0022-3565/02/3023-1113–1122$7.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics
JPET 302:1113–1122, 2002
Vol. 302, No. 3
33852/1005695
Printed in U.S.A.
Bupropion Inhibits Nicotine-Evoked [3H]Overflow from Rat
Striatal Slices Preloaded with [3H]Dopamine and from Rat
Hippocampal Slices Preloaded with [3H]Norepinephrine
DENNIS K. MILLER, SANGEETHA P. SUMITHRAN, and LINDA P. DWOSKIN
College of Pharmacy, University of Kentucky, Lexington, Kentucky
Received January 28, 2002; accepted May 17, 2002
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
This research was supported by Pharmacia Corporation (Kalamazoo, MI).
Article, publication date, and citation information can be found at
http://jpet.aspetjournals.org.
DOI: 10.1124/jpet.102.033852.
lease assay. However, none of the concentrations (1 nM–100
␮M) examined evoked [3H]NE overflow and, thus, were without
intrinsic activity in this assay. Moreover, bupropion inhibited
both nicotine-evoked [3H]DA overflow (IC50 ⫽ 1.27 ␮M) and
nicotine-evoked [3H]NE overflow (IC50 ⫽ 323 nM) at bupropion
concentrations well below those eliciting intrinsic activity. Results from Schild analyses suggest that bupropion competitively inhibits nicotine-evoked [3H]DA overflow, whereas evidence for receptor reserve was obtained upon assessment of
bupropion inhibition of nicotine-evoked [3H]NE overflow. Thus,
bupropion acts as an antagonist at ␣3␤2ⴱ and ␣3␤4ⴱ nAChRs
in rat striatum and hippocampus, respectively, across the same
concentration range that inhibits DAT and NET function. The
combination of nAChR and transporter inhibition produced by
bupropion may contribute to its clinical efficacy as a smoking
cessation agent.
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 ␮M) into rat striatal synaptosomes, [3H]norepinephrine ([3H]NE) uptake (IC50
⫽ 5 ␮M) into rat hypothalamic synaptosomes, and, less potently (IC50 ⫽ 58 ␮M), [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 mem-
ABBREVIATIONS: DAT, dopamine transporter; NET, norepinephrine transporter; DA, dopamine; NE, norepinephrine; nAChR, nicotinic acetylcholine receptor; ACh, acetylcholine; ANOVA, analysis of variance; ⴱ, putative nAChR subtype assignment.
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ABSTRACT
Bupropion, an efficacious antidepressant and smoking cessation agent, inhibits dopamine and norepinephrine transporters
(DAT and NET, respectively). Recently, bupropion has been
reported to noncompetitively inhibit ␣3␤2, ␣3␤4, and ␣4␤2
nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus oocytes or established cell lines. The present study evaluated bupropion-induced inhibition of native ␣3␤2ⴱ and ␣3␤4ⴱ
nAChRs using functional neurotransmitter release assays, nicotine-evoked [3H]overflow from superfused rat striatal slices
preloaded with [3H]dopamine ([3H]DA), and nicotine-evoked
[3H]overflow from hippocampal slices preloaded with [3H]norepinephrine ([3H]NE). The mechanism of inhibition was evaluated
using Schild analysis. To eliminate the interaction of bupropion
with DAT or NET, nomifensine or desipramine, respectively,
was included in the superfusion buffer. A high bupropion concentration (100 ␮M) elicited intrinsic activity in the [3H]DA re-
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Miller et al.
Materials and Methods
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-[N3
H]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 ␮m; 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 ␮M 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 ␮M [3H]DA or 0.1 ␮M
[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 ␮M) 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 ␮M) 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 ␮M) 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 ␮M), 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 ␮M) 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 (t1/2 ⫽ 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
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branes (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 ␮M) carbamylcholine (1 mM)-induced 86Rb⫹ efflux from human neuroblastoma cells expressing the ␣3␤4 ganglionic nAChR subtype and more potently inhibited (IC50 ⫽ 1.51 ␮M) 86Rb⫹
efflux from human clonal cells expressing the ␣1 muscle
nAChR subtype (Fryer and Lukas, 1999). Bupropion also
inhibited acetylcholine (ACh; 1 ␮M) activation of rat ␣3␤2
(IC50 ⫽ 1.3 ␮M) and ␣4␤2 (IC50 ⫽ 8 ␮M) subtypes expressed
in Xenopus oocytes (Slemmer et al., 2000). Furthermore,
bupropion inhibited the ␣7 subtype, but with lower affinity
(IC50 ⫽ 60 ␮M). 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 ␮M) did not displace [3H]nicotine binding to whole rat brain membranes, also consistent with noncompetitive inhibition of ␣4␤2 nAChRs (Slemmer et al.,
2000). Thus, bupropion noncompetitively inhibits ␣3␤2,
␣4␤2, and ␣3␤4 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 nicotineevoked [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 ␣3␤2ⴱ and ␣3␤4ⴱ 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).
Bupropion Inhibits Nicotinic Receptors
ability of mecamylamine (0.01 – 100 ␮M) 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
␮M) and nomifensine (10 ␮M). 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 ␮M),
which remained in the buffer until the end of the experiment. Each
slice was exposed to only one concentration of mecamylamine. Subsequently, bupropion (100 ␮M) 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 ␮M) 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.
The intrinsic activity of bupropion on [3H]overflow and the ability
of bupropion to inhibit [3H]overflow evoked by 10 ␮M nicotine were
analyzed via one-way repeated measures analysis of variance
(ANOVA) with bupropion concentration as a within-subjects factor
(SPSS, Version 9.0; SPSS Science, Chicago, IL). Separate analyses
were performed for [3H]DA overflow and [3H]NE overflow experiments. Where appropriate, Tukey post hoc tests (p ⬍ 0.05) were
performed. Time course data were analyzed via two-way repeated
measures ANOVA with time and concentration as within-subject
factors. EC50 or IC50 values were determined via nonlinear regression to fit the mean data points to a sigmoidal concentration-response curve (Prism,Version 3.0; GraphPad, San Diego, CA).
The mechanism by which bupropion inhibited nicotine-evoked
[3H]DA overflow and [3H]NE overflow was determined using separate Schild analyses (Goldstein et al., 1974; Kenakin, 1997). For each
experiment using slices from a single rat, the concentration-response
for nicotine was determined in both the absence and presence of
bupropion, and nonlinear regression was used to fit the concentration-response curves. For each experiment, the dose ratio of the
equiactive concentration of nicotine in the presence of bupropion to
the nicotine concentration in the absence of bupropion was calculated for total [3H]overflow at 0.5% and at 1.0% of tissue tritium
content. The log(dose ratio ⫺ 1) was plotted as a function of log(bupropion concentration), and linear regression was performed to provide the Schild regression. Additionally, bupropion-induced inhibition of nicotine-induced [3H]overflow was analyzed via three-way
repeated measures ANOVA with nicotine concentration and the
absence or presence of bupropion as within-subject factors, and with
bupropion concentration as a between-groups factor.
The ability of bupropion to inhibit electrically evoked [3H]overflow
was analyzed via two-way repeated measures ANOVA with bupropion
concentration as a within-subjects factor and number of electrical
pulses as a between-group factor. Mecamylamine-induced inhibition of
bupropion (100 ␮M)-evoked [3H]overflow was analyzed via one-way
repeated measures ANOVA with mecamylamine concentration as a
within-subjects factor.
Results
Bupropion Competitively Inhibits Nicotine-Evoked
[3H]DA Overflow. The ability of bupropion (10 nM–100 ␮M)
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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).
The mechanism of the bupropion-induced inhibition of nicotineevoked [3H]overflow was determined using a Schild analysis. For
each experiment, the complete concentration-response for nicotine (1
nM–100 ␮M) was determined in the absence or presence of a single
concentration of bupropion using brain slices from a single rat. The
inhibitory effect of at least three concentrations of bupropion (1–10
␮M for [3H]DA experiments or 0.1–10.0 ␮M for [3H]NE experiments)
were determined. Bupropion concentrations were selected based on
the respective IC50 values for inhibition of nicotine (10 ␮M)-evoked
[3H]overflow. For [3H]DA or [3H]NE overflow experiments, striatal
or hippocampal slices were superfused with buffer containing pargyline and nomifensine (10 ␮M) or desipramine (10 ␮M), respectively, for 75 min. Subsequently, slices were superfused in the absence or presence of a single concentration of bupropion (10 nM–10
␮M), which remained in the buffer throughout the experiment. After
30 min of superfusion with bupropion, one of six concentrations of
nicotine (1 nM–100 ␮M) was added to the buffer, and superfusion
continued for an additional 60 min. Each slice from a single animal
was exposed to only one concentration of nicotine and one concentration of bupropion. These experiments utilized a repeated measures design, such that the concentration-response for nicotine was
determined in both the absence and presence of each concentration of
bupropion using either striatum or hippocampus from a single rat.
Tissue and superfusate samples were processed as previously described.
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 ␮M) 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 ␮M) and nomifensine (10
␮M). 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 ␮M), 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 60min period. For these experiments, the number of pulses was a
between-group factor and the bupropion concentration was a withinsubjects factor.
Mecamylamine-Induced Inhibition of Bupropion-Evoked
[3H]Overflow. To assess whether bupropion (100 ␮M)-evoked
[3H]overflow (intrinsic activity) is mediated by nAChRs, striatal
slices were preloaded with [3H]DA as previously described, and the
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Miller et al.
TABLE 1
Bupropion produces no intrinsic activity in the [3H]NE release assay
using hippocampal slices; and only at high concentrations (100 ␮M),
bupropion evoked [3H]DA release from superfused striatal slices
In separate series of experiments, rat striatal slices were preloaded with [3H]DA and
rat hippocampal slices were preloaded with [3H]NE. Superfusion buffer contained
pargyline (10 ␮M) and nomifensine (10 ␮M) or desipramine (10 ␮M). Slices were
superfused with various concentrations of bupropion, which was added to the buffer.
Each slice was exposed to only one concentration of bupropion. Data represent
[3H]overflow during the 30-min period of superfusion in the absence and presence of
bupropion.
Bupropion Concentration
0M
1 nM
10 nM
100 nM
1 ␮M
10 ␮M
100 ␮M
[3H]DA Overflow
[3H]NE Overflow
0.01 ⫾ 0.01a
ND
0.00 ⫾ 0.00
0.049 ⫾ 0.041
0.092 ⫾ 0.077
0.035 ⫾ 0.028
0.40 ⫾ 0.16*
0.043 ⫾ 0.02
0.080 ⫾ 0.042
0.020 ⫾ 0.019
0.050 ⫾ 0.020
0.11 ⫾ 0.058
0.12 ⫾ 0.035
0.069 ⫾ 0.033
ND, not determined.
a
Data are mean ⫾ S.E.M. of total [3H]overflow expressed as a percentage of
tissue tritium.
* p ⬍ 0.05, compared to 0 to 10 ␮M bupropion in the [3H]DA series of experiments.
n ⫽ 6 rats/experiment.
was surmounted. When the Schild analysis was performed
using a [3H]overflow response of 0.5% total tissue tritium,
the Schild regression revealed a linear relationship (F1,14 ⫽
16.4, p ⬍ 0.01; r2 ⫽ 0.54) with a slope of 0.90 (Fig. 2, inset).
A linear relationship was also observed for a [3H]overflow
response of 1% total tissue tritium (slope ⫽ 0.87; F1,15 ⫽ 4.52,
p ⫽ 0.0506; r2 ⫽ 0.23; regression not shown), indicative of a
competitive interaction.
The data from the Schild analysis were also analyzed using
ANOVA. A significant main effect of nicotine concentration
was found (F6,60 ⫽ 15.66, p ⬍ 0.001), indicating a concentration-dependent increase in nicotine-evoked [3H]overflow
across the series of experiments determining inhibition induced by one of the three concentrations of bupropion. The
lowest (1 ␮M) concentration of bupropion did not significantly inhibit nicotine-evoked [3H]overflow (F1,5 ⫽ 0.51, p ⫽
0.51), whereas higher bupropion concentrations (3 and 10
␮M) inhibited nicotine-evoked [3H]overflow (F1,5 ⫽ 7.14, p ⬍
0.05 and F1,5 ⫽ 20.86, p ⬍ 0.01, respectively). Importantly,
the inhibition produced by bupropion (3–10 ␮M) was surmounted by superfusion with increasing concentrations of
nicotine.
Bupropion Inhibits Nicotine-Evoked [3H]NE Overflow. The ability of bupropion (1 nM–100 ␮M) 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 (F6,30 ⫽
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.
In a concentration-dependent manner, bupropion inhibited
nicotine (10 ␮M)-evoked [3H]overflow from superfused hippocampal slices preloaded with [3H]NE, with an IC50 value of
323 nM (Fig. 3). Analysis of total [3H]overflow following
superfusion with bupropion and nicotine revealed a significant main effect of bupropion concentration (F6,30 ⫽ 5.25, p ⬍
0.001). Post hoc tests indicated a significant inhibition (48 –
94%) of nicotine-evoked [3H]overflow following superfusion
with 100 nM to 100 ␮M bupropion. Analysis of the time
course data also revealed a significant main effect of bupropion concentration (F60,416 ⫽ 2.12, p ⬍ 0.001; Fig. 3, inset).
Bupropion (1–100 ␮M) significantly inhibited nicotineevoked fractional release at each 5-min time point during the
60-min period of exposure to nicotine compared with control.
Bupropion (0.1 ␮M) significantly inhibited [3H]overflow only
at the 60-, 75-, and 80-min time points compared with control. Thus, bupropion potently inhibited nicotine-evoked
[3H]NE overflow from rat hippocampal slices.
Schild analysis was utilized to determine the mechanism of
bupropion-induced inhibition of nicotine-evoked [3H]NE
overflow from hippocampal slices by generating concentration-response curves for nicotine (10 nM–100 ␮M) in the
absence and presence of one of four bupropion concentrations
(100 nM–10 ␮M; Fig. 4). Figure 4 illustrates that increasing
bupropion concentrations (100 nM–1 ␮M) shifted the nicotine
concentration-response curve parallel and to the right; however, the highest bupropion concentration (10 ␮M) shifted
the nicotine concentration-response curve to the right and
down. Thus, increasing concentrations of nicotine did not
surmount the inhibitory effect of the highest bupropion concentration. When a Schild analysis was performed on the
data generated from bupropion concentrations of 100 nM to 1
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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 (F5,25 ⫽
5.52, p ⬍ 0.001). Post hoc tests revealed that 100 ␮M 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 ␮M) of bupropion examined produced intrinsic activity
in this assay and, therefore, was not included in the determination of the bupropion-induced inhibition of nicotineevoked [3H]DA overflow.
In a concentration-dependent manner, bupropion inhibited
nicotine (10 ␮M)-evoked [3H]overflow from [3H]DA-preloaded striatal slices, with an IC50 value of 1.27 ␮M (Fig. 1).
A significant main effect of bupropion concentration was
found (F5,25 ⫽ 9.49, p ⬍ 0.001). Post hoc tests revealed that
10 ␮M bupropion inhibited (⬃72%) nicotine-evoked [3H]overflow compared with control (i.e., nicotine-evoked [3H]overflow in the absence of bupropion). Analysis of the time course
data (Fig. 1, inset) revealed a significant main effect of bupropion concentration (F20,756 ⫽ 63.3, p ⬍ 0.001). A high
concentration (10 ␮M) of bupropion significantly decreased
fractional release evoked by nicotine across the 60-min period of superfusion with nicotine, relative to control. A lower
concentration (1 ␮M) of bupropion significantly inhibited
fractional release at the 5 to 35 min of superfusion with
nicotine. Thus, at concentrations (1–10 ␮M) that did not
intrinsically evoke [3H]overflow, bupropion inhibited nicotine-evoked [3H]overflow from rat striatal slices preloaded
with [3H]DA.
To determine the mechanism of the bupropion-induced
inhibition of nicotine-evoked [3H]overflow from rat striatal
slices preloaded with [3H]DA, a Schild analysis was performed. In each series of experiments, the concentrationresponse for nicotine (1 nM–100 ␮M)-evoked [3H]overflow
from [3H]DA-preloaded striatal slices was determined in both
the absence and presence of one of three bupropion concentrations (1–10 ␮M; Fig. 2). Figure 2 illustrates that increasing concentrations of bupropion shifted the nicotine concentration-response curve parallel and to the right; and at high
nicotine concentrations, the bupropion-induced inhibition
Bupropion Inhibits Nicotinic Receptors
1117
␮M at a [3H]overflow response of 1.0% total tissue tritium, a
linear relationship (F1,15 ⫽ 35.42, p ⬍ 0.01; r2 ⫽ 0.70) with a
slope of 1.37 (Fig. 4, inset) was revealed. A linear relationship
was also observed for a [3H]overflow response of 0.5% total
tissue tritium (slope ⫽ 1.04; F1,16 ⫽ 7.23, p ⬍ 0.05; r2 ⫽ 0.31;
regression not shown) across the 100 nM to 1 ␮M concentration range.
Data from this Schild analysis were analyzed also via
repeated-measures ANOVA, which revealed a significant
three-way interaction of nicotine concentration, the absence
or presence of bupropion, and bupropion concentration
(F18,120 ⫽ 5.53, p ⬍ 0.001). Simple main effect analyses and
post hoc tests revealed that nicotine produced a concentration-dependent increase in [3H]overflow across the series of
experiments (F1,20 ⫽ 70.3, p ⬍ 0.001). The lowest concentration (100 nM) of bupropion examined did not significantly
inhibit nicotine-evoked [3H]overflow. Bupropion (300 nM)
significantly inhibited [3H]overflow evoked by nicotine (100
nM-3 ␮M), whereas higher concentrations of nicotine (10 –
100 ␮M) surmounted the inhibition induced by 300 nM bupropion (F6,30 ⫽ 86.8, p ⬍ 0.001). Bupropion (1 ␮M) significantly inhibited [3H]overflow evoked by nicotine (100 nM-10
␮M), whereas nicotine (100 ␮M) surmounted the inhibition
induced by 1 ␮M bupropion (F6,30 ⫽ 76.1, p ⬍ 0.001). The
highest concentration (10 ␮M) of bupropion inhibited
[3H]overflow evoked by 100 nM to 100 ␮M nicotine (F6,36 ⫽
65.7, p ⬍ 0.001), and inhibition produced by this concentration of bupropion was not surmounted by increasing concentrations of nicotine.
Bupropion Does Not Inhibit Field StimulationEvoked [3H]Overflow. [3H]DA-preloaded striatal slices
were superfused for 60 min in the absence or presence of
bupropion (0.1–10 ␮M), 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 ␮M) 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 ␮M)-Evoked [3H]DA Overflow Is Not
Mecamylamine-Sensitive.
[3H]DA-preloaded
striatal
slices were superfused in the absence or presence of
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 15, 2017
Fig. 1. Bupropion inhibits nicotine (10 ␮M)-evoked [3H]overflow from rat striatal slices preloaded with [3H]DA. Buffer contained pargyline (10 ␮M)
and nomifensine (10 ␮M) 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). 多, p ⬍ 0.05, different from control (0 ␮M bupropion, 10 ␮M 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.
1118
Miller et al.
mecamylamine (0.01–100 ␮M) for 30 min. Subsequently, bupropion (100 ␮M) 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
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 ␮M) 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 ␮M, respectively), suggesting antagonist ac-
tivity at ␣3␤2ⴱ and ␣3␤4ⴱ 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
␮M; 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 (ⱕ1 mM) did not evoke 86Rb⫹
efflux from human neuroblastoma cells expressing the ␣3␤4
ganglionic nAChR subtype or from human clonal cells expressing the ␣1 muscle nAChR subtype (Fryer and Lukas, 1999), nor
did bupropion (ⱕ50 ␮M) elicit current in rat ␣7, ␣3␤2, or ␣4␤2
subtypes expressed in Xenopus oocytes (Slemmer et al., 2000).
Similarly, in the present study, bupropion (1 nM–100 ␮M) did
not evoke [3H]NE overflow from rat hippocampal slices; however, the highest (100 ␮M) 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, desi-
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 15, 2017
Fig. 2. Concentration response for
nicotine (1 nM–100 ␮M) to evoke
[3H]DA overflow from rat striatal
slices in the absence and presence of
1 to 10 ␮M bupropion. Buffer contained pargyline (10 ␮M) and nomifensine (10 ␮M) 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 Nicotinic Receptors
1119
pramine, nisoxetine, citalopram, and nomifensine, inhibited
nicotine (100 ␮M)-evoked [3H]NE overflow from rat hippocampal slices (Hennings et al., 1997, 1999). IC50 values
(0.36 –1.8 ␮M) 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, antidepressantinduced 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 ␣3␤4 ganglionic
nAChR subtype expressed in human neuroblastoma cells (Fryer and Lukas, 1999), as well as rat ␣3␤2 and ␣4␤2 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 ␣3␤2
subtype (Luetje et al., 1990; Cartier et al., 1996). These ␣3␤2
subtype-selective antagonists inhibit nicotine-evoked [3H]DA
overflow from rodent striatal preparations (Schulz and Zig-
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 15, 2017
Fig. 3. Bupropion inhibits nicotine (10 ␮M)-evoked [3H]overflow from rat hippocampal slices preloaded with [3H]NE. Buffer contained pargyline (10
␮M) and desipramine (10 ␮M) 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). 多, p ⬍ 0.05, different from control (0 ␮M bupropion, 10 ␮M 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.
1120
Miller et al.
TABLE 2
Bupropion does not inhibit electrical field stimulation-evoked
[3H]overflow from [3H]DA-preloaded striatal slices
TABLE 3
Bupropion (100 ␮M)-evoked [3H]overflow from [3H]DA-preloaded rat
striatal slices is not inhibited by mecamylamine (0.01–100 ␮M)
Superfusion buffer contained pargyline (10 ␮M) and nomifensine (10 ␮M). Slices
were superfused with a range of bupropion concentrations added to the buffer. Each
slice was exposed to only one concentration of bupropion and was subsequently
stimulated by either 120 or 300 pulses (1 Hz). Data represent [3H]overflow during
the 60-min period of superfusion following electrical field stimulation.
Superfusion buffer contained pargyline (10 ␮M) and nomifensine (10 ␮M). Slices
were superfused with mecamylamine for 30 min. Subsequently, bupropion (100 ␮M)
was added to buffer, and superfusion continued for 60 min. Each slice was exposed
to only one concentration of mecamylamine. Data represent [3H]overflow during the
60-min period of superfusion with bupropion.
Bupropion (␮M)
Control (0 M)
0.1
1
3
10
120 Pulses
300 Pulses
2.23 ⫾ 0.22a
1.61 ⫾ 0.32
2.33 ⫾ 0.19
1.72 ⫾ 0.37
1.65 ⫾ 0.27
4.19 ⫾ 0.79
4.56 ⫾ 0.55
4.69 ⫾ 0.88
4.18 ⫾ 1.12
3.09 ⫾ 1.25
a
Data are mean ⫾ S.E.M. of total [3H]overflow expressed as a percentage of
tissue tritium. n ⫽ 6 –9 rats/experiment.
mond, 1989; Grady et al., 1992; Kaiser et al., 1998), suggesting that nicotine stimulates presynaptic ␣3␤2ⴱ 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 ␣4and/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 ␣3␤2 to
stimulate DA release in striatum.
Mecamylamine (␮M)
Control (0 M)
0.01
0.01
1
10
100
Bupropion (100 ␮M)
8.83 ⫾ 1.67a
6.95 ⫾ 2.13
11.20 ⫾ 1.35
8.39 ⫾ 1.96
7.90 ⫾ 1.79
8.78 ⫾ 2.52
a
Data are mean ⫾ S.E.M. of total [3H]overflow expressed as a percentage of
tissue tritium. n ⫽ 4 rats.
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 ␣3␤4 nAChRs with 100-fold greater potency
than it inhibited responses in oocytes expressing ␣3␤2 or other
subunit combinations, indicating ␣-conotoxin AuIB selectivity
for ␣3␤4 (Luo et al., 1998). ␣-Conotoxin AuIB inhibited nicotineevoked [3H]NE release from rat hippocampal synaptosomes,
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 15, 2017
Fig. 4. Concentration response for
nicotine (1 nM–100 ␮M) to evoke
[3H]NE overflow from hippocampal
slices in the absence and presence of
100 nM to 10 ␮M bupropion. Buffer
contained pargyline (10 ␮M) and desipramine (10 ␮M) 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 ␮M)-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 Inhibits Nicotinic Receptors
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
␣3␤4ⴱ nAChR reserve. Across low concentrations of bupropion (100 nM–1 ␮M), 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 ␮M bupropion was clearly not surmounted by increasing concentrations of nicotine, even at
100 ␮M 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 ␣3␤4ⴱ 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 carbamylcholineinduced 86Rb⫹ efflux from cells expressing ␣3␤4 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 ␣3␤4ⴱ and recombinant ␣3␤4
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 nicotineevoked [3H]DA and [3H]NE release by ␣3␤2ⴱ and ␣3␤4ⴱ
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.
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