Visual Overview
Abstract
Bupropion, a Food and Drug Administration–approved antidepressant and smoking cessation aid, blocks dopamine and norepinephrine reuptake transporters and noncompetitively inhibits nicotinic acetylcholine and serotonin (5-HT) type 3A receptors (5-HT3ARs). 5-HT3 receptors are pentameric ligand-gated ion channels that regulate synaptic activity in the central and peripheral nervous system, presynaptically and postsynaptically. In the present study, we examined and compared the effect of bupropion and its active metabolite hydroxybupropion on mouse homomeric 5-HT3A and heteromeric 5-HT3AB receptors expressed in Xenopus laevis oocytes using two-electrode voltage clamp experiments. Coapplication of bupropion or hydroxybupropion with 5-HT dose dependently inhibited 5-HT–induced currents in heteromeric 5-HT type 3AB receptors (5-HT3ABRs) (IC50 = 840 and 526 μM, respectively). The corresponding IC50s for bupropion and hydroxybupropion for homomeric 5-HT3ARs were 10- and 5-fold lower, respectively (87 and 113 μM). The inhibition of 5-HT3ARs and 5-HT3ABRs was non–use dependent and voltage independent, suggesting bupropion is not an open channel blocker. The inhibition by bupropion was reversible and time-dependent. Of note, preincubation with a low concentration of bupropion that mimics therapeutic drug conditions inhibits 5-HT–induced currents in 5-HT3A and 5-HT3AB receptors considerably. In summary, we demonstrate that bupropion inhibits heteromeric 5-HT3ABRs as well as homomeric 5-HT3ARs. This inhibition occurs at clinically relevant concentrations and may contribute to bupropion’s clinical effects.
SIGNIFICANCE STATEMENT Clinical studies indicate that antagonizing serotonin (5-HT) type 3AB (5-HT3AB) receptors in brain areas involved in mood regulation is successful in treating mood and anxiety disorders. Previously, bupropion was shown to be an antagonist at homopentameric 5-HT type 3A receptors. The present work provides novel insights into the pharmacological effects that bupropion exerts on heteromeric 5-HT3AB receptors, in particular when constantly present at low, clinically attainable concentrations. The results advance the knowledge on the clinical effects of bupropion as an antidepressant.
Introduction
The 5-hydroxytryptamine-3, or serotonin (5-HT) type 3, receptor is an ionotropic receptor and a member of the Cys-loop family of pentameric ligand-gated ion channels, and thereby, differs from G-protein-coupled serotonin receptors (Thompson and Lummis, 2007). The 5-HT type 3 receptor (5-HT3R) is similar in structure and function to other members of the pentameric ligand-gated ion channel family, including cation-selective nicotinic acetylcholine (nACh) receptors (nAChRs) and anion-selective GABAA and glycine receptors. Malfunction in these receptors has been linked to several neurologic disorders (Lemoine et al., 2012). Together, they are responsible for fast neurotransmission in the central and peripheral nervous system (Thompson and Lummis, 2013) and are involved in virtually all brain functions (Hassaine et al., 2014).
To date, five different 5-HT3 subunits have been identified (5-HT3A – 5-HT3E). The first subunit to be cloned, 5-HT3A (Maricq et al., 1991), is the only subunit among these that can form functional homo-oligomeric receptors on the cell membrane when expressed in Xenopus oocytes or cell lines (Hussy et al., 1994). Introduction of the 5-HT3B subunit yields functional heteromers with altered properties compared with the homo-oligomer and with heteromer function more closely resembling the functional responses observed in native tissues (Hussy et al., 1994; Davies et al., 1999). When compared with 5-HT3A, the 5-HT type 3AB receptor (5-HT3ABR) differs in agonist concentration-response curves, shows increased single-channel conductance and desensitization, and an altered current-voltage relationship (Davies et al., 1999; Dubin et al., 1999; Kelley et al., 2003b).
The 5-HT3R is widely distributed in the central and peripheral nervous systems and on extraneuronal cells, such as monocytes, chondrocytes, T-cells, and synovial tissue (Fiebich et al., 2004). In the periphery, 5-HT3Rs are found in the autonomic, sensory, and enteric nervous systems (Faerber et al., 2007), where they are involved in regulating gastrointestinal functions, such as motility, emesis, visceral perception, and secretion (Niesler et al., 2003; Lummis, 2012). The highest density of 5-HT3Rs in the central nervous system is in the hindbrain, particularly the dorsal vagal complex involved in the vomiting reflex, and in limbic structures, notably the amygdala, hippocampus, nucleus accumbens, and striatum (Jones et al., 1992; Miyake et al., 1995). Substantial 5-HT3B expression was identified in the human brain with high levels in the amygdala, hippocampus, and the nucleus caudate (Dubin et al., 1999; Tzvetkov et al., 2007). A high amount of 5-HT3Rs are found on presynaptic nerve fibers (Nayak et al., 2000; Miquel et al., 2002), through which they can modulate the release of other neurotransmitters, such as dopamine, cholecystokinin, GABA, substance P, and acetylcholine (Chameau and van Hooft, 2006; Faerber et al., 2007). Owing to its involvement in many brain functions, the 5-HT3R represents an attractive therapeutic target.
5-HT3R antagonists are used to effectively treat patients experiencing irritable bowel syndrome and chemotherapy-/radiotherapy-induced and postoperative nausea and vomiting (Thompson and Lummis, 2007). Some antidepressants (Choi et al., 2003; Eisensamer et al., 2003) and antipsychotic drugs (Rammes et al., 2004) also antagonize 5-HT3Rs, which, together with other preclinical and clinical studies, suggests the relevance of 5-HT3R antagonism for treating psychiatric disorders (Walstab et al., 2010; Bétry et al., 2011). We recently discovered that bupropion (Bup), another antidepressant, antagonizes 5-HT type 3A receptors (5-HT3ARs) (Pandhare et al., 2017).
Bupropion was first approved as an “atypical” antidepressant over 30 years ago, and today it is one of the most commonly prescribed antidepressants. Despite its recognized clinical efficacy for both depression and smoking cessation, a comprehensive picture of how bupropion modulates neurotransmission is still emerging. Bupropion’s therapeutic effect is thought to be mediated by the blocked reuptake of dopamine and norepinephrine (Stahl et al., 2004) and the noncompetitive inhibition of neuronal and muscular AChRs (Slemmer et al., 2000). Most recently, the discovery that bupropion also noncompetitively inhibits 5-HT3ARs (Pandhare et al., 2017) raises the questions of whether this inhibition takes place at clinically relevant concentrations and if bupropion also inhibits heteromeric members of the 5-HT3 family. Therefore, we investigated the effect of bupropion and its major metabolite, hydroxybupropion (HydroB), on the function of heteromeric 5-HT3ABRs as compared with the homomeric 5-HT3ARs expressed in Xenopus oocytes. Here, we demonstrate that 5-HT3ABRs, like 5-HT3ARs, are dose-dependently inhibited by bupropion and its metabolite. This inhibition is voltage-independent and non–use dependent (i.e., affected by preincubation) and occurs at physiologically relevant concentrations.
Materials and Methods
Materials
Stock of serotonin (2 mM 5-HT, serotonin creatinine sulfate monohydrate; Acros Organics, New Jersey, NJ) and bupropion (100 mM, Toronto Research Chemicals, Inc., North York, Canada) were prepared in distilled water. Hydroxybupropion (100 mM, Toronto Research Chemicals, Inc., North York, Canada) was dissolved in DMSO. All solutions were made in oocyte ringer solution (OR-2) immediately before conducting experiments.
Molecular Biology.
Complementary DNA encoding the mouse 5-HT3AR (AAT37716) containing a V5-tag (GKPIPNPLLGLDSTQ) close to the N-terminus (Jansen et al., 2008) and the mouse 5-HT3B receptor (NP_064670) in the pGEMHE vector were used for oocyte expression (Reeves et al., 2001). Plasmids were linearized with the NheI restriction enzyme and in vitro transcribed with the T7 RNA polymerase kit (mMESSAGE mMACHINE T7 Kit; Applied Biosystems/Ambion, Austin, TX). Capped complementary RNA (cRNA) was purified with the MEGAclear Kit (Applied Biosystems/Ambion) and precipitated using 5 M ammonium acetate. cRNA dissolved in nuclease-free water was stored at −80°C.
X. laevis Oocyte Preparation.
Oocytes were isolated, enzymatically defolliculated, and stored as previously described (Goyal et al., 2011). X. laevis frogs were handled and maintained following procedures approved by the local animal welfare committee (Institutional Animal Care and Use Committee, IACUC no. 08014, PHS Assurance no. A 3056-01). In brief, the isolated oocytes were incubated with collagenase (collagenase from Clostridium histolyticum Type IA; Sigma-Aldrich) for 1 hour in OR-2 (115 mM NaCl, 2.5 mM KCl, 1.8 mM MgCl2, 10 mM HEPES, pH 7.5), which was followed by extensive washing with OR-2. Oocytes were then rinsed three times with OR-2 + 2 mM Ca2+ for 45 minutes each and maintained in standard oocyte saline medium [100 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 5 mM HEPES, pH 7.5, supplemented with 1% Antibiotic-Antimycotic (100×, 10,000 U/ml of penicillin, 10,000 mg/ml of streptomycin and 25 mg/ml amphotericin B; Gibco, Thermo Fisher Scientific), 5% horse serum] for up to 7 days at 16°C. Oocytes were microinjected with 10 ng of in vitro synthesized cRNA (200 ng/μl) using an automatic oocyte injector (Nanoject II; Drummond Scientific Co., Broomall, PA) up to 48 hours after isolation. For optimal expression of the heteromeric 5-HT3ABR, the A and B subunits were coinjected in a 1:3 ratio. This ratio has been shown to be optimal for 5-HT3ABR expression because a lower ratio results in 5-HT3AR mimicked current response and a higher ratio would impact overall receptor expression (Thompson and Lummis, 2013; Corradi et al., 2015).
Electrophysiology.
Two-electrode voltage clamp recordings were performed and analyzed using a TEV-200A amplifier (Dagan Instruments, Minneapolis, MN), a Digidata 1440A data interface (Molecular Devices, Sunnyvale, CA), a MiniDigi 1B (Molecular Devices), and the pClamp 10.7 software (Molecular Devices). Recordings were conducted 1–4 days after microinjection. All experiments were performed at room temperature (22–24°C) and at a holding potential of −60 mV, unless otherwise stated. The oocytes were held in a 250 µl chamber and perfused with OR-2 using gravity flow at an approximate rate of 5 ml/min. Drugs and serotonin were dissolved in the same solution and applied by gravity perfusion. The glass microelectrodes were filled with 3 M KCl and had a resistance of below 2 MΩ. Agonists/antagonists were applied until stable response or desensitization was observed to record maximal current responses.
Data Analysis.
All electrophysiological data were analyzed with pClamp, Origin (OriginLab Corporation, Northampton, MA) and GraphPad Prism 6 (GraphPad SoftwareSoft, La Jolla, CA). Data are represented as the mean ± S.D., and maximal current induced by 5-HT was used as the normalizing standard (100% current response) for other current responses in the same oocyte. Statistical significance was determined with paired or unpaired t test (in Origin) with a cutoff for significance of 0.05 (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001) or one-way ANOVA followed by Dunnett’s multiple comparisons test (in Prism). The 5-HT (agonist stimulation—eq. 1a), bupropion, or hydroxybupropion (antagonist inhibition—eq. 1b) concentration dependence on 5-HT3 currents was fitted using the variable-slope sigmoidal dose response curve equations:
(1a)
(1b)Within these equations, Imax is the current activated at saturating 5-HT concentration, EC50 is the agonist concentration producing 50% of the Imax, IC50 is the concentration of antagonist producing 50% inhibition of Imax, X is the logarithm of agonist (eq. 1a) or antagonist (eq. 1b) concentration, and nH is the Hill coefficient. All figures and graphs were made in Origin and Adobe Illustrator CC 2018.
Results
Differentiating between 5-HT3AR and 5-HT3ABR.
To evaluate the effect of bupropion and its major metabolite hydroxybupropion (Fig. 1) on homomeric and heteromeric 5-HT3 receptors, we expressed 5-HT3ARs and 5-HT3ABRs [in a 1:3 A to B ratio (Thompson and Lummis, 2013; Corradi et al., 2015)] in Xenopus oocytes. First, we substantiated the obvious difference between the two receptor types (Fig. 1). The application of the agonist 5-HT to Xenopus oocytes expressing 5-HT3ARs (Fig. 1A, top trace) or 5-HT3ABRs (Fig. 1A, bottom trace) elicits a rapid inward current with a concentration-dependent amplitude. The currents resulting from a range of 5-HT concentrations were used to calculate the concentrations that produce a half-maximal response (Fig. 1B), yielding EC50 values of 0.80 μM (n = 5, Hill slope nH = 2.53 ± 0.58) for the 5-HT3AR and 4.30 μM (n = 8; nH = 1.04 ± 0.02) for the 5-HT3ABR (Table 1). Our EC50 values are comparable to those reported previously (Jansen et al., 2008; Lochner and Lummis, 2010), with the heteromeric receptor showing a right-shift in potency (Fig. 1B) and fast characteristic desensitization kinetics (Fig. 1C). As reported in the literature, the Hill coefficients are indicative of highly cooperative agonist sites for homopentameric channels and of a single site for heteropentameric channels (Thompson et al., 2011).
Comparing 5-HT3ARs to 5-HT3ABRs. (A) Sample traces of 5-HT3A (black) and 5-HT3AB (green) at varying concentrations of 5-HT. (B) Concentration-response curves show a higher potency of 5-HT at 5-HT3ARs as compared with 5-HT3AB, as well as a steeper Hill slope. Parameters from these curves: 5-HT3A: EC50 = 0.8 µM, nH = 2.53 ± 0.58, n = 5, 5-HT3AB: EC50 = 4.30 µM, nH = 1.04 ± 0.02, n = 8. Data are represented as the mean ± S.D. (C) Direct comparison of 5-HT3A and 5-HT3AB inward current evoked by 1 μM 5-HT for 30 seconds.
EC50 (5-HT) and IC50 values (bupropion and hydroxybupropion) determined in X. laevis oocytes expressing 5-HT3ARs and 5-HT3ABRs.
Data represented as mean ± S.D. of n experiments. Statistical significance of A as compared with AB was determined with unpaired t test (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001). pEC50 and pIC50 are the negative logarithms of EC50 and IC50, respectively.
Effect of Bupropion on 5-HT3AR and 5-HT3ABR.
5-HT and a wide range of bupropion concentrations (A: 10–1000 µM; AB: 30–4000 µM) were coapplied to Xenopus oocytes expressing homomeric 5-HT3ARs (Fig. 2A, top) or heteromeric 5-HT3ABRs (Fig. 2A, bottom) under two-electrode voltage clamp. 5-HT was applied at a concentration that elicits approximately 30% of the maximal response (EC30) (5-HT3AR: 0.5 µM, 5-HT3ABR: 2 µM). Both traces in Fig. 2A show representative current responses at −60 mV. The first inward current represents the control current evoked by 5-HT alone, and this is followed by subsequent currents obtained by the coapplication of 5-HT (EC30) and increasing concentrations of bupropion, which dose-dependently inhibited 5-HT-induced currents for 5-HT3A and 5-HT3AB receptors. IC50 values were 87.1 µM (nH = 1.28 ± 0.15, n = 5) and 840 µM (nH = 1.78 ± 0.15, n = 7) for A and AB, respectively (Fig. 2B; Table 1). Bupropion’s potency at 5-HT3ABR was 10-fold lower when compared with 5-HT3AR (unpaired t test, t(10) = 8.49, P = 6.99 × 10−6). Bupropion alone did not elicit an inward current for either receptor (Fig. 2C).
Bupropion’s antagonistic activity at homomeric and heteromeric 5-HT3Rs. (A) Sample traces of oocytes expressing 5-HT3A or 5-HT3AB in response to 5-HT (∼EC30) alone and in combination with bupropion. 5-HT–evoked inward currents (gray, 5-HT3A = 0.3 μM, 5-HT3AB = 2 μM) were used for the control current. Following, the 5-HT concentration was kept constant and coapplied with increasing concentrations of bupropion (5-HT3A: 10–1000 μM, 5-HT3AB: 30–4000 μM). (B) Currents were normalized to the control currents and yielded the following IC50 values: 5-HT3A: IC50 = 87.1 µM (nH = 1.28 ± 0.15, n = 5, mean ± S.D.) and 5-HT3AB: IC50 = 840 µM (nH = 1.78 ± 0.15, n = 7, mean ± S.D.). (C) Oocytes expressing 5-HT3A and 5-HT3AB did not elicit an inward current in response to bupropion alone.
Effect of Hydroxybupropion on 5-HT3AR and 5-HT3ABR.
Hydroxybupropion, a major metabolite of bupropion, is known to contribute to the biologic efficacy of the parent drug because it also inhibits dopamine/norepinephrine transporters, nAChRs, and 5-HT3ARs (Bondarev et al., 2003; Damaj et al., 2004; Pandhare et al., 2017). Similar to bupropion, hydroxybupropion inhibited 5-HT3ARs and 5-HT3ABRs dose-dependently when coapplied with 5-HT (Fig. 3A). The hydroxybupropion concentrations that reduced the 5-HT–evoked currents to 50% of the initial response were 113 µM (n = 5, nH = 1.17 ± 0.14) for 5-HT3ARs and 526 µM (n = 8, nH = 1.80 ± 0.16) for 5-HT3ABRs (Fig. 3B; Table 1). Similar to bupropion, the potency of hydroxybupropion for 5-HT3ABRs was right-shifted, resulting in a higher IC50 value as compared to 5-HT3ARs (unpaired t, t(11) = 28.9, P = 1.01 × 10−11). Hydroxybupropion did not elicit a response in 5-HT3A or 5-HT3AB expressing oocytes when applied alone (Fig. 3C).
Hydroxybupropion is an antagonist for 5-HT3ARs and 5-HT3ABRs. (A) Sample traces of oocytes expressing 5-HT3A or 5-HT3AB in response to 5-HT (∼EC30) alone and in combination with hydroxybupropion. 5-HT–evoked inward currents (gray, 5-HT3A = 0.3 μM, 5-HT3AB = 2 μM) were used for the control current. Following, the 5-HT concentration was kept constant and coapplied with increasing concentrations of hydroxybupropion (5-HT3A: 10–1000 μM, 5-HT3AB: 50–2000 μM). (B) Currents were normalized to the control currents and yielded the following IC50 values: 5-HT3A: IC50 = 113 µM (nH = 1.17 ± 0.15, n = 5, mean ± S.D.) and 5-HT3AB: IC50 = 526 µM (nH = 1.80 ± 0.16, n = 8, mean ± S.D.). (C) Oocytes expressing 5-HT3A and 5-HT3AB did not elicit an inward current in response to hydroxybupropion alone.
Effect of Preincubation with Bupropion and Hydroxybupropion on 5-HT3A and 5-HT3AB Receptors.
Bupropion’s allosteric inhibition of 5-HT3ARs is not dependent on the opening of the receptor’s channel; it is non–use dependent (Pandhare et al., 2017). To evaluate the extent of inhibition evoked by preincubating oocytes expressing 5-HT3A and 5-HT3ABRs with bupropion or its metabolite, results were compared to the current amplitudes resulting from coapplication of 5-HT and bupropion/hydroxybupropion. First, oocytes were perfused with 5-HT (∼EC30, 5-HT3AR: 0.5 μM, 5-HT3ABR: 2 μM) and bupropion (∼IC50, 5-HT3AR: 100 μM, 5-HT3ABR: 1 mM) to obtain the control current (Fig. 4A). Once a stable response was achieved, a constant IC50 concentration of bupropion was exposed to the receptors for exactly 5 min before another coapplication of the same 5-HT and bupropion solutions. Preincubation decreased the current amplitude of 5-HT3ARs to 76.2% ± 7.16% (Fig. 4C, left panel) of control, consistent with previous findings (Pandhare et al., 2017). On the contrary, under the same experimental conditions, the 5-HT3ABR was greatly affected by preincubation, which resulted in a current amplitude reduced to 35.5% ± 5.62% of the control current (Fig. 4C, right panel). Similar results were obtained from preincubation with hydroxybupropion (Fig. 4B). Compared with coapplication alone, preapplication resulted in a greater depression of current for 5-HT3ARs and 5-HT3ABRs with hydroxybupropion (Fig. 4C, 5-HT3AR: 93.0% ± 6.12% and 5-HT3ABR: 46.1% ± 4.95% of control current).
Non–use dependent allosteric inhibition of 5-HT3ARs and 5-HT3ABRs. (A) Sample traces of oocytes expressing 5-HT3A (black, left panel) and 5-HT3AB (green, right panel). The first 5-HT–evoked currents were used for the control currents (gray bars, ∼EC30, 5-HT3A: 0.5 µM, 5-HT3AB: 2 µM) that were obtained by coapplication with bupropion (magenta bars, ∼IC50, 5-HT3A: 100 µM, 5-HT3AB: 1 mM). Following the stable 5-HT response, bupropion (∼EC50) was perfused for 5 min before another coapplication of 5-HT and bupropion. (B) Same experimental design as in (A) but with hydroxybupropion (blue bars, ∼IC50, 5-HT3A: 100 µM, 5-HT3AB: 500 µM). (C) Quantification of fractional inhibition of currents when the oocyte was preincubated in bupropion (magenta) or hydroxybupropion (blue) normalized to the control current (100%). Preincubation reduced current amplitudes for 5-HT3A (Bup: 76.1% ± 7.16%, n = 5; HydroB: 93.0% ± 6.12%, n = 6) and 5-HT3AB (Bup: 35.5% ± 5.62%, n = 6; HydroB: 46.1% ± 4.95%, n = 4) as compared with coapplication. Statistical significance was determined with paired t test (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001), comparing coapplication (representing 100% of the current) to preapplication + coapplication (Pre+Co) with each drug independently. Data are represented as the mean ± S.D.
Recovery Times for Bupropion Inhibition.
Bupropion’s antagonistic effect on 5-HT–evoked inward currents has been shown to be reversible (Pandhare et al., 2017). To evaluate the recovery times of bupropion’s inhibition of 5-HT–induced currents at homomeric and heteromeric receptors, bupropion was applied to the oocytes for 60 s at a 400 μM concentration. For these experiments, the ∼EC50 concentration of 5-HT (gray bars, 5-HT3AR: 0.8 μM, 5-HT3ABR: 5.0 μM) was applied episodically after washing in between each application (∼2 min). These agonist-induced currents led to minimal run-down, and the difference in current amplitudes was less than 10% (Fig. 5A). Sample traces of current recovery following bupropion application and removal are shown in Fig. 5B (left: black, 5-HT3AR, right: green, 5-HT3ABR). The first current response is the control current evoked by the agonist alone. Once a stable current response was obtained, bupropion was applied without the agonist for 60 s (magenta bars). Subsequently, the agonist was either applied immediately or after 10, 30, or 60 s (Fig. 5B, top trace: 0 s, middle: 30 s, bottom: 60 s) after bupropion exposure. The largest decrease in current amplitude for both receptors was immediately after the bupropion application, leaving 82.4% ± 3.08% and 38.4% ± 15.8% of the initial current for A and AB, respectively (Fig. 5C). Rapid recovery of current amplitude was achieved by increasing the wash times between bupropion and 5-HT applications. 5-HT3ARs and 5-HT3ABRs both show time-dependent recovery from bupropion inhibition with full reversal after 7+ min wash time.
Recovery times for bupropion. (A and B) Sample traces of bupropion application (magenta bar) and the recovery times for 5-HT3A (left panel, black) and 5-HT3AB (right panel, green). (A) In two-electrode voltage clamp experiments, oocytes expressing 5-HT3A and 5-HT3AB showed a stable response to repeated applications of 0.8 and 5 μM 5-HT at −60 mV, with an approximate wash time of 2 min. (B) The first 5-HT–evoked response represents the control current for the recovery experiment. Bupropion (400 μM) was applied alone for 60 s at −60 mV, followed by an immediate application of 5-HT. The gray and magenta bars represent the time of application of 5-HT and bupropion, respectively. Moving down the panel, the wash times after bupropion application were 0, 30, and 60 s. (C) Quantitative representation of current amplitudes and results in (B) (n = 3). 5-HT3A was maximally reduced to 82.4% ± 3.08% and 5-HT3AB to 38.4% ± 15.8% of the control current when the agonist was applied immediately after bupropion, followed by a stepwise recovery. All currents could be recovered to ∼95% after ∼7.5-min wash. Statistical significance of each wash time as compared with the control current (the 5-HT–induced current response before exposure to Bup or HydroB) was determined with one-way ANOVA, Dunnett’s multiple comparisons test (*P ≤ 0.05; ***P ≤ 0.001). Data are represented as the mean ± S.D.
Voltage-Independent Binding of Bupropion.
To determine if bupropion binds to 5-HT3Rs in a voltage-dependent manner, 5-HT–induced currents (∼EC50; 5-HT3AR: 0.8 µM; 5-HT3ABR: 5.0 µM) were evoked in oocytes expressing 5-HT3ARs and 5-HT3ABRs at two different holding potentials, +40 and −40 mV (Fig. 6A). First, the control current was obtained at positive and negative voltages before the coapplication of 5-HT and bupropion (∼IC50; 5-HT3AR: 100 µM; 5-HT3ABR: 1 mM). Bupropion reduced the current amplitudes of homomeric and heteromeric receptors at both voltages. The mean fractional block was recorded at each voltage and normalized to the control current (Fig. 6B; 5-HT3AR: 55.8% ± 0.11%, 59.8% ± 0.10%; 5-HT3ABR: 56.6% ± 0.04%, 59% ± 0.08%; −40 and +40 mV, respectively, n = 4). For 5-HT3AR and 5-HT3ABR, this fractional inhibition is similar at positive and negative voltages (paired t test, 3A: t(3) = 1.106, P = 0.349; 3AB: t(3) = 0.291, P = 0.790). Based on these results, inhibition of 5-HT–induced currents by bupropion is independent of voltage.
Voltage-independent block of 5-HT3–mediated currents by bupropion. (A) Sample traces of 5-HT3A– and 5-HT3AB–expressing oocytes (5-HT3A: left, black; 5-HT3AB: right, green) in response to 5-HT (∼EC50; top and bottom traces, 5-HT3A: 0.8 µM; 5-HT3AB: 5.0 µM) in the absence and presence of bupropion (magenta traces, ∼IC50; 5-HT3A: 100 µM; 5-HT3AB: 1 mM) at different voltages. (B) Quantification of fractional inhibition, currents were normalized to the control currents at each voltage (n = 4). Data are shown as mean ± S.D. Statistical significance between the inhibition at positive and negative voltages was determined with paired t test (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001).
Bupropion at Physiologic Concentrations and Its Effect on 5-HT3AR and 5-HT3ABR.
To better understand the clinical significance of the bupropion-induced inhibition of 5-HT3Rs, 5-HT–induced currents were measured in the presence of a clinically relevant bupropion concentration [∼20 µM (Schroeder, 1983; Vázquez-Gómez et al., 2014)]. First, oocytes expressing 5-HT3ARs and 5-HT3ABRs were exposed to three different 5-HT concentrations (0.5, 1.0, 5.0 µM) in the absence of bupropion to obtain the initial current amplitudes (Fig. 7A, black, left panel: 5-HT3AR, green, right panel: 5-HT3ABR). Next, the oocytes were continuously perfused with 20 µM bupropion, and the same 5-HT concentrations were reapplied; the oocytes were preincubated with bupropion for at least 2 minprior to 5-HT application (Fig. 7A, magenta bars indicating bupropion presence). The results indicate that the continuous presence of a low concentration of bupropion in the bath solution partially inhibits 5-HT–induced currents of 5-HT3ARs and 5-HT3ABRs at all 5-HT concentrations tested (Fig. 7B, paired t test, P ≤ 0.05 or lower). Bupropion inhibited 5-HT–induced currents by ∼18% for 5-HT3ARs (n = 4), whereas 5-HT3ABRs showed a ∼23% decrease in current (n = 5, Fig. 7B).
Bupropion at clinically achievable concentrations and its effect on 5-HT3ARs and 5-HT3ABRs. (A) Sample traces of oocytes expressing 5-HT3A (black, left panel) and 5-HT3AB (green, right panel) in response to 0.5, 1.0, and 5.0 μM 5-HT (gray bars) followed by the same concentrations coapplied with 20 μM bupropion. Following the initial exposure to the three 5-HT concentrations (control current), the oocytes were exposed to 20 μM bupropion for at least 2 min before coapplication with the agonist. (B) Quantitative representation of current amplitudes and results in (A) (A: n = 4, AB: n = 5). Data are shown as mean ± S.D. Statistical significance between each 5-HT concentration without and with bupropion at the same 5-HT concentration was determined with paired t test (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001).
Discussion
Our results, for the first time, demonstrate that bupropion antagonizes heteromeric 5-HT3AB receptors and that the kinetics of inhibition are distinct from 5-HT3A. Two-electrode voltage clamp experiments indicate that bupropion reversibly inhibits 5-HT–induced currents of Xenopus oocytes expressing 5-HT3A and 5-HT3ABRs in a concentration-dependent manner, with inhibitory potencies of 87.1 μM [same as previously reported (Pandhare et al., 2017)] and 840 μM, respectively. Similar to other noncompetitive antagonists [such as picrotoxin (Das and Dillon, 2003)], bupropion has a lower potency (∼10-fold) at 5-HT3AB as compared to 5-HT3A receptors and, therefore, could be used to discriminate between these two receptors (Thompson and Lummis, 2013).
Similarly to bupropion, the metabolite inhibits nAChRs and 5-HT3ARs in a noncompetitive manner (Damaj et al., 2004; Pandhare et al., 2017) and also shares a comparable potency (unpaired t test, P value = 0.06704) for the homomeric receptor [5-HT3AR: IC50 = 113 μM, similar to previously reported data (Pandhare et al., 2017)]. Hydroxybupropion exhibits a ∼4.5-fold shift in IC50 for the heteromeric channel (5-HT3ABR: IC50 = 526 μM). The estimated Hill slopes (nH values) for both bupropion and hydroxybupropion for both receptors were greater than unity (1.17–1.80), suggesting the presence of multiple binding sites with a cooperative mechanism. The Hill coefficients for the 5-HT3ABR were ∼1.4 times larger for both bupropion and hydroxybuporpion as compared with for the 5-HT3AR, which may indicate a concerted conformational change or cooperativity of binding (Colquhoun, 1998).
Bupropion-mediated inhibition of 5-HT3ARs is non–use dependent (Pandhare et al., 2017). In general, use-dependent block, or inhibition that would require a channel to be open to occur, is not influenced by preapplication. We evaluated the effect of a 5-min preincubation with bupropion and its metabolite hydroxybupropion on the homomeric and the heteromeric receptor (Fig. 4). Preincubation with antagonistled to an increased inhibition in all cases when compared with coapplication, indicating that the block is non–use dependent for both receptors. Our observation that bupropion’s inhibition of 5-HT3Rs is voltage-independent additionally concurs with it not acting as an open channel blocker (Slemmer et al., 2000; Choi et al., 2003; Gumilar et al., 2003). Similar results are shown with other antidepressants at 5-HT3Rs (Eisensamer et al., 2003) and with bupropion at nAChRs (e.g., α3β2, α4β2, α3β4) with predictions for an external binding site for bupropion (Slemmer et al., 2000; García-Colunga et al., 2011). Considering different binding sites within the family of AChRs (Pandhare et al., 2012), bupropion may have distinct binding sites in each channel (Arias et al., 2009).
We saw a greater depression of current amplitudes when bupropion or its metabolite was preincubated as compared with coapplication with 5-HT [Fig. 5, 5-HT3AR: 76.2% ± 7.16%, 93.0% ± 6.12%; 5-HT3ABR: 35.5% ± 5.62%, 46.1% ± 4.95% of control current, Bup and HydroB, respectively]. During the preincubation experiments, bupropion binds and inhibits the receptor prior to the opening of the channel, therefore presumably interacting with the closed channel and potentially inhibiting the channel from opening (Choi et al., 2003; Arias et al., 2009). Consistent with other data, greater potencies of inhibition have been reported for bupropion and tricyclic antidepressants on Cys-loop receptors in the resting state than the open state (Choi et al., 2003; Gumilar and Bouzat, 2008; Arias et al., 2009). This phenomenon may be due to the accumulation of antidepressants and antipsychotics in the cell membrane during preincubation, which may be important for the functional antagonistic effects of these drugs at the 5-HT3 receptor (Eisensamer et al., 2005). Overall, this may indicate that bupropion has access to its binding site(s) from the membrane environment. We find that inhibition after preincubation is more pronounced in the 5-HT3AB as compared with the 5-HT3A receptor.
Bupropion’s inhibition of 5-HT–mediated currents is reversible after a substantial amount of washing. In this study, we investigated the time it takes for 5-HT3R to recover from preincubation with bupropion at high concentrations (400 µM Bup). Similar to our preincubation experiments, bupropion reduced 5-HT3AR currents but to a lesser extent than 5-HT3ABR currents. The largest reduction of current was observed with the shortest amount of wash time between the bupropion and agonist applications (5-HT3AR: 82.4% ± 3.08%; 5-HT3ABR: 38.4% ± 15.8% of the control current after 0-s wash). 5-HT3A and 5-HT3AB receptors show a time-dependent recovery from bupropion’s inhibition, and their currents could be fully recovered after ∼7.5 min of washing.
The clinical relevance of 5-HT3 inhibition by bupropion is currently unknown. Bupropion, but not its metabolites, concentrates in many tissues with a brain to plasma ratio of 25:1 (Schroeder, 1983), which results in brain concentrations of ∼20 µM (Vázquez-Gómez et al., 2014). Coapplication of 20 μM bupropion with agonist minimally inhibits 5-HT–induced currents of 5-HT3ARs and does not affect HT3ABRs. On the contrary, preincubation with bupropion has a drastic impact on its inhibitory effect (Fig. 7). Our results indicate that a preincubation time of 5 min with 20 µM bupropion is enough to inhibit 5-HT3 receptors (5-HT3AR: ∼82.7% 5-HT3ABR: ∼74.9% of control current). Moreover, hydroxybupropion reaches ∼10-fold higher plasma concentrations in humans as compared with the parent drug (Findlay et al., 1981; Hsyu et al., 1997). With an average of ∼100 µM (based on clinical data, test ID: FBUMT; Mayo Clinic, MN), hydroxybupropion’s plasma concentrations are equivalent to 5-HT3AR’s IC50 value. Additionally, considering the increased inhibitory effect due to preincubation of 5-HT3Rs, we conclude that bupropion and hydroxybupropion have the potential to inhibit these receptors at clinically attainable concentrations.
The comprehensive mechanism by which bupropion achieves its therapeutic efficacy is multifactorial. At therapeutic dosages, bupropion inhibits nACh receptors in the ventral tegmental area, dorsal raphe nucleus neurons, and interneurons in the hippocampal CA1 area (Alkondon and Albuquerque, 2005; Vázquez-Gómez et al., 2014). There, nAChRs can modulate serotonergic projections (Aznar et al., 2005; Chang et al., 2011) and alter GABAergic transmission (Ji and Dani, 2000), in turn increasing dopamine levels, contributing to bupropion’s antidepressant activity (Arias, 2009; Vázquez-Gómez et al., 2014). 5-HT3 receptors also show strong interactions with GABAergic neurons in the hippocampus and neocortical cells (Morales et al., 1996) and mediate stress-dependent activation of dopaminergic neurotransmission (Devadoss et al., 2010; Bhatt et al., 2013). Animal studies have demonstrated that 5-HT3 antagonists have anxiolytic activity, possibly because of the inhibition of limbic hyperactivity responses (Bhatt et al., 2013), and this is supported by the finding that 5-HT3AR gene deletion produces an anxiolytic phenotype in mice (Kelley et al., 2003a). Furthermore, 5-HT3 antagonists have implications on hippocampal long-term potentiation (Bétry et al., 2011), increase synaptic norepinephrine levels, facilitate 5-HT neurotransmission of other 5-HT receptors (Rajkumar and Mahesh, 2010), and even enhance the antidepressant action of bupropion (Devadoss et al., 2010).
In conclusion, 5-HT3 and nACh receptors have shown many implications in the neurobiology of depression and a highly complex interplay can be expected between these systems. Currently, it is not known if bupropion- or hydroxybupropion-mediated inhibition of 5-HT3 receptors is clinically relevant for their antidepressant activity. Further studies focused on characterizing bupropion’s accumulation in membranes, identification of its binding sites, and delineation of its molecular mechanism of action are warranted. We show here that bupropion inhibits 5-HT3 receptors at clinically relevant concentrations and that this inhibition may contribute to bupropion’s clinical effects.
Acknowledgments
We thank Dr. Myles Akabas (Einstein, NY) for providing us with the 5-HT3 plasmids. We thank the Texas Tech University Health Sciences Center (TTUHSC) Core Facilities: some of the images and/or data were generated in the Image Analysis Core Facility & Molecular Biology Core Facility supported by TTUHSC.
Authorship Contributions
Participated in research design: Stuebler, Jansen.
Conducted experiments: Stuebler.
Performed data analysis: Stuebler, Jansen.
Wrote or contributed to the writing of the manuscript: Stuebler, Jansen.
Footnotes
- Received September 25, 2019.
- Accepted December 13, 2019.
Research reported in this publication was supported in part by the National Institute of Neurologic Disorders and Stroke of the National Institutes of Health [R01 NS077114 (to M.J.)]. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Abbreviations
- 5-HT
- serotonin
- 5-HT3R
- 5-HT type 3 receptor
- 5-HT3AR
- 5-HT type 3A receptor
- 5-HT3ABR
- 5-HT type 3AB receptor
- Bup
- bupropion
- cRNA
- complementary RNA
- EC30
- concentration that elicits approximately 30% of the maximal response
- HydroB
- hydroxybupropion
- nACh
- nicotinic acetylcholine
- nAChR
- nACh receptor
- OR-2
- oocyte ringer solution
- Copyright © 2020 by The Author(s)
This is an open access article distributed under the CC BY-NC Attribution 4.0 International license.
References
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Bupropion’s Effect on 5-HT3ABRs
