Bupropion Inhibits Nicotine-Evoked [H]Overflow from Rat Striatal Slices Preloaded with [H]Dopamine and from Rat Hippocampal Slices Preloaded with [H]Norepinephrine

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 2 and 3 4 nAChRs using functional neurotransmitter release assays, nicotine-evoked [H]overflow from superfused rat striatal slices preloaded with [H]dopamine ([H]DA), and nicotine-evoked [H]overflow from hippocampal slices preloaded with [H]norepinephrine ([H]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 [H]DA release assay. However, none of the concentrations (1 nM–100 M) examined evoked [H]NE overflow and, thus, were without intrinsic activity in this assay. Moreover, bupropion inhibited both nicotine-evoked [H]DA overflow (IC50 1.27 M) and nicotine-evoked [H]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 [H]DA overflow, whereas evidence for receptor reserve was obtained upon assessment of bupropion inhibition of nicotine-evoked [H]NE overflow. Thus, bupropion acts as an antagonist at 3 2 and 3 4 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. 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 [H]dopamine ([H]DA) uptake (IC50 2 M) into rat striatal synaptosomes, [H]norepinephrine ([H]NE) uptake (IC50 5 M) into rat hypothalamic synaptosomes, and, less potently (IC50 58 M), [ H]serotonin uptake into rat hypothalamic synaptosomes (Ferris and Cooper, 1993; Ascher et al., 1995). A competitive interaction with DAT has been demonstrated using [H]mazindol binding to rat striatal memThis 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. ABBREVIATIONS: DAT, dopamine transporter; NET, norepinephrine transporter; DA, dopamine; NE, norepinephrine; nAChR, nicotinic acetylcholine receptor; ACh, acetylcholine; ANOVA, analysis of variance; , putative nAChR subtype assignment. 0022-3565/02/3023-1113–1122$7.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 302, No. 3 Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics 33852/1005695 JPET 302:1113–1122, 2002 Printed in U.S.A. 1113 at A PE T Jornals on D ecem er 3, 2017 jpet.asjournals.org D ow nladed from 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 Rb efflux from human neuroblastoma cells expressing the 3 4 ganglionic nAChR subtype and more potently inhibited (IC50 1.51 M) Rb 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 [H]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 [H]DA and [H]NE release assays. Specifically, bupropion inhibition of nicotineevoked [H]overflow from superfused rat striatal slices preloaded with [H]DA and rat hippocampal slices preloaded with [H]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 2 and 3 4 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 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). [H]NE (levo-[7-H]norepinephrine; specific activity 14.4 Ci/mmol) and [H]DA (3,4-ethyl-2-[NH]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). [H]Overflow Assay. [H]overflow from striatal slices preloaded with [H]DA or [H]overflow from hippocampal slices preloaded with [H]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 [H]DA or 0.1 M [H]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 [H]overflow represented primarily [H]DA or [H]NE, rather than their metabolites (Zumstein et al., 1981). In [H]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 [H]DA uptake into rat striatal synaptosomes was 150 nM (Hunt et al., 1974). In [H]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 [ H]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 [H]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 [H]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 [H]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 1114 Miller et al. at A PE T Jornals on D ecem er 3, 2017 jpet.asjournals.org D ow nladed from 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 [H]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,