Introduction

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Clinical studies have revealed a strong correlation between the incidence of tobacco smoking and mood disorders (Glassman et al., 1990; Pomerleau et al., 2000). Individuals with clinical depression are more likely to be tobacco smokers, dependent on nicotine, and to experience difficulty quitting with greater withdrawal symptoms upon cessation (Covey et al., 1997; Covey, 1999). Smokers undergoing cessation experience symptoms of depression, occurring more frequently among smokers with a history of major depression (Covey et al., 1997). The antidepressant, bupropion, has therapeutic benefit as a smoking cessation agent (Hurt et al., 1997; Jorenby et al., 1999; Shiffman et al., 2000); however, the mechanism by which bupropion reduces smoking is not fully understood.

Interestingly, acute administration of a low dose of bupropion increased nicotine self-administration, whereas a high dose of bupropion decreased nicotine self-administration in rats, suggesting that bupropion alters nicotine reinforcement (Rauhut et al., 2002). These results are consistent with a recent report that acute bupropion administration increases smoking in non-treatment-seeking smokers (Cousins et al., 2001), while reducing smoking during cessation (Hurt et al., 1997; Jorenby et al., 1999; Shiffman et al., 2000). This biphasic response to bupropion suggests that it has a complex mechanism of action.

The antidepressant effects of bupropion result from inhibition of dopamine and norepinephrine transporters (DAT and NET, respectively); however, its mechanism of action is not fully understood (Ascher et al., 1995). Bupropion inhibits [³H]dopamine ([³H]DA) uptake (IC₅₀ = 2 µM) into rat striatal synaptosomes, [³H]norepinephrine ([³H]NE) uptake (IC₅₀ = 5 µM) into rat hypothalamic synaptosomes, and, less potently (IC₅₀ = 58 µM), [³H]serotonin uptake into rat hypothalamic synaptosomes (Ferris and Cooper, 1993; Ascher et al., 1995). A competitive interaction with DAT has been demonstrated using [³H]mazindol binding to rat striatal membranes (Dersch et al., 1994). Bupropion-induced inhibition of DAT and NET function and associated increases in extracellular DA and NE concentrations, respectively, may substitute for nicotine-evoked neurotransmitter release during smoking, although nicotine reinforcement primarily has been associated with increased DA release (Corrigall et al., 1992). Thus, bupropion inhibition of transporter function likely contributes to its therapeutic efficacy as a smoking cessation agent.

Another mechanism potentially contributing to the efficacy of bupropion as a smoking cessation agent is inhibition of nicotinic acetylcholine receptors (nAChRs). The ability of bupropion to interact with specific nAChR subtypes has been investigated. Bupropion inhibited (IC₅₀ = 10.5 µM) carbamylcholine (1 mM)-induced ⁸⁶Rb⁺ efflux from human neuroblastoma cells expressing the 34 ganglionic nAChR subtype and more potently inhibited (IC₅₀ = 1.51 µM) ⁸⁶Rb⁺ efflux from human clonal cells expressing the 1 muscle nAChR subtype (Fryer and Lukas, 1999). Bupropion also inhibited acetylcholine (ACh; 1 µM) activation of rat 32 (IC₅₀ = 1.3 µM) and 42 (IC₅₀ = 8 µM) subtypes expressed in Xenopus oocytes (Slemmer et al., 2000). Furthermore, bupropion inhibited the 7 subtype, but with lower affinity (IC₅₀ = 60 µM). Bupropion-induced inhibition of the above nAChR subtypes was not surmounted by increasing agonist concentrations, indicative of a noncompetitive interaction (Fryer and Lukas, 1999; Slemmer et al., 2000). Interestingly, bupropion (1 and 10 µM) did not displace [³H]nicotine binding to whole rat brain membranes, also consistent with noncompetitive inhibition of 42 nAChRs (Slemmer et al., 2000). Thus, bupropion noncompetitively inhibits 32, 42, and 34 subtypes when studied using a variety of nAChR expression systems.

Since alterations in both DA and NE neurotransmission likely contribute to the antidepressant effects of bupropion, the present study evaluated the ability of bupropion to inhibit native nAChR subtypes using both [³H]DA and [³H]NE release assays. Specifically, bupropion inhibition of nicotine-evoked [³H]overflow from superfused rat striatal slices preloaded with [³H]DA and rat hippocampal slices preloaded with [³H]NE was determined under conditions in which DAT and NET function was inhibited by inclusion of nomifensine and desipramine, respectively, in the superfusion buffer. The exact subunit composition of native nAChRs has not been elucidated conclusively (Lukas et al., 1999). Subtype assignment has been based primarily on the demonstration of inhibition of nicotine response by subtype-selective antagonists in native tissue preparations. However, subtype selectivity of the antagonists has been determined using cell expression systems in which the nAChR subunit composition is known. Nevertheless, converging lines of evidence suggest that nicotine-evoked DA release from striatum and NE release from hippocampus are mediated by 32* and 34* nAChRs, respectively, although several different nAChR subtypes may be involved in these responses (Kaiser et al., 1998; Luo et al., 1998; Fu et al., 1999; Reuben et al., 2000).

	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 in the Division of Laboratory Animal Resources at the College of Pharmacy at the University of Kentucky. Experimental protocols involving the animals were in strict 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.

Chemicals. (±)-Bupropion was kindly provided as a gift from Dr. John Reinhard (GlaxoSmithKline, Research Triangle Park, NC). S-()-Nicotine ditartrate was purchased from Sigma/RBI (Natick, MA). Desipramine hydrochloride, mecamylamine hydrochloride, nomifensine maleate, and pargyline hydrochloride were obtained from Sigma-Aldrich (St. Louis, MO). [³H]NE (levo-[7-³H]norepinephrine; specific activity 14.4 Ci/mmol) and [³H]DA (3,4-ethyl-2-[N-³H]dihydroxyphenylethylamine; specific activity 25.6 Ci/mmol) were purchased from PerkinElmer Life Sciences (Boston, MA). -D-Glucose, L-ascorbic acid, and TS-2 tissue solubilizer were purchased from Aldrich Chemical (Milwaukee, WI), AnalaR (BHD Ltd., Poole, Dorset, U.K.), and Research Products International (Mount Prospect, IL), respectively. All other chemicals were purchased from Fisher Scientific (Pittsburgh, PA).

[³H]Overflow Assay. [³H]overflow from striatal slices preloaded with [³H]DA or [³H]overflow from hippocampal slices preloaded with [³H]NE was determined using separate groups of rats using modifications of a previously published method (Dwoskin and Zahniser, 1986). Briefly, coronal striatal or hippocampal slices (500 µm; 6-8 mg for striatum; 3-4 mg for 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 L-ascorbic acid, and 4.0 µM disodium ethylenediamine tetraacetate, pH 7.4, saturated with 95% O₂/5% CO₂) in a metabolic shaker at 34°C for 30 min. Slices (six to eight slices/3 ml) were incubated in fresh buffer containing 0.1 µM [³H]DA or 0.1 µM [³H]NE for an additional 30 min. After rinsing, each individual slice was transferred to a glass superfusion chamber containing two platinum electrodes and maintained at 34°C, and superfused at 1 ml/min with oxygenated Krebs' buffer, containing pargyline (10 µM) to ensure that [³H]overflow represented primarily [³H]DA or [³H]NE, rather than their metabolites (Zumstein et al., 1981). In [³H]DA overflow experiments, nomifensine (10 µM) was included in the superfusion buffer to inhibit DAT function. The concentration of nomifensine was based on previous research in which the IC₅₀ value to inhibit [³H]DA uptake into rat striatal synaptosomes was ~150 nM (Hunt et al., 1974). In [³H]NE overflow experiments, desipramine (10 µM) was included in the superfusion buffer to inhibit NET function. The concentration of desipramine was based on previous research in which the IC₅₀ value to inhibit [³H]NE uptake into rat hippocampal synaptosomes was 20 nM (Lindbrink et al., 1971; Miller et al., 2002). After 60 min of superfusion, superfusate was collected across the entire sampling period in 5-min fractions (5 ml/sample). Three superfusate samples 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 bupropion (1 nM-100 µM), and samples were collected to determine intrinsic activity (i.e., ability of bupropion to evoke [³H]overflow). Each slice was exposed to only one concentration of bupropion. Bupropion remained in the buffer throughout the experiment. After 30 min of superfusion in the absence or presence of bupropion, nicotine (10 µM) was added to the buffer of each chamber and superfusion continued; samples were collected for an additional 60 min to determine the ability of bupropion to inhibit nicotine-evoked [³H]overflow. A control slice from each rat was superfused for 30 min with buffer (in the absence of bupropion) followed by superfusion for 60 min with nicotine. The 60-min duration of exposure of the slices to nicotine was chosen based on our previous superfusion experiments determining the effect of nicotine on neurotransmitter release (Dwoskin et al., 1993; Teng et al., 1997), on the observed residence time of nicotine in rat brain (t_1/2 = 52 min) following a single s.c. injection of nicotine (Crooks et al., 1997; Ghosheh et al., 1999), and on observations from the literature that tobacco smokers maintain a relatively constant plasma nicotine concentration across the day (Jacob et al., 1999). The present experiments utilized a repeated measures design, such that the bupropion concentration-response for intrinsic activity and for inhibition of nicotine-evoked [³H]overflow were determined using brain slices from a single animal. 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 samples was determined by liquid scintillation spectroscopy (Packard model B1600 TR scintillation counter; Packard, Downer's Grove, IL).

Electrically Evoked [³H]Overflow. To assess the selectivity of the bupropion-induced inhibition of the effect of nicotine, striatal slices were preloaded with [³H]DA, as previously described, and the ability of bupropion (0.1-10 µM) to inhibit electrical field stimulation-evoked [³H]overflow was determined. Field stimulation consisted of a train of unipolar, rectangular pulses (1 Hz; 2-ms duration for 2 or 5 min; 120 or 500 pulses, respectively, applied by a model SD9 stimulator; Grass Instruments, Quincy, MA). The number of pulses was chosen to provide [³H]overflow equivalent to that evoked by superfusion with the range of nicotine concentrations utilized in the Schild analysis. Each slice was exposed to only one concentration of bupropion and was field stimulated by either 120 or 500 pulses. Additionally, one slice in each experiment was superfused in the absence of bupropion and stimulated at either 120 or 500 pulses, serving as the control condition. Each slice was transferred to a glass superfusion chamber containing two platinum electrodes and maintained at 34°C. Chambers were superfused at 1 ml/min with oxygenated Krebs' buffer containing pargyline (10 µM) and nomifensine (10 µM). After 60 min of superfusion, three 5-min samples (5 ml) were collected to determine basal [³H]overflow. After collection of the third basal sample, striatal slices from an individual rat were superfused for 60 min in the absence or presence of a single concentration of bupropion (0.1-10 µM), which remained in the buffer until the end of the experiment. Subsequently, electrical field stimulation was applied, and superfusate samples were collected for an additional 60-min period. For these experiments, the number of pulses was a between-group factor and the bupropion concentration was a within-subjects factor.

Mecamylamine-Induced Inhibition of Bupropion-Evoked [³H]Overflow. To assess whether bupropion (100 µM)-evoked [³H]overflow (intrinsic activity) is mediated by nAChRs, striatal slices were preloaded with [³H]DA as previously described, and the ability of mecamylamine (0.01 - 100 µM) to inhibit the [³H]overflow evoked by bupropion was determined. Each slice was transferred to a glass superfusion chamber maintained at 34°C and was superfused at 1 ml/min with oxygenated Krebs' buffer containing pargyline (10 µM) and nomifensine (10 µM). After 60 min of superfusion, three 5-min samples (5 ml) were collected to determine basal [³H]overflow. After collection of the third basal sample, striatal slices from an individual rat were superfused for 30 min in the absence or presence of one of several concentrations of mecamylamine (0.01 - 100 µM), which remained in the buffer until the end of the experiment. Each slice was exposed to only one concentration of mecamylamine. Subsequently, bupropion (100 µM) was added to buffer and superfusate samples were collected for an additional 60 min. In each experiment, one slice was superfused in the absence of mecamylamine, and determined the effect of bupropion (100 µM) alone (control condition). For this experiment, mecamylamine concentration was a within-subjects factor.

Data Analysis. Fractional release was calculated by dividing the tritium collected in each sample by the total tritium present in the tissue at the time of sample collection. Fractional release is expressed as a percentage of total tissue tritium (dpm). Basal [³H]outflow was calculated from the average fractional release in the three 5-min samples just prior to the addition of bupropion to the superfusion buffer. Bupropion and nicotine-evoked total [³H]overflow were calculated by summing the increases in fractional release that resulted from exposure to drug and subtracting the basal [³H]outflow across an equivalent period of time.

	Results

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Bupropion Competitively Inhibits Nicotine-Evoked [³H]DA Overflow. The ability of bupropion (10 nM-100 µM) to evoke [³H]overflow from superfused rat striatal slices preloaded with [³H]DA was determined (Table 1). A significant main effect of bupropion concentration was found (F_5,25 = 5.52, p < 0.001). Post hoc tests revealed that 100 µM bupropion significantly increased [³H]overflow above that in the absence of bupropion or during superfusion with lower bupropion concentrations. Thus, only the highest concentration (100 µM) of bupropion examined produced intrinsic activity in this assay and, therefore, was not included in the determination of the bupropion-induced inhibition of nicotine-evoked [³H]DA overflow.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Bupropion inhibits nicotine (10 µM)-evoked [3H]overflow from rat striatal slices preloaded with [3H]DA. Buffer contained pargyline (10 µM) and nomifensine (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 bupropion plus nicotine (n = 6 rats). , p < 0.05, different from control (0 µM bupropion, 10 µM nicotine). The inset illustrates the data presented as fractional release across the entire sampling period. The left arrow on the x-axis designates addition of bupropion to buffer and the right arrow designates addition of nicotine to buffer.

View larger version (29K): [in this window] [in a new window]   Fig. 2.   Concentration response for nicotine (1 nM-100 µM) to evoke [3H]DA overflow from rat striatal slices in the absence and presence of 1 to 10 µM bupropion. Buffer contained pargyline (10 µM) and nomifensine (10 µM) from the start of superfusion. Bupropion was included in the superfusion buffer for 30 min prior to the addition of nicotine to the buffer, and superfusion continued for an additional 60 min. Data are presented as mean ± S.E.M. of total [3H]overflow during the 60-min period of exposure to nicotine in the absence or presence of bupropion. Since the controls (i.e., concentration response for nicotine in the absence of bupropion) were not significantly different (F2,15 = 0.582, p = 0.57) among the series of experiments, these control data were pooled for graphical presentation (n = 6 rats/group). The inset illustrates the Schild regression (slope = 0.90) for bupropion-induced inhibition of the nicotine-evoked [3H]DA overflow at 0.5% tissue tritium. Data in the inset are presented as the log of (dose ratio  1) as a function of log bupropion concentration.

Bupropion Inhibits Nicotine-Evoked [³H]NE Overflow. The ability of bupropion (1 nM-100 µM) to evoke [³H]overflow from superfused rat hippocampal slices preloaded with [³H]NE was determined (Table 1). The main effect of bupropion concentration was not significant (F_6,30 = 1.38, p = 0.25). Thus, across a concentration range of 5 orders of magnitude, bupropion does not stimulate [³H]NE overflow from superfused hippocampal slices.

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Bupropion inhibits nicotine (10 µM)-evoked [3H]overflow from rat hippocampal slices preloaded with [3H]NE. 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 bupropion plus nicotine (n = 6 rats). , p < 0.05, different from control (0 µM bupropion, 10 µM nicotine). The inset illustrates the data presented as fractional release across the entire sampling period. The left arrow on the x-axis designates addition of bupropion to buffer and the right arrow designates addition of nicotine to buffer.

View larger version (33K): [in this window] [in a new window]   Fig. 4.   Concentration response for nicotine (1 nM-100 µM) to evoke [3H]NE overflow from hippocampal slices in the absence and presence of 100 nM to 10 µM bupropion. Buffer contained pargyline (10 µM) and desipramine (10 µM) from the start of superfusion. Bupropion was included in the superfusion buffer for 30 min prior to the addition of nicotine to the buffer, and superfusion continued for an additional 60 min. Data from the four series of experiments are presented as mean ± S.E.M. of total [3H]overflow during the 60-min period of exposure to nicotine in the absence or presence of bupropion. Since the controls (i.e., concentration response for nicotine in the absence of bupropion) were not significantly different (F2,14 = 0.11, p = 0.90) among the series of experiments, these control data were pooled for graphical presentation (n = 6 rats/group). The inset illustrates the Schild regression (slope = 1.37) for bupropion (100 nM-1 µM)-induced inhibition of the nicotine-evoked [3H]NE overflow at 1.0% tissue tritium. Data in the inset are presented as the log of (dose ratio  1) as a function of log bupropion concentration.

Bupropion Does Not Inhibit Field Stimulation-Evoked [³H]Overflow. [³H]DA-preloaded striatal slices were superfused for 60 min in the absence or presence of bupropion (0.1-10 µM), and were subsequently field stimulated with 120 or 300 electrical pulses (1 Hz stimulation for 2 or 5 min, respectively). Prior to electrical stimulation, bupropion did not increase [³H]overflow (data not shown), indicating that these concentrations (0.1-10 µM) of bupropion did not exhibit intrinsic activity. Table 2 provides the results demonstrating that electrical field stimulation-evoked [³H]overflow was not inhibited by bupropion. Electrical field stimulation resulted in total [³H]overflow of 1.6 to 4.7% of [³H]tissue content), which was within the range of [³H]overflow evoked by nicotine in the Schild analysis. ANOVA revealed that neither the main effect of bupropion concentration nor the bupropion concentration × number of pulses interaction was significant (p > 0.05). Thus, bupropion did not inhibit electrical stimulation-evoked [³H]overflow.

Bupropion (100 µM)-Evoked [³H]DA Overflow Is Not Mecamylamine-Sensitive. [³H]DA-preloaded striatal slices were superfused in the absence or presence of mecamylamine (0.01-100 µM) for 30 min. Subsequently, bupropion (100 µM) was added to the buffer and superfusion continued for 60 min. Prior to the addition of bupropion, mecamylamine did not significantly increase [³H]overflow (p > 0.05, data not shown). Table 3 provides the results demonstrating that bupropion-evoked [³H]overflow was not inhibited by mecamylamine. ANOVA revealed that mecamylamine did not inhibit the bupropion-evoked increase in [³H]overflow (p > 0.05; Table 3). Thus, bupropion-evoked [³H]overflow was not mecamylamine-sensitive.

	Discussion

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The present study demonstrates that bupropion inhibited nicotine-evoked [³H]DA overflow and [³H]NE overflow from superfused striatal and hippocampal slices, respectively. The interaction of bupropion with DAT or NET was eliminated by inclusion of nomifensine or desipramine, respectively, in the buffer. Thus, bupropion-induced inhibition of the transporters was not involved in the inhibition of nicotine-evoked neurotransmitter release. The highest bupropion concentration (100 µM) evoked [³H]DA overflow but had no intrinsic activity in the [³H]NE release assay. Bupropion concentrations well below those eliciting intrinsic activity inhibited nicotine-evoked [³H]DA and [³H]NE overflow (IC₅₀ values = 1.27 and 0.323 µM, respectively), suggesting antagonist activity at 32* and 34* nAChRs in rat striatum and hippocampus, respectively. Thus, inhibition of these nAChR subtypes was observed across a similar range of concentrations reported to inhibit DAT and NET function (IC₅₀ = 2-5 µM; Ferris and Cooper, 1993).

Although bupropion acted as a nAChR antagonist in several in vitro assays, no intrinsic activity was observed in previous reports. Specifically, bupropion (1 mM) did not evoke ⁸⁶Rb⁺ efflux from human neuroblastoma cells expressing the 34 ganglionic nAChR subtype or from human clonal cells expressing the 1 muscle nAChR subtype (Fryer and Lukas, 1999), nor did bupropion (50 µM) elicit current in rat 7, 32, or 42 subtypes expressed in Xenopus oocytes (Slemmer et al., 2000). Similarly, in the present study, bupropion (1 nM-100 µM) did not evoke [³H]NE overflow from rat hippocampal slices; however, the highest (100 µM) bupropion concentration evoked [³H]DA overflow from striatal slices. Importantly, the bupropion-evoked [³H]DA overflow was not inhibited by mecamylamine, indicating that bupropion-induced intrinsic activity is not mediated by nAChRs. Furthermore, since nomifensine was included in the superfusion buffer in the latter experiments, bupropion-induced intrinsic activity also was not the result of its interaction with DAT.

The observation that bupropion inhibits nicotine-evoked [³H]DA and [³H]NE overflow is consistent with previous findings that other antidepressants, including fluoxetine, desipramine, nisoxetine, citalopram, and nomifensine, inhibited nicotine (100 µM)-evoked [³H]NE overflow from rat hippocampal slices (Hennings et al., 1997, 1999). IC₅₀ values (0.36-1.8 µM) for these antidepressants were similar to those obtained for bupropion in the present study. Studies by Hennings et al. (1997, 1999) did not include an inhibitor of NET in the superfusion buffer throughout the experiment, such that NET likely played a role in the inhibition of nicotine-evoked [³H]NE overflow. Nevertheless, antidepressant-induced inhibition of nicotine-evoked [³H]NE overflow was not correlated with inhibition of NET function, indicating that NET was not involved in the inhibition of NE release (Hennings et al., 1997, 1999). Inclusion of nomifensine or desipramine in the buffer throughout the present experiments was aimed at eliminating DAT or NET function to allow a more direct investigation of the role of nAChRs in bupropion-induced inhibition of nicotine-evoked neurotransmitter release.

To determine whether the inhibitory effect of bupropion on nicotine-evoked [³H]overflow was specific, striatal slices were depolarized by electrical field stimulation, and the effect of bupropion was determined. The field stimulation parameters chosen provided [³H]overflow equivalent to that evoked by superfusion across the range of nicotine concentrations utilized in the Schild analysis. Bupropion did not inhibit [³H]DA overflow evoked by electrical field stimulation. Thus, these results suggest that the bupropion-induced inhibition of the effect of nicotine is mediated by a specific effect at nAChR sites on dopaminergic terminals in striatum. Bupropion interaction with specific nAChR subtypes has been investigated using cell expression systems. Bupropion inhibited the 34 ganglionic nAChR subtype expressed in human neuroblastoma cells (Fryer and Lukas, 1999), as well as rat 32 and 42 expressed in Xenopus oocytes (Slemmer et al., 2000). The current study extends the latter work by demonstrating that bupropion inhibits native nAChRs; however, the exact subunit composition of native nAChRs has not been elucidated conclusively (Lukas et al., 1999).

Subtype assignment of native receptors has been based primarily on inhibition of nicotinic agonist response by subtype-selective antagonists, defined by their inhibitory activity in cell systems expressing nAChR subunits of known composition. Regarding DA release, neuronal bungarotoxin and -conotoxin-MII selectively inhibit ACh electrophysiological responses in Xenopus oocytes expressing the 32 subtype (Luetje et al., 1990; Cartier et al., 1996). These 32 subtype-selective antagonists inhibit nicotine-evoked [³H]DA overflow from rodent striatal preparations (Schulz and Zigmond, 1989; Grady et al., 1992; Kaiser et al., 1998), suggesting that nicotine stimulates presynaptic 32* nAChRs to evoke striatal [³H]DA overflow. However, -conotoxin-MII inhibited only 50% of nicotine-evoked [³H]DA overflow, indicating that additional nAChR subtypes are involved (Kaiser et al., 1998). These additional subtypes may contain 4- and/or 4-subunits (Rapier et al., 1990; Grady et al., 1992; Kaiser et al., 1998; Sharples et al., 2000), although numerous nAChR subunit mRNAs are expressed in substantia nigra (Deneris et al., 1989; LeNovère et al., 1996; Göldner et al., 1997; Charpantier et al., 1998), suggesting that numerous subunits may be candidates for combination with 32 to stimulate DA release in striatum.

Evidence has accumulated that different nAChR subtypes are responsible for agonist stimulation of DA and NE release. Rank order of potency differed for nAChR agonists to evoke [³H]DA and [³H]NE release from rat striatal and hippocampal synaptosomes, respectively, suggesting involvement of different nAChR subtypes (Reuben et al., 2000). -Conotoxin AuIB inhibited electrophysiological responses to ACh in Xenopus oocytes expressing 34 nAChRs with 100-fold greater potency than it inhibited responses in oocytes expressing 32 or other subunit combinations, indicating -conotoxin AuIB selectivity for 34 (Luo et al., 1998). -Conotoxin AuIB inhibited nicotine-evoked [³H]NE release from rat hippocampal synaptosomes, but not nicotine-evoked [³H]DA release from striatal synaptosomes, suggesting that 34* is responsible for nicotine-evoked [³H]NE release (Luo et al., 1998). However, -conotoxin AuIB only partially inhibited (84%) nicotine-evoked [³H]NE release, suggesting that other subunit combinations may also be implicated. -Conotoxin MII did not inhibit nicotine-evoked [³H]NE release, indicating that 32* nAChRs are not involved (Luo et al., 1998). Thus, strong evidence was obtained for a role for 34* in agonist-stimulated NE release from hippocampus. However, other investigators have reported that the 32-selective antagonists neuronal bungarotoxin and -conotoxin MII inhibited nicotine-evoked [³H]NE overflow from superfused rat hippocampal slices and nicotine-evoked NE efflux during hippocampal microdialysis, respectively, suggesting that 32* may be involved (Sershen et al., 1997; Fu et al., 1999). Furthermore, 3, 6, 2, and 4 subunit mRNA localization to NE-containing neurons (Wada et al., 1989; Dineley-Miller and Patrick, 1992; LeNovère et al., 1996) suggests their combination with 34 to modulate nicotine-evoked hippocampal NE release.

In the present study, bupropion inhibited nicotine-evoked [³H]NE overflow ~4-fold more potently than it inhibited [³H]DA overflow. Since different nAChR subtypes are likely responsible for these responses, the present results indicate that bupropion lacks selectivity in its inhibition of native nAChRs. Furthermore, the present results suggest similar potency for inhibition of 32* and 34* subtypes and are in good agreement with studies showing that bupropion inhibited 32 and 34 with similar potency when these subunits were expressed in cell systems (Fryer and Lukas, 1999; Slemmer et al., 2000). Although the exact subunit combination for native receptors is unknown, agreement between the present results and those from expressions systems provides evidence that bupropion inhibits these nAChR subtypes.

The present results from Schild analyses revealed that bupropion-induced inhibition of nicotine-evoked [³H]DA overflow from striatal slices was via a competitive interaction with 32* nAChRs. The Schild regression was not significantly different from linearity and had a slope of unity, indicating competitive antagonism at this nAChR subtype. Bupropion-induced inhibition of nicotine-evoked [³H]DA overflow was surmountable with increasing concentrations of nicotine. The competitive nature of bupropion inhibition using native 32* nAChRs in the current study contrasts with the noncompetitive interaction of bupropion at recombinant 32 (Slemmer et al., 2000). In the present study, the highest concentration (10 µM) of bupropion nearly completely inhibited the effect of 1 nM to 10 µM nicotine in the [³H]DA release assay, whereas inhibition was completely surmounted by the highest concentration (100 µM) of nicotine examined. However, a caveat of the interpretation that bupropion acts as a competitive 32* nAChR antagonist should be considered based on previous findings. Using either superfused mouse striatal synaptosomes or rat striatal slices, [³H]DA overflow evoked by 100 µM nicotine was only partially inhibited by the classical nAChR antagonist mecamylamine, indicating that the striatal response at this high nicotine concentration is not completely dependent on nAChR activation (Grady et al., 1992; Teng et al., 1997). Therefore, the competitive nature of bupropion interaction with 32* nAChRs is obscured to some extent by non-nicotinic actions of the highest nicotine concentration utilized.

Results from the Schild analysis of bupropion-induced inhibition of nicotine-evoked [³H]NE overflow from rat hippocampal slices are consistent with an interpretation of 34* nAChR reserve. Across low concentrations of bupropion (100 nM-1 µM), inhibition was surmounted with increasing concentrations of nicotine, and the nicotine concentration-response curves appeared shifted in a rightward parallel fashion. Across these bupropion concentrations, Schild regression yielded linearity with a slope of unity, appearing to indicate competitive antagonism. However, inhibition produced by 10 µM bupropion was clearly not surmounted by increasing concentrations of nicotine, even at 100 µM nicotine. These data are consistent with the classic definition of spare receptors (Goldstein et al., 1974). Classically, maximal response is obtained only when a fraction of the total receptor pool is occupied, such that parallel rightward shifts of the curve give the appearance of competitive antagonism; however, when the antagonist has eliminated the receptor reserve, further inhibition of functional receptors decreases the agonist-evoked maximal response because not enough free receptors are available for interaction with agonist. The presence of spare 34* nAChRs in the current assay is consistent with a previous report indicating spare 4-containing nAChRs on adrenal chromaffin cells (Wenger et al., 1997). Furthermore, the present results are consistent with those from previous studies indicating that bupropion acts in a noncompetitive manner to inhibit carbamylcholine-induced ⁸⁶Rb⁺ efflux from cells expressing 34 ganglionic nAChRs, since this inhibition was not surmounted by increasing carbamylcholine concentrations (Fryer and Lukas, 1999). Thus, the noncompetitive nature of the bupropion interaction with native 34* and recombinant 34 nAChRs appears similar; however, receptor reserve apparent under native conditions complicates and makes the determination of the mechanism more difficult in hippocampus.

In summary, bupropion-induced inhibition of nicotine-evoked [³H]DA and [³H]NE release by 32* and 34* subtypes, respectively, was observed across a range of bupropion concentrations similar to those that inhibit DAT and NET function. The effect of bupropion to decrease nicotine self-administration may be the result of its inhibition of one or both of these nAChR subtypes. Thus, the combination of bupropion-induced inhibition of native nAChR subtypes and neurotransmitter transporters may provide a beneficial pharmacological profile affording clinical efficacy as both a smoking cessation agent and an antidepressant.