Introduction

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The efficacy of reboxetine, a well tolerated antidepressant in clinical use in Europe (Berzewdski et al., 1997; Montgomery, 1997), has been linked to inhibition of neurotransmitter uptake, specifically at the norepinephrine (NE) transporter (NET). Reboxetine is a highly selective inhibitor of NET, relative to the serotonin transporter (SERT) and the dopamine transporter (DAT; Montgomery, 1997; Wong et al., 2000). Like other antidepressants, reboxetine displays a characteristic delay in onset of action (Versiani et al., 1999), due to prerequisite neural adaptations (Harkin et al., 2000; Invernizzi et al., 2001). Adaptive changes in transporter function seem to be dependent on the specific antidepressant administered and transporter affected. For example, repeated administration of the SERT inhibitors citalopram and fluoxetine did not produce adaptive modifications of SERT function (Gobbi et al., 1997). In contrast, repeated administration of the DAT inhibitor bupropion, but not nomifensine, resulted in up-regulation of DAT (Tella et al., 1997). Thus, one purpose of the present study was to determine whether repeated reboxetine produced adaptive modifications in NET, SERT, or DAT function.

Many studies have correlated mood disorders with tobacco smoking (Glassman et al., 1990). Individuals who are depressed are more likely to be smokers, dependent on nicotine, experience difficulty quitting, and have greater withdrawal symptoms (Covey, 1999). Depression may also be a symptom of nicotine withdrawal, and depression associated with cessation occurs more frequently among smokers with a history of depression (Covey, 1999). Recent clinical studies report that bupropion, an antidepressant that inhibits DAT and NET function, is a useful smoking cessation therapy (Hurt et al., 1997; Jorenby et al., 1999; Shiffman et al., 2000).

The intrinsic rewarding properties of nicotine, the major alkaloid in tobacco believed to be responsible for maintenance of the smoking habit, result from activation of neuronal DA pathways (Corrigall et al., 1992). Nicotinic acetylcholine receptors (nAChRs) are located on cell bodies and terminals of the hippocampal NE pathway (Sershen et al., 1997) and the nigrostriatal DA pathway (Wonnacott, 1997). Nicotine has been shown to evoke [³H]NE overflow from rat hippocampal slices (Sershen et al., 1997) and [³H]DA overflow from rat striatal slices (Teng et al., 1997). The diversity of mRNA for nAChR subunits isolated in NE- and DA-containing neurons (Wada et al., 1989; Dineley-Miller and Patrick, 1992; Charpantier et al., 1998) supports the speculation that several nAChR subtypes may be responsible for nicotine-evoked NE and DA release. Furthermore, evidence has been obtained suggesting that nicotine-evoked NE release from hippocampus is the result of the stimulation of presynaptic 34* nAChRs (Clarke and Reuben, 1996; Sershen et al., 1997; Reuben et al., 2000), whereas at least in part, nicotine-evoked striatal DA release is via presynaptic 32* nAChRs (Schulz and Zigmond, 1989; Grady et al., 1992; Cartier et al., 1996; Kaiser et al., 1998). Convincing evidence suggests that nicotine-evoked ⁸⁶Rb⁺ efflux from thalamic synaptosomes is the result of stimulation of 42* nAChRs. The rank order of potency of nAChR agonists to increase ⁸⁶Rb⁺ efflux from thalamic synaptosomes was highly correlated with that for inhibition of [³H]nicotine binding to thalamic membranes (Marks et al., 1995).

Recently, several antidepressants have been reported to act as nAChR antagonists in assays assessing receptor function. Specifically, desipramine, nisoxetine, and citalopram inhibited nicotine-evoked [³H]NE overflow from rat hippocampal slices (Hennings et al., 1997, 1999) and bupropion inhibited acetylcholine-induced currents in rat 32, 42, and 7 nAChRs expressed in Xenopus oocytes (Slemmer et al., 2000). Also, bupropion and several other antidepressants, e.g., fluoxetine, sertraline, paroxetine, and nefazodone, were shown to inhibit carbamylcholine-evoked ⁸⁶Rb⁺ efflux from human muscle-type nAChRs (1) in TE671/RD cells, to inhibit carbamylcholine-evoked ⁸⁶Rb⁺ efflux from human autonomic nAChRs (345 ± 2) in SH-SY5Y neuroblastoma cells, and to inhibit nicotine-evoked ⁸⁶Rb⁺ efflux from chick V274T mutant 7-nAChR heterologously expressed in native nAChR-null SH-EP1 epithelial cells (Fryer and Lukas, 1999a,b). Although reboxetine has been established as a potent and selective inhibitor of NET, its interaction with nAChRs has not been investigated previously. Thus, the second purpose of the present study was to evaluate the interaction of reboxetine with native nAChRs containing 7, 4, and 3 subunits by determining reboxetine-induced inhibition of [³H]methyllycaconitine (MLA), [³H]cytisine, and [³H]epibatidine binding, respectively, using whole rat brain membranes, inhibition of nicotine-evoked ³H overflow from superfused hippocampal and striatal slices preloaded with [³H]NE or [³H]DA, respectively, and inhibition of nicotine-evoked ⁸⁶Rb⁺ efflux from thalamic synaptosomes.

	Materials and Methods

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Subjects. Male Sprague-Dawley rats (200-250 g) were obtained from Harlan (Indianapolis, IN) and were housed two per cage with free access to food and water. Experimental protocols were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at the University of Kentucky and at Pharmacia Corporation.

Chemicals. Reboxetine CH₃SOCH (a racemic mixture of R,R- and S,S- ([2-[[2-ethoxyphenoxy]benzyl]-morpholine sulfate]) was synthesized and provided by Pharmacia Corporation (Kalamazoo, MI). (±)-Bupropion HCl, ()-cytisine, desipramine HCl, (±)-epibatidine dihydrochloride, fluoxetine HCl, GBR-12909 HCl, methyllycaconitine citrate, nomifensine maleate, S-()-nicotine ditartrate, pargyline HCl, tetrodotoxin (TTX), HEPES, bovine serum albumin, and catechol were purchased from Sigma-Aldrich (St. Louis, MO). [³H]Cytisine HCl [hexahydro-1,5-methano-8H-pyrido-[1,2-a][1,5]diazocin-8-one, 3,5-³H(N); specific activity, 35 Ci/mmol], [³H]epibatidine [[5,6-bicycloheptyl-³H]-(±)-epibatidine; specific activity, 48 Ci/mmol], [³H]NE [norepinephrine, levo-[7-³H]; specific activity, 14.4 Ci/mmol], [³H]DA [dihydroxyphenylethylamine 3,4-ethyl-2-[N-³H]; specific activity, 25.6 Ci/mmol], [³H]5-HT [hydroxytryptamine creatine sulfate 5-[1,2-³H(N)]; specific activity, 27.5 Ci/mmol], and ⁸⁶RbCl (specific activity, 55.2 mCi/mmol) were purchased from PerkinElmer Life Sciences (Boston, MA). [³H]MLA [1,(S),6,14,16]-20-ethyl-1,6,14,16-tetramethoxy-4-[[[2-([3-³H]-[3-³H]-methyl-2,5-dioxo-1-pyrrolidinyl)benzoyl]oxy]methyl]aconitane-7,8-diol; specific activity, 25 Ci/mmol] was purchased from Tocris Cookson (Ballwin, MO). -D-Glucose was purchased from Aldrich Chemical Co. (Milwaukee, WI), L-ascorbic acid was purchased from AnalaR-BHD Ltd. (Poole, Dorset, UK), potassium phosphate monobasic was purchased from Mallinckrodt (Paris, KY), and TS-2 tissue solubilizer was purchased from Research Products International (Mount Prospect, IL). All other chemicals were purchased from Fisher Scientific (Pittsburgh, PA).

[³H]Neurotransmitter Uptake Assays. [³H]NE and [³H]5-HT uptake assays were performed using hippocampal synaptosomes, and [³H]DA uptake assays were performed using rat striatal synaptosomes. Striata or hippocampi from an individual rat were homogenized in 20 ml of ice-cold sucrose solution (0.32 M sucrose and 5 mM NaHCO₃, pH 7.4) with 16 passes of a Teflon pestle homogenizer (clearance 0.003 inches). The homogenate was centrifuged (2000g, 10 min, 4°C), and the resulting supernatant was centrifuged (20,000g, 15 min, 4°C). The resulting pellet was resuspended in 2.4 ml of ice-cold assay buffer (125 mM NaCl, 5 mM KCl, 1.5 mM MgSO₄, 1.25 mM CaCl₂, 1.5 mM KH₂PO₄, 10 mM -D-glucose, 25 mM HEPES, 0.1 mM EDTA, 0.1 mM pargyline, and 0.1 mM ascorbic acid, saturated with 95% O₂/5% CO₂, pH 7.4). The final protein concentration was 400 µg/ml, as determined by protein-dye bonding using bovine serum albumin as a standard (Bradford, 1976). Assays were performed in duplicate in a total volume of 500 µl. Aliquot parts of synaptosomal suspension (50 µl) were added to tubes containing 350 µl of buffer and 50 µl of one of nine concentrations of drug (reboxetine, desipramine, fluoxetine, or GBR-12909) followed by 50 µl of [³H]NE, [³H]5-HT, or [³H]DA (final concentration of 0.1 µM). Accumulation proceeded for 10 min at 34°C. Accumulation was terminated by addition of 3 ml of ice-cold buffer containing catechol (1 mM) and rapid filtration through Whatman GF/B glass fiber filters presoaked with catechol (1 mM). Filters were washed three times with ice-cold buffer, transferred to scintillation vials, cocktail was added, and radioactivity was determined by liquid scintillation spectroscopy (model B1600 TR scintillation counter; Packard Instrument Company, Inc., Downer's Grove, IL).

[³H]MLA, [³H]Cytisine, and [³H]Epibatidine Binding Assays. [³H]MLA was used to probe the 7* nAChR (Davies et al., 1999) and [³H]cytisine to probe the 4* nAChR (Pabreza et al., 1990). Binding of [³H]epibatidine in the presence of cytisine (30 nM) was used to probe the 3* nAChR subtype (Xiao et al., 1998). Rat whole brain, minus cerebellum, was dissected quickly, weighed, and homogenized in 9 vol/g wet weight of ice-cold 0.32 M sucrose with 10 passes of a rotating Teflon pestle homogenizer (clearance 0.003 inches; setting 50, 099CK4; Glas-Col, Terre Haute, IN). The homogenate was centrifuged (1000g, 10 min, 4°C). The resulting supernatant was centrifuged (20,000g, 20 min, 4°C). The pellet was resuspended to a final protein concentration of 1 to 8 mg/ml. The homogenate (5 ml) was frozen at 80°C until use in the binding assay. On the day of assay, aliquot parts of membrane suspension were thawed at room temperature and diluted with Krebs-HEPES buffer (20 mM HEPES, 4.16 mM NaHCO₃, 0.44 mM KH₂PO₄, 127 mM NaCl, 5.36 mM KCl, 1.26 mM CaCl₂, and 0.98 mM MgCl₂, pH 7.0) to afford 25 to 150 µg of protein/0.4 ml, which was added to each assay tube.

³H Overflow Superfusion Assay. ³H Overflow from rat striatal slices preloaded with [³H]DA or rat hippocampal slices preloaded with [³H]NE was determined using modifications of a previously published method (Dwoskin and Zahniser, 1986). Coronal striatal or hippocampal slices (500 µm; 6-8 mg from striatum; 3-4 mg from hippocampus) were incubated in Krebs' buffer (118 mM NaCl, 4.7 mM KCl, 1.2 mM MgCl₂, 1.0 mM NaH₂PO₄, 1.3 mM CaCl₂, 11.1 mM -D-glucose, 25 mM NaHCO₃, 0.11 mM ascorbic acid, and 4.0 µM EDTA, saturated with 95% O₂/5% CO₂, pH 7.4) in a metabolic shaker at 34°C for 30 min. Slices were incubated in fresh buffer containing 0.1 µM [³H]DA or 0.1 µM [³H]NE (6-8 slices/3 ml) for an additional 30 min. After rinsing, each slice was transferred to a glass superfusion chamber maintained at 34°C and superfused at 1 ml/min with oxygenated Krebs' buffer containing pargyline (10 µM) and either nomifensine (10 µM) in [³H]DA release experiments or desipramine (10 µM) in [³H]NE release experiments to inhibit metabolism by monoamine oxidase and to inhibit uptake into the respective presynaptic terminals, ensuring that ³H overflow represented primarily the parent ³H-neurotransmitter rather than metabolites. After 60 min of superfusion, three 5-min samples (5 ml/sample) were collected to determine basal ³H outflow. After collection of the third basal sample, slices from an individual rat were superfused for 30 min in the absence or presence of six to seven concentrations of reboxetine (1 nM-100 µM), which remained in the buffer until the end of the experiment. Intrinsic activity (i.e., the ability of reboxetine to evoke ³H overflow) was assessed during the 30-min period of superfusion. The complete concentration response for reboxetine in the [³H]NE release assay was determined in one series of experiments. However, two series of experiments were performed to obtain the complete concentration response in the [³H]DA release assay; the first series determined the effect of 1 nM to 10 µM, and the second series determined the effect of 10 nM to 100 µM. Subsequently, nicotine (10 µM) was added to the buffer containing reboxetine, and superfusion continued for an additional 60 min. The ability of reboxetine to inhibit nicotine-evoked ³H overflow was determined during the latter 60-min period of superfusion. Each striatal slice was exposed to only one concentration of reboxetine. Each experiment was replicated a minimum of six times. A repeated measures design was used, such that the concentration effect of reboxetine was determined using brain slices from a single rat, and a minimum of six rats. At the end of the experiment, each slice was solubilized with TS-2. The pH and volume of the solubilized tissue samples were adjusted to those of the superfusate samples. Radioactivity in the superfusate and tissue was determined by liquid scintillation spectroscopy (model B1600 TR scintillation counter; Packard Instrument Company, Inc.).

⁸⁶Rb⁺ Efflux Assay. The effects of reboxetine on ⁸⁶Rb⁺ efflux were determined using previously published methods (Miller et al., 2000). Thalamus was homogenized and centrifuged (1000g, 10 min, 4°C). The supernatant fraction was centrifuged (12,000g, 20 min, 4°C) to obtain the synaptosomal fraction. Synaptosomes were incubated for 30 min in 35 µl of buffer (140 mM NaCl, 1.5 mM KCl, 2.0 mM CaCl₂, 1.0 mM MgSO₄, and 20 mM -D-glucose, pH 7.5) containing 4 µCi of ⁸⁶Rb⁺. ⁸⁶Rb⁺ uptake was terminated by filtration of the synaptosomes onto glass fiber filters (6 mm, type A/E; Gelman Instrument Co., Ann Arbor, MI) under gentle vacuum (0.2 atm), followed by three washes with buffer (0.5 ml each). Subsequently, each filter with ⁸⁶Rb⁺-loaded synaptosomes (39 ± 4.8 µg of protein/µl) was placed on a 13-mm glass fiber filter (type A/E) mounted on a polypropylene platform. ⁸⁶Rb⁺ efflux assay buffer (125 mM NaCl, 5 mM CsCl, 1.5 mM KCl, 2 mM CaCl₂, 1 mM MgSO₄, 25 mM HEPES, 20 mM -D-glucose, 0.1 µM TTX, and 1.0 g/l bovine serum albumin, pH 7.5) was superfused at a rate of 2.5 ml/min. TTX and CsCl were included in the assay buffer to block voltage-gated Na⁺ and K⁺ channels, respectively, and to reduce the rate of basal ⁸⁶Rb⁺ efflux. The ability of reboxetine (1 nM-100 µM) to inhibit ⁸⁶Rb⁺ efflux evoked by 1 µM nicotine was determined. In previous experiments, this concentration of nicotine was the lowest concentration that produced maximal ⁸⁶Rb⁺ efflux (Miller et al., 2000). After 8 min of superfusion, basal samples (sample/18 s) were collected for 2 min. Subsequently, synaptosomes were superfused for 3 min in the absence or presence of one of six concentrations (1 nM-100 µM) of reboxetine. Then, nicotine (1 µM) was added to buffer, and superfusion was continued for an additional 3 min, followed by superfusion for 3 min with buffer in the absence of either drug. Each aliquot part of thalamic synaptosomes was exposed to only one concentration of reboxetine. In each experiment, one synaptosomal aliquot part was also exposed to nicotine (1 µM) in the absence of reboxetine, and one synaptosomal aliquot part was superfused in the absence of either drug to determine basal ⁸⁶Rb⁺ efflux during the course of the experiment. Samples were analyzed by liquid scintillation spectroscopy (model B1600 TR scintillation counter; Packard Instrument Company, Inc.). To determine the basal rate of ⁸⁶Rb⁺ efflux, an exponential decay curve was used to fit the data points proceeding and following superfusion with reboxetine and nicotine (SigmaPlot 2000; SPSS, Inc., Chicago, IL). Drug-evoked increases in ⁸⁶Rb⁺ efflux were calculated as the fractional increase above baseline. Increases were summed to obtain total ⁸⁶Rb⁺ efflux during the period of superfusion with reboxetine and/or nicotine, and normalized to ⁸⁶Rb⁺ content in the corresponding synaptosomal sample to reduce variability within and between experiments.

Data Analysis. For the ³H neurotransmitter uptake assays, IC₅₀ values for each individual concentration-response curve were generated using nonlinear regression and a sigmoidal curve fit (GraphPad Prism, version 3.0; GraphPad Software, San Diego, CA). For the ³H neurotransmitter uptake assays, comparisons of IC₅₀ values for reboxetine with desipramine, fluoxetine, or GBR-12909 were analyzed by independent group t tests. To assess the effect of repeated reboxetine treatment on inhibition of ³H neurotransmitter uptake, two-way analysis of variance (ANOVA) was performed on the IC₅₀ values with repeated drug treatment (reboxetine or saline) and inhibitor (reboxetine versus desipramine, fluoxetine, or GBR-12909) as between-group factors (SPSS version 9.0; SSPS, Inc.). Where appropriate, Tukey post hoc tests and simple main effect analyses (p < 0.05) were performed. In competition binding studies, the inhibition constant (K_i) was calculated from the binding isotherms for [³H]MLA, [³H]cytisine, and [³H]epibatidine binding using a nonlinear regression curve fit program according to the Cheng and Prusoff equation. For the ³H overflow assays, intrinsic activity of reboxetine and the ability of reboxetine to inhibit nicotine-evoked ³H overflow were analyzed via one-way repeated measures ANOVA with reboxetine concentration as a within-subject factor. EC₅₀ values for intrinsic activity and IC₅₀ values for inhibition of nicotine-evoked ³H overflow were determined via nonlinear regression, which fit the mean data to a sigmoidal concentration-response curve. The mechanism of inhibition of [³H]NE overflow was determined via Schild analysis and analyzed via three-way repeated measures ANOVA with nicotine concentration and the absence or presence of reboxetine as within-subject factors, and reboxetine concentration as a between-groups factor. The intrinsic activity of reboxetine on ⁸⁶Rb⁺ efflux and the ability of reboxetine to inhibit nicotine-evoked ⁸⁶Rb⁺ efflux were analyzed via one-way repeated measures ANOVA with reboxetine concentration as a within-subject factor.

	Results

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Repeated Administration of Reboxetine Does Not Alter Selectivity as a NET Inhibitor. The concentration of reboxetine that inhibited [³H]NE uptake by 50% (IC₅₀) was 8.5 nM. IC₅₀ values for reboxetine inhibition of [³H]DA and [³H]5-HT uptake were 89 and 6.9 µM, respectively (Fig. 1). Thus, reboxetine was 3 to 4 orders of magnitude more potent as an inhibitor of [³H]NE uptake than [³H]DA and [³H]5-HT uptake. Significant differences were not found between reboxetine and desipramine (IC₅₀ value = 17.0 nM) to inhibit [³H]NE uptake [t₍₆₎ = 0.20, p = 0.85; Fig. 1]. GBR-12909 (IC₅₀ value = 1.5 µM) was significantly more potent [t₍₄₎ = 2.62, p < 0.05] than reboxetine in inhibiting [³H]DA uptake, and fluoxetine (IC₅₀ value = 0.4 µM) was significantly more potent [t₍₄₎ = 15.84, p < 0.001] than reboxetine in inhibiting [³H]5-HT uptake (Fig. 1). Thus, reboxetine is a highly potent and selective [³H]NE uptake inhibitor.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Reboxetine inhibits [3H]NE and [3H]5-HT uptake into rat hippocampal synaptosomes and [3H]DA uptake into rat striatal synaptosomes. Data are expressed as the mean (±S.E.M.) represented in picomoles per minute per milligram of specific [3H]NE, [3H]5-HT, or [3H]DA uptake (n = 4-6 rats).

Reboxetine Does Not Inhibit [³H]MLA, [³H]Cytisine, or [³H]Epibatidine Binding to Rat Whole Brain Membrane Preparations. To validate the nAChR binding assays, concentration-dependent inhibition of binding was determined using several standard competitors, MLA, nicotine, cytisine, and epibatidine (Table 2). Neither reboxetine nor bupropion significantly inhibited binding of these three nAChR radioligands (Table 2).

Reboxetine Noncompetitively Inhibits Nicotine-Evoked ³H Overflow from Superfused [³H]NE-Preloaded Hippocampal Slices. In a concentration-dependent manner, reboxetine increased ³H overflow from superfused rat hippocampal slices preloaded with [³H]NE (Table 3). A significant main effect of reboxetine concentration was found [F_(6,30) = 10.67, p < 0.001], and post hoc tests revealed greater ³H overflow after superfusion with 100 µM reboxetine compared with control (0 M) or compared with lower concentrations (1 nM-10 µM) of reboxetine. Furthermore, ³H overflow from slices superfused with 1 nM to 10 µM reboxetine was not different from control (Table 3), indicating that these concentrations of reboxetine produced no intrinsic activity.

View larger version (28K): [in this window] [in a new window]   Fig. 2.   Reboxetine inhibits nicotine (10 µM)-evoked 3H overflow from rat hippocampal slices preloaded with [3H]NE. Superfusion buffer contained pargyline (10 µM) and desipramine (10 µM) from the start of superfusion. Data are presented as the mean (±S.E.M.) of total 3H overflow during the 60-min period of superfusion with reboxetine plus nicotine. , p < 0.05, different from 0 µM reboxetine (n = 6 rats). Inset, data are presented as mean ± S.E.M. of fractional [3H]NE release during the entire collection period. Left arrow designates beginning of superfusion with reboxetine, and right arrow designates addition of nicotine to the buffer.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Reboxetine noncompetitively inhibits nicotine-evoked 3H overflow from rat hippocampal slices preloaded with [3H]NE. The superfusion buffer contained pargyline (10 µM) and desipramine (10 µM) from the start of the experiment. The concentration response for nicotine (10 nM-1 µM) was determined in the absence and presence of 10 nM, 100 nM, or 1 µM reboxetine. Reboxetine was included in the superfusion buffer for 30 min before the addition of nicotine to the buffer, and superfusion continued for an additional 60 min. Data from these three series of experiments are presented as the mean (±S.E.M.) of total 3H overflow during the 60-min period of exposure to nicotine in the absence or presence of reboxetine. Because no significant differences [F(2,15) = 0.17, p = 0.85] were found among the control groups (i.e., the concentration response for nicotine in the absence of reboxetine) for the three series of experiments, these data were collapsed for graphical presentation. , p < 0.05, different from control (0 M reboxetine) across the corresponding nicotine concentration (n = 6 rats/group).

Reboxetine Evokes ³H Overflow from Rat Striatal Slices Preloaded with [³H]DA; However, Reboxetine Does Not Inhibit Nicotine-Evoked ³H Overflow. Two series of experiments were performed to determine the effect of reboxetine across 6 orders of magnitude concentration. The effect of reboxetine to increase ³H overflow from [³H]DA-preloaded striatal slices is shown in Table 4. Significant main effects of reboxetine concentration were found in the two series of experiments [F_(5,25) = 3.77, p < 0.05 and F_(6,30) = 119.86, p < 0.001, respectively]. Post hoc tests revealed a concentration-dependent increase in ³H overflow after superfusion with 3 to 100 µM reboxetine compared with control; 1 nM to 1 µM reboxetine did not increase ³H overflow compared with control. Concentrations of reboxetine that did not produce intrinsic activity, also did not inhibit nicotine-evoked ³H overflow (Table 4; F_(1,11) = 3.00, p = 0.11 and F_(1,11) = 1.84, p = 0.49). Thus, reboxetine did not inhibit nicotine-evoked ³H overflow from superfused striatal slices preloaded with [³H]DA.

Reboxetine Inhibits Nicotine-Evoked ⁸⁶Rb⁺ Efflux from Rat Thalamic Synaptosomes. Only the highest concentration (100 µM) of reboxetine significantly [F_(6,18) = 13.95, p < 0.001] evoked ⁸⁶Rb⁺ efflux above basal levels (Table 5). Thus, this high concentration was not included in the analysis of reboxetine-induced inhibition of nicotine-evoked ⁸⁶Rb⁺ efflux (Fig. 4). Reboxetine significantly inhibited nicotine-evoked ⁸⁶Rb⁺ efflux [F_(6,18) = 6.77, p < 0.01] with an IC₅₀ value of 650 nM. Post hoc tests revealed that 1 and 10 µM reboxetine inhibited nicotine-evoked ⁸⁶Rb⁺ efflux, compared with control.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Reboxetine inhibits nicotine-evoked 86Rb+ efflux from superfused rat thalamic synaptosomes. Data are presented as the mean (±S.E.M.) of the percentage of 86Rb+ tissue content during the 3-min period of superfusion with reboxetine plus nicotine. , p < 0.05, different from control (0 M reboxetine) (n = 4 rats).

	Discussion

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The present study demonstrates that reboxetine potently and selectively inhibits [³H]NE uptake into rat hippocampal synaptosomes. [³H]NE uptake was inhibited 3 to 4 orders of magnitude more potently than either [³H]DA or [³H]5-HT uptake. The present results demonstrating that reboxetine is a highly selective inhibitor of NET are consistent with previous findings (Montgomery, 1997; Wong et al., 2000). The current results, demonstrating that repeated injection of reboxetine does not alter its potency or selectivity to inhibit [³H]NE uptake, suggest that reboxetine does not produce adaptive modifications of NET, SERT, or DAT function under the current experimental conditions. Furthermore, tolerance did not develop to the reboxetine-induced inhibition of NET after repeated reboxetine administration.

The same reboxetine injection regimen (10 mg/kg/day i.p., twice daily for 14 days) used in the present ³H neurotransmitter uptake study was previously demonstrated to produce characteristic neural adaptive changes, e.g., down-regulation of -adrenergic receptors (Harkin et al., 2000), indicating that this reboxetine injection regimen was sufficient to induce neural adaptation. Because reboxetine requires 2 to 3 weeks of treatment before its antidepressant effects are observed, direct inhibition of NET, which occurs immediately upon reboxetine treatment, cannot account entirely for its clinical efficacy. Rather, the neural adaptation must be, at least in part, responsible for the antidepressant effect of reboxetine. One possible neural adaptation may be a change in the surface expression of NET; however, an alteration in inhibition of [³H]NE uptake in the current study was not observed. Thus, the present results taken together with previous findings of Harkin et al. (2000) suggest that continued inhibition of NET by reboxetine may be necessary for the neural adaptation to be manifest.

Studies have demonstrated that the antidepressant bupropion has efficacy for smoking cessation and ameliorates some nicotine withdrawal symptoms (Hurt et al., 1997; Jorenby et al., 1999; Shiffman et al., 2000). Recently, bupropion and other antidepressants were reported to be antagonists of nAChRs (Hennings et al., 1997, 1999; Fryer and Lukas, 1999a,b; Slemmer et al., 2000). Bupropion inhibited rat 32, 42, and 7 nAChRs expressed in Xenopus oocytes (Slemmer et al., 2000). Bupropion, fluoxetine, sertraline, paroxetine, and nefazodone inhibited the 1 subtype expressed in TE671/RD cells, the 345 ± 2 subtype expressed in SH-SY5Y neuroblastoma cells, and the chick V274T mutant 7 subtype expressed in SH-EP1 epithelial cells (Fryer and Lukas, 1999a,b). Desipramine, nisoxetine, and citalopram inhibited nicotine-evoked [³H]NE overflow from rat hippocampal slices (Hennings et al., 1997, 1999), suggesting an interaction with the 34* nAChR subtype. Based on these findings, it seems that different antidepressants may have different inhibitory profiles across the diversity of nAChR subtypes. Importantly, the specific nAChR subtype(s) inhibited may impart the pharmacological properties necessary for the inhibition of the reinforcing effect of nicotine. Thus, selected antidepressants may prove efficacious as smoking cessation pharmacotherapies, in part depending on their pharmacological profile as inhibitors of specific nAChR subtypes.

The present study provides an initial profile for the functional antagonism produced by reboxetine at several nAChR subtypes. Reboxetine potently inhibited (IC₅₀ value = 7.3 nM) nicotine-evoked [³H]NE overflow from superfused hippocampal slices, suggesting antagonism at the 34* subtype. Furthermore, the reboxetine-induced inhibition was not surmounted by increasing concentrations of nicotine, indicative of a noncompetitive mechanism of action. It is notable that reboxetine inhibited the function of this nAChR subtype within the same concentration range that it inhibited NET function. Inclusion of desipramine in the superfusion buffer in the [³H]NE release experiments eliminated the contribution of NET function to the observed effect of reboxetine to inhibit nicotine-evoked [³H]NE release. The functional elimination of NET provided the necessary conditions to focus on the contribution of nAChR function to the observed effect of reboxetine. Thus, the present observation that reboxetine potently inhibited nicotine-evoked [³H]NE release in the presence of desipramine strongly suggests the involvement of the 34* nAChR. In agreement, other investigators have reported that IC₅₀ values for inhibition of nicotine-evoked [³H]NE overflow from rat hippocampal slices are not correlated with K_i values for inhibition of NET function (Hennings et al., 1999). Thus, both the current studies and the work of others suggest that there is no relationship between antidepressant-induced inhibition of NET function and antidepressant-induced inhibition of the effect of nicotine to release NE from its presynaptic terminal in hippocampus.

Furthermore, the present results suggest that reboxetine selectively inhibits the 34* nAChR subtype because reboxetine failed to inhibit nicotine-evoked [³H]DA release from superfused rat striatal slices in the presence of nomifensine, indicating a lack of interaction with the 32* subtype. Additionally, reboxetine did not inhibit binding of several nAChR radioligands, including [³H]epibatidine and [³H]MLA, indicating that reboxetine does not interact at the ligand binding site on either the 32* or 7* nAChR subtypes. Thus, neither functional inhibition nor inhibition of ligand binding induced by reboxetine was observed at the 32* nAChR site, suggesting pharmacological selectivity for reboxetine at the 34* subtype.

Regarding the 42* nAChR subtype, reboxetine also inhibited nicotine-evoked ⁸⁶Rb⁺ efflux (IC₅₀ value = 650 nM) from thalamic synaptosomes, a functional assay for the 42* nAChR. The reboxetine-induced inhibition of the 42* subtype was 90-fold less potent than inhibition of the 34* subtype. Importantly, reboxetine did not inhibit [³H]cytisine binding to rat whole brain membranes, indicating that it does not interact at the ligand binding site on the 42* nAChR. Taken together, the present results from the ⁸⁶Rb⁺ efflux and [³H]cytisine binding experiments suggest that reboxetine interacts with the 42* nAChR subtype in a noncompetitive manner, and does not directly interact with the specific site to which [³H]cytisine binds on the 42* protein. The present results do not rule out the involvement of NET in the reboxetine-induced inhibition of nicotine-evoked ⁸⁶Rb⁺ efflux because desipramine was not included in the superfusion buffer at concentrations maximally inhibiting NET function. However, the concentration required to for inhibition of 42* function was 100-fold greater than that which inhibits NET function, mitigating against the role of NET in this effect of reboxetine. Furthermore, the present results obtained using native receptors are consistent with previous results demonstrating that antidepressants noncompetitively inhibit 42 nAChRs expressed in Xenopus oocytes in the absence of NET coexpression (Slemmer et al., 2000).

Pharmacological interventions as smoking cessation agents are restricted in number. Currently, the noncompetitive nAChR antagonist mecamylamine, various forms of nicotine replacement therapy, and the nonselective DA/NE transport inhibitor and nAChR antagonist bupropion have been reported to show limited efficacy. Recent studies demonstrate that reboxetine inhibits nicotine self-administration in rats and that tolerance does not develop to the inhibitory properties of reboxetine after repeated administration (A. S. Rauhut, S. N. Mullins, L. P. Dwoskin, and M. T. Bardo, submitted for publication). The latter results suggest that reboxetine may be a good candidate for the treatment of nicotine dependence and may have utility as a smoking cessation agent. Furthermore, the present study suggests that the 34* nAChR may be a novel target for therapeutic intervention in smoking cessation, and that the noradrenergic system may play a more important role than previously appreciated in nicotine-induced reward. Thus, given the concordance of smoking behavior and dysphoric mood disorders, reboxetine may provide an alternative effective treatment for tobacco smoking cessation.