methadone and mecamylamine in an
sible for this selectivity [25]. The initial activation of the lowaffinity (a4)3(b2)2 nAChRs could lead to receptor desensitization as reflected by our finding that in methadone pre-treated cells, the effect of nicotine on [Ca2+]i was abolished. These data suggest that methadone acts as a partial agonist of a4b2 nAChRs with low activation efficacy and also as a selective desensitizer of these a4b2 nAChRs. However, we cannot rule out the possibility that the introduction of a4b2 nAChRs into the SH-EP1 cells changes the expression levels of the endogenous opioid receptors, which could explain why the opioid antagonists, naloxone and naltrexone, do not have greater effect. Previous in vitro experiments have concluded that methadone is a non-competitive antagonist at rat a3b4 nAChRs [12] and an agonist at human a7 nAChR [11]. Our findings are in line with these results. Methadone inhibits [3H]epibatidine binding in SH-SY5Y cells and SH-EP1-ha7 cells. SH-SY5Y cells express the a3, a5, a7, b2 and b4 nAChR subunits, which form different combinations, for example homopentameric a7 and heteropentameric a3* nAChR subtypes, of which the b2 subunit-containing receptors have a higher affinity for [3H]epibatidine than the b4 or a7 subunit-containing receptors [18,19]. Because SH-EP1-ha7 cells express only homopentameric a7 nAChRs, it is most likely that methadone binds to a7 nAChRs in both of these cell lines. It is well known that a7 nAChR function cannot be studied with an 86Rb+ efflux assay due to the rapid channel kinetics of this receptor subtype [24]. The a7 nAChRs have a high permeability to Ca2+ [27], which enables functional studies with calcium fluorometry, although the rapid channel kinetics of the receptor also need to be taken into account here. Metha-
Table 2. Rb+ efflux assay in the SH-EP1-ha4b2 and SH-SY5Y cell lines.
86
SH-EP1-ha4b2 Nicotine 10 lM Compound
IC50 (lM)
Methadone Mecamylamine
7.4 2.9
LogIC50 S.D. 5.1 0.03 5.5 0.02
SH-SY5Y Nicotine 1 lM
IC50 (lM) 5.6 2.6
LogIC50 S.D. 5.3 0.08 5.6 0.03
Nicotine 30 lM IC50 (lM) 0.8 0.2
LogIC50 S.D. 6.1 0.03 6.7 0.03
Nicotine 10 lM IC50 (lM) 1.0 0.1
LogIC50 S.D. 6.0 0.05 7.0 0.04
IC50, the half-maximal inhibitory concentration; logIC50, the half-maximal inhibitory concentration in logarithmic scale; S.D., standard deviation.
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THE ACTION OF METHADONE AT a4b2, a3 AND a7 nAChRs
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C
D
Fig. 3. Effects of nAChR and opioid antagonists on methadone-evoked increases in [Ca2+]i in SH-EP1-ha4b2 (A), SH-SY5Y (B), SH-EP1-ha7 (C) and SH-EP1 (D) cells. The Fluo-3 AM-loaded cells were incubated for 10 min. with vehicle, mecamylamine (Meca), methyllycaconitine (MLA), naloxone (Nalo) or naltrexone (Nalt) and then stimulated with 100 lM methadone. The responses are expressed as the percentage of the methadone response. The values are the mean S.E.M. from three to six separate assays. In each experiment, there were at least six replicates for each condition. Significantly different from methadone response *p < 0.05, **p < 0.01, and ***p < 0.001 using one-way ANOVA.
Our conclusion that methadone is a non-competitive antagonist at a4b2 nAChRs is based on several findings. Firstly, methadone does not possess binding affinity for the epibatidine-binding site of the receptor. Secondly, it has no effect on the 86Rb+ efflux when administered in buffer alone. Thirdly, methadone inhibits the effects of nicotine on a4b2 nAChRs in the functional assays used. The methadone-induced antagonism is non-competitive in nature, as it does not shift the EC50 value for nicotine, and the inhibition curve is not surmountable in the 86Rb+ efflux assay. Furthermore, the methadone-induced inhibition of a4b2 receptor function is comparable to that of the non-selective nicotinic antagonist mecamylamine, as both of them produce functional inhibition of a4b2 nAChRs without binding to the epibatidine-binding site. We have previously shown that morphine has affinity for a4b2 nAChRs and acts as a partial agonist at this subtype [13]. Thus, different opioids seem to have somewhat different effects on nAChRs. A high concentration of methadone (100 lM) increases [Ca2+]i in SH-EP1-ha4b2 cells, although to a considerably lesser extent than nicotine. This effect can be antagonized by mecamylamine, but not by opioid antagonists. In SH-EP1ha4b2 cells, the a4b2 nAChRs exist in two stoichiometries, that is the low-affinity (a4)3(b2)2 and the high-affinity (a4)2(b2)3 nAChRs [25]. The effect of methadone on [Ca2+]i may be due to the initial activation of the low-affinity (a4)3(b2)2 nAChRs because these receptors mediate agonistinduced responses when agonists are used at high concentrations [26]. Furthermore, racemic mecamylamine has been shown to be more selective in antagonizing the low-affinity a4b2 nAChRs, the (S)-(+) enantiomer being primarily respon-
IC50 and logIC50 values for methadone and mecamylamine in an
sible for this selectivity [25]. The initial activation of the lowaffinity (a4)3(b2)2 nAChRs could lead to receptor desensitization as reflected by our finding that in methadone pre-treated cells, the effect of nicotine on [Ca2+]i was abolished. These data suggest that methadone acts as a partial agonist of a4b2 nAChRs with low activation efficacy and also as a selective desensitizer of these a4b2 nAChRs. However, we cannot rule out the possibility that the introduction of a4b2 nAChRs into the SH-EP1 cells changes the expression levels of the endogenous opioid receptors, which could explain why the opioid antagonists, naloxone and naltrexone, do not have greater effect. Previous in vitro experiments have concluded that methadone is a non-competitive antagonist at rat a3b4 nAChRs [12] and an agonist at human a7 nAChR [11]. Our findings are in line with these results. Methadone inhibits [3H]epibatidine binding in SH-SY5Y cells and SH-EP1-ha7 cells. SH-SY5Y cells express the a3, a5, a7, b2 and b4 nAChR subunits, which form different combinations, for example homopentameric a7 and heteropentameric a3* nAChR subtypes, of which the b2 subunit-containing receptors have a higher affinity for [3H]epibatidine than the b4 or a7 subunit-containing receptors [18,19]. Because SH-EP1-ha7 cells express only homopentameric a7 nAChRs, it is most likely that methadone binds to a7 nAChRs in both of these cell lines. It is well known that a7 nAChR function cannot be studied with an 86Rb+ efflux assay due to the rapid channel kinetics of this receptor subtype [24]. The a7 nAChRs have a high permeability to Ca2+ [27], which enables functional studies with calcium fluorometry, although the rapid channel kinetics of the receptor also need to be taken into account here. Metha-
Table 2. Rb+ efflux assay in the SH-EP1-ha4b2 and SH-SY5Y cell lines.
86
SH-EP1-ha4b2 Nicotine 10 lM Compound
IC50 (lM)
Methadone Mecamylamine
7.4 2.9
LogIC50 S.D. 5.1 0.03 5.5 0.02
SH-SY5Y Nicotine 1 lM
IC50 (lM) 5.6 2.6
LogIC50 S.D. 5.3 0.08 5.6 0.03
Nicotine 30 lM IC50 (lM) 0.8 0.2
LogIC50 S.D. 6.1 0.03 6.7 0.03
Nicotine 10 lM IC50 (lM) 1.0 0.1
LogIC50 S.D. 6.0 0.05 7.0 0.04
IC50, the half-maximal inhibitory concentration; logIC50, the half-maximal inhibitory concentration in logarithmic scale; S.D., standard deviation.
© 2014 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society)
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Fig. 6. Mechanisms of antagonism of nAChR function by methadone and mecamylamine using an 86Rb+ efflux assay. The nicotine-stimulated specific 86Rb+ efflux was determined in the absence or presence of various concentrations of either methadone or mecamylamine in SH-EP1-ha4b2 cells (A, C) or SH-SY5Y (B, D) cells. The specific efflux for each drug concentration was calculated by subtracting the non-specific efflux (buffer controls) from the total efflux (1 mM carbamylcholine stimulation). The cells were exposed to the ligands for 5 min. The data were fit to the variable slope model. The values are the mean S.E.M. from three to five separate assays.
methadone is a non-competitive antagonist. In addition, the nicotine-induced increase in [Ca2+]i is significantly reduced in SH-SY5Y cells exposed to methadone, which further supports methadone’s ability to block a3* nAChRs and desensitize a7 nAChRs. Methadone elevates [Ca2+]i levels in SH-SY5Y cells, most likely through a7 nAChRs, and this elevation is inhibited by both nicotinic and opioid antagonists similarly in the SHEP1-ha7 cells. Our previous results show that morphine also acts as an antagonist at a3* nAChRs, and this action is independent of its agonist properties at opioid receptors [13]. In untransfected SH-EP1 cells, methadone dose-dependently increases [Ca2+]i, and the increase is antagonized by opioid antagonists, but not by nAChR antagonists. The inhibition results most likely from the antagonism of opioid receptors because these cells do not contain nAChRs [20]. Moreover, the methadone-induced increase in [Ca2+]i is negligible in untransfected SH-EP1 cells in comparison with that observed in SH-EP1 cells expressing recombinant nAChRs [11]. Methadone is in clinical use for treating moderate to severe pain and as a maintenance replacement treatment for opioid addiction. Most methadone maintenance treatment patients are smokers, and the use of methadone increases smoking [6–9]. A circulus vitiosus is apparent because nicotine has been shown to increase methadone self-administration and reinforcing properties [30]. Thus, it can be suggested that methadone maintenance treatment patients smoke more because of the more satisfying effects that they get from their methadone dose or vice versa (they might find smoking more satisfying because of their methadone use). However, our data would support the alternative hypothesis that these patients do not get the normal effects from nicotine because of methadone antagonism at a4b2 and a3* nAChRs or the desensitisation of a7 nAChRs, and thus, they smoke more to overcome this interaction. Although a direct correlation between in vitro and human data might not be relevant, our findings support the
hypothesis of significant nicotine–opioid interactions at the level of nAChRs. When comparing in vitro experiments and the clinical use of compounds, one can always criticize the use of relevant concentrations. In clinical studies, the plasma concentration of methadone in maintenance patients can reach the level of 1 lM. In contrast, in animal studies, brain tissue methadone concentrations have been reported to exceed those of the plasma [31]. In our in vitro experiments, methadone had IC50 values of 0.8–9.4 lM. The steady-state nicotine blood concentration in smokers on average is 0.07–0.2 lM [32], and every puff from cigarette accumulates the nicotine concentration in the brain [33]. Therefore, the mechanism of in vivo interactions of methadone and nicotine may at least partially be explained by our findings in vitro. The principal finding of this study is that methadone acts as a non-competitive antagonist at clinically relevant concentrations at a4b2 and a3* nAChRs expressed in SH-EP1-ha4b2 and SH-SY5Y cells, respectively. This study also confirms that methadone is a human a7 nAChR agonist. Acknowledgements We thank Ronald J. Lukas and Paul Whiteaker for valuable comments, Kati Rautio, Anna Niemi and Marjo Vaha for technical assistance, Mari Havia and Juri Meijer for conducting parts of the experiments and Jyrki Kukkonen for enabling the use of FlexStation. This study was funded by the Academy of Finland Research Programme on Substance Use and Addictions [Grant 1118535] and by the Research Funds of the University of Helsinki (OS). References 1 Nutter TJ, Adams DJ. Monovalent and divalent cation permeability and block of neuronal nicotinic receptor channels in rat parasympathetic ganglia. J Gen Physiol 1995;105:701–23.
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2 Gotti C, Clementi F, Fornari A, Gaimarri A, Guiducci S, Manfredi I et al. Structural and functional diversity of native brain neuronal nicotinic receptors. Biochem Pharmacol 2009;78:703–11. 3 Gotti C, Clementi F. Neuronal nicotinic receptors: from structure to pathology. Prog Neurobiol 2004;74:363–96. 4 Dietis N, Rowbotham DJ, Lambert DG. Opioid receptor subtypes: fact or artifact? Br J Anaesth 2011;107:8–18. 5 Kristensen K, Christensen CB, Christrup LL. The mu1, mu2, delta, kappa opioid receptor binding profiles of methadone stereoisomers and morphine. Life Sci 1995;56:PL45–50. 6 Clemmey P, Brooner R, Chutuape MA, Kidorf M, Stitzer M. Smoking habits and attitudes in a methadone maintenance treatment population. Drug Alcohol Depend 1997;44:123–32. 7 Elkader AK, Brands B, Selby P, Sproule BA. Methadone-nicotine interactions in methadone maintenance treatment patients. J Clin Psychopharmacol 2009;29:231–8. 8 Chait LD, Griffiths RR. Effects of methadone on human cigarette smoking and subjective ratings. J Pharmacol Exp Ther 1984;229:636–40. 9 Story J, Stark MJ. Treating cigarette smoking in methadone maintenance clients. J Psychoactive Drugs 1991;23:203–15. 10 Lemon SC, Friedmann PD, Stein MD. The impact of smoking cessation on drug abuse treatment outcome. Addict Behav 2003;28:1323–31. 11 Pakkanen JS, Nousiainen H, Yli-Kauhaluoma J, Kyl€anlahti I, M€oykkynen T, Korpi ER et al. Methadone increases intracellular calcium in SH-SY5Y and SH-EP1-halpha7 cells by activating neuronal nicotinic acetylcholine receptors. J Neurochem 2005;94:1329–41. 12 Xiao Y, Smith RD, Caruso FS, Kellar KJ. Blockade of rat alpha3beta4 nicotinic receptor function by methadone, its metabolites, and structural analogs. J Pharm Exp Ther 2001;299:366–71. 13 Talka R, Salminen O, Whiteaker P, Lukas RJ, Tuominen RK. Nicotine – morphine interactions at a4b2, a7 and a3* nicotinic acetylcholine receptors. Eur J Pharmacol 2013;701:57–64. 14 Peng X, Gerzanich V, Anand R, Wang F, Lindstr€om J. Chronic nicotine treatment upregulates alpha3 and alpha7 acetylcholine receptor subtypes expressed by the human neuroblastoma cell line SH-SY5Y. Mol Pharmacol 1997;51:776–84. 15 Eaton JB, Peng JH, Schroeder KM, George AA, Fryer JD, Krishnan C et al. Characterization of human alpha 4 beta 2-nicotinic acetylcholine receptors stably and heterologously expressed in native nicotinic receptor-null SH-EP1 human epithelial cells. Mol Pharmacol 2003;64:1283–94. 16 Zhao L, Kuo YP, George AA, Peng JH, Purandare MS, Schr€oeder KM. Functional properties of homomeric, human alpha 7-nicotinic acetylcholine receptors heterologously expressed in the SH-EP1 human epithelial cell line. J Pharm Exp Ther 2003;305:1132–41. 17 Kazmi SM, Mishra RK. Comparative pharmacological properties and functional coupling of mu and delta opioid receptor sites in human neuroblastoma SH-SY5Y cells. Mol Pharmacol 1987;32:109–18. 18 Wang F, Gerzanich V, Wells GB, Anand R, Peng X, Keyser K et al. Assembly of human neuronal nicotinic receptor alpha5 subunits with alpha3, beta2, and beta4 subunits. J Biol Chem 1996;271:17656–65.
19 Gerzanich V, Peng X, Wang F, Wells G, Anand R, Fletcher S et al. Comparative pharmacology of epibatidine: a potent agonist for neuronal nicotinic acetylcholine receptors. Mol Pharmacol 1995;48:774–82. 20 Lukas RJ, Norman SA, Lucero L. Characterization of nicotinic acetylcholine receptors expressed by cells of the SH-SY5Y human neuroblastoma clonal line. Mol Cell Neurosci 1993;4: 1–12. 21 Baumhaker Y, Wollman Y, Goldstein MN, Sarne Y. Evidence for mu-, delta-, and kappa-opioid receptors in a human neuroblastoma cell line. Life Sci 1993;52:PL205–10. 22 Zwart R, Vijverberg HP. Four pharmacologically distinct subtypes of alpha4beta2 nicotinic acetylcholine receptor expressed in xenopus laevis oocytes. Mol Pharmacol 1998;54:1124–31. 23 Marks MJ, Laverty DS, Whiteaker P, Salminen O, Grady SR, McIntosh JM et al. John Daly’s compound, epibatidine, facilitates identification of nicotinic receptor subtypes. J Mol Neurosci 2010;40:96–104. 24 Lukas RJ, Fryer JD, Eaton JB, Gentry CL. Some methods for studies of nicotinic acetylcholine receptor pharmacology. In: Levin ED (ed.). Nicotinic Receptors and the Nervous System. CRC Press, Boca Raton, FL, 2002; 3–27. 25 Fedorov NB, Benson LC, Graef J, Lippiello PM, Bencherif M. Differential pharmacologies of mecamylamine enantiomers: positive allosteric modulation and noncompetitive inhibition. J Pharmacol Exp Ther 2009;328:525–32. 26 Marks MJ, Meinerz NM, Brown RW, Collins AC. 86Rb+ efflux mediated by alpha4beta2*-nicotinic acetylcholine receptors with high and low-sensitivity to stimulation by acetylcholine display similar agonist-induced desensitization. Biochem Pharmacol 2010;80:1238–51. 27 Castro NG, Albuquerque EX. a-Bungarotoxin-sensitive hippocampal nicotinic receptor channel has a high calcium permeability. Biophys J 1995;68:516–24. 28 Papke RL, Sanberg PR, Shytle RD. Analysis of mecamylamine stereoisomers on human nicotinic receptor subtypes. J Pharmacol Exp Ther 2001;297:646–56. 29 Almeida LE, Pereira EF, Alkondon M, Fawcett WP, Randall WR, Albuquerque EX. The opioid antagonist naltrexone inhibits activity and alters expression of alpha7 and alpha4beta2 nicotinic receptors in hippocampal neurons: implications for smoking cessation programs. Neuropharmacology 2000;39:2740–55. 30 Spiga R, Schmitz J, Day J 2nd. Effects of nicotine on methadone self-administration in humans. Drug Alcohol Depend 1998;50:157– 65. 31 Sung CY, Way EL. The metabolic fate of the optical isomers of methadone. J Pharmacol Exp Ther 1953;109:244–54. 32 Schneider NG, Olmstead RE, Franzon MA, Lunell E. The nicotine inhaler: clinical pharmacokinetics and comparison with other nicotine treatments. Clin Pharmacokinet 2001;40:661–84. 33 Rose JE, Mukhin AG, Lokitz SJ, Turkington TG, Herskovic J, Behm FM et al. Kinetics of brain nicotine accumulation in dependent and nondependent smokers assessed with PET and cigarettes containing 11C-nicotine. Procof Natl Acad Sci USA 2010;107:5190–5.
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Doi: 10.1111/bcpt.12317
Methadone is a Non-Competitive Antagonist at the a4b2 and a3* Nicotinic Acetylcholine Receptors and an Agonist at the a7 Nicotinic Acetylcholine Receptor Reeta Talka, Outi Salminen and Raimo K. Tuominen Division of Pharmacology and Pharmacotherapy, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland (Received 21 March 2014; Accepted 26 August 2014) Abstract: Nicotine–methadone interactions have been studied in human beings and in various experimental settings regarding addiction, reward and pain. Most methadone maintenance treatment patients are smokers, and methadone administration has been shown to increase cigarette smoking. Previous in vitro studies have shown that methadone is a non-competitive antagonist at rat a3b4 nicotinic acetylcholine receptors (nAChR) and an agonist at human a7 nAChRs. In this study, we used cell lines expressing human a4b2, a7 and a3* nAChRs to compare the interactions of methadone at the various human nAChRs under the same experimental conditions. A [3H]epibatidine displacement assay was used to determine whether methadone binds to the nicotinic receptors, and 86Rb+ efflux and changes in intracellular calcium [Ca2+]i were used to assess changes in the functional activity of the receptors. Methadone displaced [3H]epibatidine from nicotinic agonist-binding sites in SH-EP1-ha7 and SH-SY5Y cells, but not in SH-EP1-ha4b2 cells. The Ki values for methadone were 6.3 lM in SH-EP1-ha7 cells and 19.4 lM and 1008 lM in SHSY5Y cells. Methadone increased [Ca2+]i in all cell lines in a concentration-dependent manner, and in SH-EP1-ha7 cells, the effect was more pronounced than the effect of nicotine treatment. In SH-EP1-ha4b2 cells, the effect of methadone was negligible compared to that of nicotine. Methadone pre-treatment abolished the nicotine-induced response in [Ca2+]i in all cell lines expressing nAChRs. In SH-EP1-ha4b2 and SH-SY5Y cells, methadone had no effect on the 86Rb+ efflux, but it antagonized the nicotine-induced 86Rb+ ion efflux in a non-competitive manner. These results suggest that methadone is an agonist at human a7 nAChRs and a non-competitive antagonist at human a4b2 and a3* nAChRs. This study adds further support to the previous findings that opioids interact with nAChRs, which may underlie their frequent co-administration in human beings and might be of interest to the field of drug discovery.
The cholinergic system in the brain is important in memory and learning as well as in the control of movement. Cholinergic neurons release acetylcholine (ACh) that binds to muscarinic and neuronal nicotinic acetylcholine receptors (nAChRs). The nAChRs are ligand-gated ion channels that are permeable to Na+, K+ and Ca2+ ions [1], and in addition to ACh, nicotine also binds with high affinity to these receptors. The channel is formed in the middle of five transmembrane subunits, which contain either a (a2–a10) or a and b (b2–b4) subunits [2]. The nAChRs modulate the function of several neurotransmitter systems, including dopaminergic transmission and endogenous opioids [3]. The opioid system modulates pain behaviour, antinociception, reward and addiction. To date, four different G-protein-coupled opioid receptors have been characterized, the l, d, j and opioid receptor like-1 receptors [4]. Nicotine–methadone interactions have been studied in human beings and in vitro settings regarding addiction, reward and pain. Methadone is a l-opioid receptor agonist that also has a low affinity for d and j receptors [5] and is in clinical use for treating moderate to severe pain and for the maintenance replacement treatment of opioid addiction. Almost all methadone maintenance treatment patients are smokers [6,7]. Methadone administration increases cigarette smoking in a Author for correspondence: Reeta Talka, Division of Pharmacology and Pharmacotherapy, Faculty of Pharmacy, University of Helsinki, Viikinkaari 5 E, P.O. Box 56, FI-00014 Helsinki, Finland (fax +358 2941 59471, e-mail [email protected]).
dose-dependent manner, and the maintenance patients have higher nicotine craving and withdrawal symptoms than smokers not receiving methadone [8,9]. Furthermore, smoking cessation is considered to be more demanding for maintenance patients, whereas quitting smoking has been shown to improve opioid abstinence [10]. In in vitro studies, methadone has been suggested to be an agonist at human a7 nAChRs [11] and a non-competitive antagonist at rat a3b4 nAChRs [12]. Furthermore, prolonged exposure to methadone up-regulates nAChRs expressed in SH-SY5Y cells [11]. We have previously shown that the opioid receptor agonist morphine has interactions with nAChRs that are independent of its agonist properties at opioid receptors [13]. The cell lines used in this study are of human origin and either have a native expression of nAChRs (SH-SY5Y cells) [14] or have been transfected with human cDNAs coding nAChR subunits (SH-EP1-ha4b2 and SH-EP1-ha7 cells) [15,16]. SH-SY5Y cells express the a3, a5, a7, b2 and b4 nAChR subunits and have native expression of functional opioid receptors [17]. The b2 subunit-containing receptors from SH-SY5Y cells have a higher affinity for [3H]epibatidine than the b4 or a7 subunit-containing receptors [18,19]. SH-EP1ha4b2 and SH-EP1-ha7 cells have no native expression of nAChRs [20], but have been transfected with human cDNAs coding the a4 and b2 subunits or a7 subunit, respectively [15,16]. SH-EP1-ha4b2 and SH-EP1-ha7 cells also have native expression of the l- and d-opioid receptors [21]. The
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a4b2 nAChRs expressed in SH-EP1-ha4b2 cells have two distinct stoichiometric arrangements, (a4)2(b2)3 (high-affinity subtype) and (a4)3(b2)2 (low-affinity subtype) [22]. Untransfected SH-EP1-cells were used as a control cell line in the intracellular calcium assay. The purpose of this study was to investigate the interactions of nicotine and methadone at the receptor level using SH-EP1ha4b2, SH-SY5Y, SH-EP1-ha7 and untransfected SH-EP1 cell lines. Binding studies were performed with [3H]epibatidine, a nicotinic acetylcholine receptor agonist that binds to several nicotinic acetylcholine receptor subtypes [23], to determine whether methadone binds competitively to the orthosteric agonist-binding site in nicotinic acetylcholine receptors. Additionally, calcium fluorometry and 86Rb+ efflux assays were used to study the functional interactions. Our results show that methadone acts as a non-competitive antagonist at human a4b2 and a3* nAChRs expressed in SH-EP1-ha4b2 and SHSY5Y cells, respectively, and as an agonist at human a7 nAChRs. Materials and Methods Drugs and reagents. The ()-methadone hydrochloride was purchased from Star, Tampere, Finland. The bovine serum albumin, ( )-nicotine hydrogen tartrate, mecamylamine hydrochloride, carbamylcholine chloride, methyllycaconitine citrate salt hydrate, naltrexone hydrochloride, Bradford reagent, poly-D-lysine and poly (ethyleneimine) solution were purchased from Sigma-Aldrich (St. Louis, MO, USA). The bovine serum albumin standards were from Thermo Fisher Scientific Inc. (Rockford, IL, USA). The rubidium-86 radionuclide and [3H]epibatidine were purchased from PerkinElmer (Waltham, MA, USA). All cell culture plasticware was from Nunc (Nunc A/S, Roskilde, Denmark). The media, serum, non-essential amino acids, selection antibiotics and Fluo-3AM calcium indicator were from Invitrogen (Invitrogen, Carlsbad, CA, USA). All buffers were prepared as described in the study by Talka et al. [13]. Cell culture. All cell lines were grown as described in the study by Talka et al. [13]. SH-EP1-ha4b2 and SH-EP1-ha7 cells (kindly provided by Dr. Ronald J. Lukas, Barrow Neurological Institute, St Joseph’s Hospital and Medical Center, Phoenix, AZ, USA) were grown in Dulbecco’s modified Eagle’s medium containing foetal bovine serum, horse serum, penicillin–streptomycin, hygromycin, amphotericin B (Fungizoneâ (Invitrogen, Carlsbad, CA, USA)) and zeocin (only for SH-EP1-ha4b2 cells). Untransfected SH-EP1-cells were grown in Dulbecco’s modified Eagle’s medium containing foetal bovine serum, horse serum and penicillin–streptomycin. SH-SY5Y cells were grown in Dulbecco’s modified Eagle’s medium; Ham’s F12 was supplemented with non-essential amino acids, foetal bovine serum, penicillin and streptomycin. Cell cultures were maintained at 37°C in 5% CO2/humidified air, and passage numbers 10–30 were used in the assays. Cells were grown to confluency in 75- and 175cm2 flasks and split twice weekly. [3H]epibatidine binding assay. The [3H]epibatidine binding assays were carried out as described in the study by Talka et al. [13]. Briefly, the cells were grown to confluency, the medium was removed, and the cells were washed with phosphate-buffered saline. The cells were mechanically harvested and homogenized using an ultrasonic homogenizer (Ringo Ultrasonics Bio 70, Romanshorn, Switzerland) (75% amplitude, 6 9 5 sec.). The homogenates were centrifuged, and the pellet was resuspended in potassium phosphate
buffer and frozen. The binding studies were performed in 96-well glass-filter plates (Millipore MultiScreen HTS FC (Merck Millipore, Billerica, MA, USA)) with samples containing 50 lg of membrane protein (SH-EP1-ha4b2 cells, ligand depletion was minimized by reducing the protein concentration to 5 lg/well). The day before the binding assay, 200 ll of 0.1% poly(ethyleneimine) solution was added to the wells and left overnight at +2 to +8°C. The protein concentrations were measured using the Bradford method. A MultiScreen Vacuum Manifold (Merck Millipore, Billerica, MA, USA) was used to aspirate the assay mixtures. Cell membranes were incubated with [3H]epibatidine for 2 hr at room temperature. Nonspecific binding was defined using 1 mM nicotine. Optiphase HiSafe 3â (Perkin Elmer, Turku, Finland) scintillant was added to the wells, and radioactivity was quantified using a scintillation counter (Wallac 1450 MicroBeta TriLux Liquid Scintillation Counter & Luminometer, Turku, Finland). The binding of [3H]epibatidine to the cell membranes of cell lines expressing nAChRs was saturable [13]. Calcium fluorometry. The calcium fluorometry studies were carried out as described in the study by Talka et al. [13]. In brief, cells were seeded in 96-well microplates (in a 100 ll volume) coated with polyD-lysine the day before the experiment. On the following day, the culture medium was removed from the wells, and the cells were washed twice with Tyrode’s salt solution (TSS). The loading medium consisted of Fluo-3 AM and probenecid acid in TSS. After incubation with loading medium, the cells were washed twice with TSS. When pre-treatments were used, the cells were pre-incubated with drugs for 10 min. Fluorescence was measured with a FlexStation microplate reader (Molecular Devices, Sunnyvale, CA, USA). The test drug was injected, and the fluorescence was monitored for 80 sec. at the 485/ 525 nM wavelength. The drug-induced signals were calculated by subtracting the background signal (measured for 10 sec. before injection) from the signal (measured during the time interval from 20 to 50 sec.). The cells were also stimulated by injecting vehicle (TSS) as a control in each experiment. The changes in [Ca2+]i were expressed as the percentage of fluorescence increased by nicotine. 86
Rb+ efflux. The 86Rb+ efflux assays were carried out as described in the study by Talka et al. [13]. Briefly, cells were seeded in 24-well plates and allowed to adhere for 4 hr in a 37°C incubator, after which the medium was replaced with medium supplemented with 86Rb+. The 86 Rb+ efflux was measured 1 day after seeding the cells with the ‘flipplate’ method [24]. Each well was rinsed with fresh 86Rb+ efflux buffer for 1 min. to remove extracellular 86Rb+. The cells were exposed to efflux buffer containing the ligands being studied for 5 min. The radioactivity was counted using Cerenkov counting (Wallac 1450 MicroBeta TriLux Liquid Scintillation Counter & Luminometer) after placing cross-talk minimizing inserts (Perkin Elmer 1450-109) into each well. The specific efflux for each drug concentration was calculated by subtracting the non-specific efflux (buffer, three wells in each plate) from the total efflux (1 mM carbamylcholine, three wells in each plate). The remaining intracellular 86Rb+ was counted periodically [13]. The maximum 86 Rb+ efflux values depended on cell type, density and the concentration of 86Rb+ in the loading medium and were typically 30,000–50,000 cpm over non-specific backgrounds of 6000– 10,000 cpm. The 86Rb+ efflux assays could not be done with the SHEP1-ha7 cell line because a7 nAChRs are rapidly inactivating channels that are not open long enough to give a significant ion flux signal [24]. Data analysis. All data were analysed using GraphPad Prismâ (version 6.02; GraphPad Software, Inc., La Jolla, CA, USA). In the competition binding assay, the equilibrium dissociation constant (Ki) values were calculated using one-site and two-site curve fitting models
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THE ACTION OF METHADONE AT a4b2, a3 AND a7 nAChRs â
with Prism , and the Ki values were determined using best fit (pvalue < 0.05, extra sum-of-squares F test). The Ki values for one-site competition binding were derived from the equation logEC50 = log (10logKi(1 + L*/Kdhot)); y = min + (max min)/(1 + 10X logEC50), where L* is the concentration of [3H]epibatidine, Kdhot is the equilibrium dissociation constant of [3H]epibatidine, max and min are the plateaus of the Y axis and logKi is the log of the molar equilibrium dissociation constant of unlabelled ligand. The Ki values for two-site competition binding were derived from the Prismâ build-in model: L = 10X + 9; KiHi = 10LogKiHi + 9; KiLow = 10LogKiLow + 9; Site 1 = L*(max min)/(L* + KdL*_Ki* (1 + L/KiHi)); Site 2 = L*(max min)/(L* +KDL*_Low(1 + L/KiLow)); Y = Site 1 9 FractionHi) + min, where L* is the labelled FractionHi + Site 2 (1 ligand, L is unlabelled ligand, max and min are the plateaus in the units of the Y axis, FractionHi is the fraction of all the sites that have a high affinity for the competitor and logKiHi and logKiLow are the logarithms of the two molar Ki values. The KiHi and KiLow values are the equilibrium dissociation constant values for the high- and lowaffinity binding sites, respectively. The calcium fluorometry responses were calculated as the percentage of the increase in [Ca2+]i produced by a close to maximally effective concentration of nicotine (10 lM for SH-EP1-ha4b2 and SH-EP1-ha7 cells and 30 lM for SH-SY5Y cells). The intracellular calcium data were analysed statistically using one-way analysis of variance (ANOVA) and Dunnett’s multiplecomparison post hoc test. Values of p < 0.05 were taken to be statistically significant. Specific 86Rb+ efflux was defined as total 86 Rb+ efflux (1 mM carbamylcholine) minus non-specific 86Rb+ efflux (buffer). The specific 86Rb+ efflux was fit to the variable slope model or the biphasic dose–response model after comparing these two different fits (p-value 3000
8.30 0.30 7.09 0.13 NA
KiHi, KiLow (lM), (%) 0.005, 4.5, 10.6%, 89.4% 19.4, 1008, 48.6%, 51.4%
SH-EP1-ha7 LogKiHi S.D. logKiLow S.D. 8.31 5.35 4.71 3.00
0.60 0.07 0.28 0.50
Ki (lM)
LogKi S.D.
0.15
6.82 0.11
6.3
5.20 0.06
KiHi/KiLow, the equilibrium dissociation constant value for the high-/low-affinity binding sites; logHi/logKiLow, the equilibrium dissociation constant value for the high-/low-affinity binding sites in logarithmic scale; S.D., standard deviation; NA, not applicable.
A
B
C
Fig. 1. [3H]Epibatidine competition binding profiles in SH-EP1-ha4b2 (A), SH-SY5Y (B) and SH-EP1-ha7 (C) cell membranes for nicotine, mecamylamine and methadone. The assay mixtures contained 150 pM [3H]epibatidine, 5–50 lg cell membrane protein and nicotine, mecamylamine or methadone in increasing concentrations. To determine the specific binding, assays were performed in the absence (total binding) or presence of 1 mM nicotine (non-specific binding). The specific binding was calculated by subtracting the non-specific binding from the total binding. The data were fit to a two-sites or one-site competition binding model, and the Ki values were determined from the better fit after comparing the two fits (pvalue < 0.05, extra sum-of-squares F test). The statistical programme determined that the best fit was the two-sites model for nicotine (p < 0.001) in SH-EP1-ha4b2 cells and for nicotine (p < 0.05) and methadone (p < 0.05) in SH-SY5Y cells. The values are the mean S.E.M. from three to five assays, with each point assayed at least in triplicate. Ki and logKi values are provided in Table 1.
A
C
E
G
B
D
F
H
Fig. 2. Effects of mecamylamine and methadone on nicotine-evoked increases in [Ca2+]i in SH-EP1-ha4b2 (A), SH-SY5Y (C), SH-EP1-ha7 (E) and SH-EP1 (G) cells and methadone-induced increases in [Ca2+]i in SH-EP1-ha4b2 (B), SH-SY5Y (D), SH-EP1-ha7 (F) and SH-EP1 (H) cells. In figs A, C, E and G, Fluo-3 AM-loaded cells were incubated for 10 min. with mecamylamine or methadone and then stimulated with 10 or 30 lM nicotine. In figs B, D, F and H, Fluo-3 AM-loaded cells were incubated for 10 min. with vehicle (TSS) and then stimulated with nicotine (10 lM or 30 lM), methadone (10 lM, 50 lM or 100 lM) or vehicle (TSS). The responses are expressed as the percentage of the nicotine response measured in parallel in the absence of other agonists. The values are the mean S.E.M. from three to six separate assays. In each experiment, there were at least six replicates for each condition. Significantly different from nicotine response *p < 0.05, **p < 0.01 and ***p < 0.001; significantly different from vehicle response # p < 0.05, ##p < 0.01 and ###p < 0.001 using one-way ANOVA. Nic, nicotine; TSS, Tyrode’s salt solution.
Discussion and Conclusions Our study shows for the first time that methadone acts as a noncompetitive antagonist at a4b2 nAChRs. Additionally, we were
able to confirm previous results that postulated methadone as an antagonist at a3* nAChRs and an agonist at a7 nAChRs. Thus, methadone’s effects seem to vary by nAChR subtype, which might be of interest to the field of drug discovery.
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B
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C
D
Fig. 3. Effects of nAChR and opioid antagonists on methadone-evoked increases in [Ca2+]i in SH-EP1-ha4b2 (A), SH-SY5Y (B), SH-EP1-ha7 (C) and SH-EP1 (D) cells. The Fluo-3 AM-loaded cells were incubated for 10 min. with vehicle, mecamylamine (Meca), methyllycaconitine (MLA), naloxone (Nalo) or naltrexone (Nalt) and then stimulated with 100 lM methadone. The responses are expressed as the percentage of the methadone response. The values are the mean S.E.M. from three to six separate assays. In each experiment, there were at least six replicates for each condition. Significantly different from methadone response *p < 0.05, **p < 0.01, and ***p < 0.001 using one-way ANOVA.
Our conclusion that methadone is a non-competitive antagonist at a4b2 nAChRs is based on several findings. Firstly, methadone does not possess binding affinity for the epibatidine-binding site of the receptor. Secondly, it has no effect on the 86Rb+ efflux when administered in buffer alone. Thirdly, methadone inhibits the effects of nicotine on a4b2 nAChRs in the functional assays used. The methadone-induced antagonism is non-competitive in nature, as it does not shift the EC50 value for nicotine, and the inhibition curve is not surmountable in the 86Rb+ efflux assay. Furthermore, the methadone-induced inhibition of a4b2 receptor function is comparable to that of the non-selective nicotinic antagonist mecamylamine, as both of them produce functional inhibition of a4b2 nAChRs without binding to the epibatidine-binding site. We have previously shown that morphine has affinity for a4b2 nAChRs and acts as a partial agonist at this subtype [13]. Thus, different opioids seem to have somewhat different effects on nAChRs. A high concentration of methadone (100 lM) increases [Ca2+]i in SH-EP1-ha4b2 cells, although to a considerably lesser extent than nicotine. This effect can be antagonized by mecamylamine, but not by opioid antagonists. In SH-EP1ha4b2 cells, the a4b2 nAChRs exist in two stoichiometries, that is the low-affinity (a4)3(b2)2 and the high-affinity (a4)2(b2)3 nAChRs [25]. The effect of methadone on [Ca2+]i may be due to the initial activation of the low-affinity (a4)3(b2)2 nAChRs because these receptors mediate agonistinduced responses when agonists are used at high concentrations [26]. Furthermore, racemic mecamylamine has been shown to be more selective in antagonizing the low-affinity a4b2 nAChRs, the (S)-(+) enantiomer being primarily respon-
IC50 and logIC50 values for methadone and mecamylamine in an
sible for this selectivity [25]. The initial activation of the lowaffinity (a4)3(b2)2 nAChRs could lead to receptor desensitization as reflected by our finding that in methadone pre-treated cells, the effect of nicotine on [Ca2+]i was abolished. These data suggest that methadone acts as a partial agonist of a4b2 nAChRs with low activation efficacy and also as a selective desensitizer of these a4b2 nAChRs. However, we cannot rule out the possibility that the introduction of a4b2 nAChRs into the SH-EP1 cells changes the expression levels of the endogenous opioid receptors, which could explain why the opioid antagonists, naloxone and naltrexone, do not have greater effect. Previous in vitro experiments have concluded that methadone is a non-competitive antagonist at rat a3b4 nAChRs [12] and an agonist at human a7 nAChR [11]. Our findings are in line with these results. Methadone inhibits [3H]epibatidine binding in SH-SY5Y cells and SH-EP1-ha7 cells. SH-SY5Y cells express the a3, a5, a7, b2 and b4 nAChR subunits, which form different combinations, for example homopentameric a7 and heteropentameric a3* nAChR subtypes, of which the b2 subunit-containing receptors have a higher affinity for [3H]epibatidine than the b4 or a7 subunit-containing receptors [18,19]. Because SH-EP1-ha7 cells express only homopentameric a7 nAChRs, it is most likely that methadone binds to a7 nAChRs in both of these cell lines. It is well known that a7 nAChR function cannot be studied with an 86Rb+ efflux assay due to the rapid channel kinetics of this receptor subtype [24]. The a7 nAChRs have a high permeability to Ca2+ [27], which enables functional studies with calcium fluorometry, although the rapid channel kinetics of the receptor also need to be taken into account here. Metha-
Table 2. Rb+ efflux assay in the SH-EP1-ha4b2 and SH-SY5Y cell lines.
86
SH-EP1-ha4b2 Nicotine 10 lM Compound
IC50 (lM)
Methadone Mecamylamine
7.4 2.9
LogIC50 S.D. 5.1 0.03 5.5 0.02
SH-SY5Y Nicotine 1 lM
IC50 (lM) 5.6 2.6
LogIC50 S.D. 5.3 0.08 5.6 0.03
Nicotine 30 lM IC50 (lM) 0.8 0.2
LogIC50 S.D. 6.1 0.03 6.7 0.03
Nicotine 10 lM IC50 (lM) 1.0 0.1
LogIC50 S.D. 6.0 0.05 7.0 0.04
IC50, the half-maximal inhibitory concentration; logIC50, the half-maximal inhibitory concentration in logarithmic scale; S.D., standard deviation.
© 2014 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society)
THE ACTION OF METHADONE AT a4b2, a3 AND a7 nAChRs
A
B
325
C
D
Fig. 3. Effects of nAChR and opioid antagonists on methadone-evoked increases in [Ca2+]i in SH-EP1-ha4b2 (A), SH-SY5Y (B), SH-EP1-ha7 (C) and SH-EP1 (D) cells. The Fluo-3 AM-loaded cells were incubated for 10 min. with vehicle, mecamylamine (Meca), methyllycaconitine (MLA), naloxone (Nalo) or naltrexone (Nalt) and then stimulated with 100 lM methadone. The responses are expressed as the percentage of the methadone response. The values are the mean S.E.M. from three to six separate assays. In each experiment, there were at least six replicates for each condition. Significantly different from methadone response *p < 0.05, **p < 0.01, and ***p < 0.001 using one-way ANOVA.
Our conclusion that methadone is a non-competitive antagonist at a4b2 nAChRs is based on several findings. Firstly, methadone does not possess binding affinity for the epibatidine-binding site of the receptor. Secondly, it has no effect on the 86Rb+ efflux when administered in buffer alone. Thirdly, methadone inhibits the effects of nicotine on a4b2 nAChRs in the functional assays used. The methadone-induced antagonism is non-competitive in nature, as it does not shift the EC50 value for nicotine, and the inhibition curve is not surmountable in the 86Rb+ efflux assay. Furthermore, the methadone-induced inhibition of a4b2 receptor function is comparable to that of the non-selective nicotinic antagonist mecamylamine, as both of them produce functional inhibition of a4b2 nAChRs without binding to the epibatidine-binding site. We have previously shown that morphine has affinity for a4b2 nAChRs and acts as a partial agonist at this subtype [13]. Thus, different opioids seem to have somewhat different effects on nAChRs. A high concentration of methadone (100 lM) increases [Ca2+]i in SH-EP1-ha4b2 cells, although to a considerably lesser extent than nicotine. This effect can be antagonized by mecamylamine, but not by opioid antagonists. In SH-EP1ha4b2 cells, the a4b2 nAChRs exist in two stoichiometries, that is the low-affinity (a4)3(b2)2 and the high-affinity (a4)2(b2)3 nAChRs [25]. The effect of methadone on [Ca2+]i may be due to the initial activation of the low-affinity (a4)3(b2)2 nAChRs because these receptors mediate agonistinduced responses when agonists are used at high concentrations [26]. Furthermore, racemic mecamylamine has been shown to be more selective in antagonizing the low-affinity a4b2 nAChRs, the (S)-(+) enantiomer being primarily respon-
IC50 and logIC50 values for methadone and mecamylamine in an
sible for this selectivity [25]. The initial activation of the lowaffinity (a4)3(b2)2 nAChRs could lead to receptor desensitization as reflected by our finding that in methadone pre-treated cells, the effect of nicotine on [Ca2+]i was abolished. These data suggest that methadone acts as a partial agonist of a4b2 nAChRs with low activation efficacy and also as a selective desensitizer of these a4b2 nAChRs. However, we cannot rule out the possibility that the introduction of a4b2 nAChRs into the SH-EP1 cells changes the expression levels of the endogenous opioid receptors, which could explain why the opioid antagonists, naloxone and naltrexone, do not have greater effect. Previous in vitro experiments have concluded that methadone is a non-competitive antagonist at rat a3b4 nAChRs [12] and an agonist at human a7 nAChR [11]. Our findings are in line with these results. Methadone inhibits [3H]epibatidine binding in SH-SY5Y cells and SH-EP1-ha7 cells. SH-SY5Y cells express the a3, a5, a7, b2 and b4 nAChR subunits, which form different combinations, for example homopentameric a7 and heteropentameric a3* nAChR subtypes, of which the b2 subunit-containing receptors have a higher affinity for [3H]epibatidine than the b4 or a7 subunit-containing receptors [18,19]. Because SH-EP1-ha7 cells express only homopentameric a7 nAChRs, it is most likely that methadone binds to a7 nAChRs in both of these cell lines. It is well known that a7 nAChR function cannot be studied with an 86Rb+ efflux assay due to the rapid channel kinetics of this receptor subtype [24]. The a7 nAChRs have a high permeability to Ca2+ [27], which enables functional studies with calcium fluorometry, although the rapid channel kinetics of the receptor also need to be taken into account here. Metha-
Table 2. Rb+ efflux assay in the SH-EP1-ha4b2 and SH-SY5Y cell lines.
86
SH-EP1-ha4b2 Nicotine 10 lM Compound
IC50 (lM)
Methadone Mecamylamine
7.4 2.9
LogIC50 S.D. 5.1 0.03 5.5 0.02
SH-SY5Y Nicotine 1 lM
IC50 (lM) 5.6 2.6
LogIC50 S.D. 5.3 0.08 5.6 0.03
Nicotine 30 lM IC50 (lM) 0.8 0.2
LogIC50 S.D. 6.1 0.03 6.7 0.03
Nicotine 10 lM IC50 (lM) 1.0 0.1
LogIC50 S.D. 6.0 0.05 7.0 0.04
IC50, the half-maximal inhibitory concentration; logIC50, the half-maximal inhibitory concentration in logarithmic scale; S.D., standard deviation.
© 2014 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society)
THE ACTION OF METHADONE AT a4b2, a3 AND a7 nAChRs
A
B
C
D
327
Fig. 6. Mechanisms of antagonism of nAChR function by methadone and mecamylamine using an 86Rb+ efflux assay. The nicotine-stimulated specific 86Rb+ efflux was determined in the absence or presence of various concentrations of either methadone or mecamylamine in SH-EP1-ha4b2 cells (A, C) or SH-SY5Y (B, D) cells. The specific efflux for each drug concentration was calculated by subtracting the non-specific efflux (buffer controls) from the total efflux (1 mM carbamylcholine stimulation). The cells were exposed to the ligands for 5 min. The data were fit to the variable slope model. The values are the mean S.E.M. from three to five separate assays.
methadone is a non-competitive antagonist. In addition, the nicotine-induced increase in [Ca2+]i is significantly reduced in SH-SY5Y cells exposed to methadone, which further supports methadone’s ability to block a3* nAChRs and desensitize a7 nAChRs. Methadone elevates [Ca2+]i levels in SH-SY5Y cells, most likely through a7 nAChRs, and this elevation is inhibited by both nicotinic and opioid antagonists similarly in the SHEP1-ha7 cells. Our previous results show that morphine also acts as an antagonist at a3* nAChRs, and this action is independent of its agonist properties at opioid receptors [13]. In untransfected SH-EP1 cells, methadone dose-dependently increases [Ca2+]i, and the increase is antagonized by opioid antagonists, but not by nAChR antagonists. The inhibition results most likely from the antagonism of opioid receptors because these cells do not contain nAChRs [20]. Moreover, the methadone-induced increase in [Ca2+]i is negligible in untransfected SH-EP1 cells in comparison with that observed in SH-EP1 cells expressing recombinant nAChRs [11]. Methadone is in clinical use for treating moderate to severe pain and as a maintenance replacement treatment for opioid addiction. Most methadone maintenance treatment patients are smokers, and the use of methadone increases smoking [6–9]. A circulus vitiosus is apparent because nicotine has been shown to increase methadone self-administration and reinforcing properties [30]. Thus, it can be suggested that methadone maintenance treatment patients smoke more because of the more satisfying effects that they get from their methadone dose or vice versa (they might find smoking more satisfying because of their methadone use). However, our data would support the alternative hypothesis that these patients do not get the normal effects from nicotine because of methadone antagonism at a4b2 and a3* nAChRs or the desensitisation of a7 nAChRs, and thus, they smoke more to overcome this interaction. Although a direct correlation between in vitro and human data might not be relevant, our findings support the
hypothesis of significant nicotine–opioid interactions at the level of nAChRs. When comparing in vitro experiments and the clinical use of compounds, one can always criticize the use of relevant concentrations. In clinical studies, the plasma concentration of methadone in maintenance patients can reach the level of 1 lM. In contrast, in animal studies, brain tissue methadone concentrations have been reported to exceed those of the plasma [31]. In our in vitro experiments, methadone had IC50 values of 0.8–9.4 lM. The steady-state nicotine blood concentration in smokers on average is 0.07–0.2 lM [32], and every puff from cigarette accumulates the nicotine concentration in the brain [33]. Therefore, the mechanism of in vivo interactions of methadone and nicotine may at least partially be explained by our findings in vitro. The principal finding of this study is that methadone acts as a non-competitive antagonist at clinically relevant concentrations at a4b2 and a3* nAChRs expressed in SH-EP1-ha4b2 and SH-SY5Y cells, respectively. This study also confirms that methadone is a human a7 nAChR agonist. Acknowledgements We thank Ronald J. Lukas and Paul Whiteaker for valuable comments, Kati Rautio, Anna Niemi and Marjo Vaha for technical assistance, Mari Havia and Juri Meijer for conducting parts of the experiments and Jyrki Kukkonen for enabling the use of FlexStation. This study was funded by the Academy of Finland Research Programme on Substance Use and Addictions [Grant 1118535] and by the Research Funds of the University of Helsinki (OS). References 1 Nutter TJ, Adams DJ. Monovalent and divalent cation permeability and block of neuronal nicotinic receptor channels in rat parasympathetic ganglia. J Gen Physiol 1995;105:701–23.
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