Abstract
Lobeline is currently being developed as a substitution therapy for tobacco smoking cessation. Activation of CNS dopamine (DA) systems results in the reinforcing properties of nicotine. The present study compared the effects of lobeline and nicotine on rat striatum. Both lobeline and nicotine evoked [3H]overflow from striatal slices superfused in the presence of pargyline and nomifensine in the buffer. Marked DA depletion (42–67%) and a concomitant 2-fold increase in dihydroxyphenylacetic acid (DOPAC) in slices superfused with high concentrations (30–100 μM) of lobeline were observed. The effect of nicotine (10 μM) was inhibited in a concentration-dependent manner by mecamylamine (1–100 μM). However, lobeline (0.1–100 μM)-evoked [3H]overflow was calcium-independent, and was not antagonized by mecamylamine (1–100 μM), suggesting a mechanism of action other than stimulation of nicotinic receptors. Lobeline inhibited [3H]DA uptake into synaptosomes (IC50 = 80 ± 12 μM) and vesicles (IC50 = 0.88 ± 0.001 μM), whereas nicotine (≤100 μM) did not inhibit synaptosomal or vesicular [3H]DA uptake. In the absence of pargyline and nomifensine in the buffer, endogenous DA was detected in superfusate only in those slices exposed to the highest concentration (100 μM) of lobeline. However, endogenous DOPAC concentration was increased in a concentration-dependent manner, indicating that lobeline exposure resulted in increased cytosolic DA which was rapidly metabolized to DOPAC. Under these conditions, lobeline (10–100 μM) also significantly depleted (66–85%) DA content; however, no change in DOPAC content was observed. The results suggest that, unlike nicotine, lobeline increases DA release by potent inhibition of DA uptake into synaptic vesicles, and a subsequent alteration in presynaptic DA storage.
Lobeline (α-lobeline) is a lipophilic, nonpyridino, alkaloidal constituent of Indian tobacco (Lobelia inflata). No obvious structural resemblance to S(−)nicotine is apparent (fig. 1), and structure-function relationships between nicotine and lobeline do not suggest a common pharmacophore (Barlow and Johnson, 1989). However, lobeline has been reported to have many nicotine-like effects, including tachycardia and hypertension (Olin et al., 1995), bradycardia and hypotension in urethane- and pentobarbital-anesthetized rats (Sloan et al., 1988), hyperalgesia (Hamann and Martin, 1994), anxiolytic activity (Brioni et al., 1993) and improvement of learning and memory (Decker et al., 1993). Moreover, lobeline has been used as a substitution therapy for tobacco smoking cessation (Nunn-Thompson and Simon, 1989; Prignot, 1989; Olinet al., 1995); its effectiveness is controversial, however, as reflected by both positive (Dorsey, 1936; Kalyuzhnyy, 1968) and negative reports (Wright and Littauer, 1937; Nunn-Thompson and Simon, 1989). Furthermore, only short-term usage of lobeline as a smoking deterrent has been recommended because of its acute toxicity (nausea, severe heart burn and dizziness) and the lack of information concerning its long-term usage (Wright and Littauer, 1937; Olin et al., 1995).
In behavioral studies, nicotine has been shown to increase locomotor activity (Clarke and Kumar, 1983a, 1983b; Clarke, 1990; Fung and Lau, 1988) and produce conditioned place preference (Shoaib et al., 1994; Fudala et al., 1985) in rats. However, the results of the latter studies are controversial (Clarke and Fibiger, 1987). In contrast, lobeline does not increase locomotor activity (Stolerman et al., 1995) or produce conditioned place preference (Fudala and Iwamoto, 1986). Although lobeline was initially shown to generalize to nicotine in discrimination studies (Gelleret al., 1971), most subsequent studies failed to replicate these original findings (Schechter and Rosecrans, 1972; Reavillet al., 1990; Romano and Goldstein, 1980). Nicotine has been reported to be avidly self-administered by rats (Corrigal et al., 1992, 1994; Donny et al., 1995); however, the ability of lobeline to support self-administration has not been investigated. The differential effects of lobeline and nicotine in behavioral studies suggest that these drugs may not be actingvia a common CNS mechanism, even though lobeline is often considered a nicotinic agonist (Decker et al., 1995).
The positive reinforcing effect of nicotine is believed to be due to the activation of central dopaminergic systems (Benwell and Balfour, 1992; Corrigal et al., 1992, 1994). Presynaptic nicotinic receptors have been found on DA-containing nerve terminals (Giorguieff-Chesselet et al., 1979; Clarke and Pert, 1985). Nicotine binds to nicotinic receptors with high affinity (Kd = 1–7 nM) (Lippiello and Fernandes, 1986; Reavillet al., 1988; Romm et al., 1990; Bhat et al., 1991; Loiacono et al., 1993; Anderson and Arneric, 1994). Also, lobeline has been reported to displace [3H]nicotine binding from central nicotinic receptors with high affinity (Ki = 5–30 nM) (Yamada et al., 1985; Lippiello and Fernades, 1986; Banerjee and Abood, 1989;Broussolle et al., 1989). Chronic treatment with nicotine results in an increase in the number of nicotinic receptors in many regions of rat and mouse brain (Collins et al., 1990; Bhatet al., 1991, 1994; Marks et al., 1992; Sandersonet al., 1993). An increase in the number of nicotinic receptors in post-mortem human brain tissue obtained from smokers also has been reported (Benwell et al., 1988). In contrast, chronic lobeline administration did not increase the number of nicotinic receptors in mouse brain regions in which increases were observed after chronic nicotine administration (Bhat et al., 1991).
Nicotine evokes DA release in in vitro superfusion studies using striatal slices (Westfall, 1974; Giorguieff-Chesselet et al., 1979; Westfall et al., 1987; Harsing et al., 1992) and striatal synaptosomes (Chesselet, 1984; Rowellet al., 1987; Rapier et al., 1988, 1990; Gradyet al., 1992, 1994; Rowell and Hillebrand, 1992, 1994;Rowell, 1995) and in in vivo studies using microdialysis in striatum (Imperato et al., 1986; Damsma et al., 1989; Brazell et al., 1990; Toth et al., 1992). Nicotine-evoked DA release is calcium-dependent, mecamylamine-sensitive and mediated by nicotinic receptors (Giorguieff-Chesselet et al., 1979; Westfall et al., 1987; Rapier et al., 1988; Grady et al., 1992). Mecamylamine is a noncompetitive nicotinic receptor antagonist that effectively blocks the ion channel of the receptor (Varanda et al., 1985;Loiacono et al., 1993; Peng et al., 1994). Like nicotine, lobeline has been reported to increase DA release from superfused rat and mouse striatal synaptosomes (Sakurai et al., 1982; Takano et al., 1983; Grady et al., 1992). Nicotine and lobeline similarly release DA and displace [3H]nicotine binding. On the basis of these neurochemical studies, it has been suggested that lobeline is an agonist at nicotinic receptors. However, the up-regulation of nicotinic receptors that is observed after chronic nicotine administration is not observed after chronic lobeline administration.
The present study was performed to determine the involvement of nicotinic receptors in lobeline-evoked DA release from rat striatal slices. Striatal DA and DOPAC content were also determined after superfusion with lobeline. The calcium dependence of the effect of lobeline and the ability of mecamylamine to inhibit the lobeline response were investigated. To assess the contribution of potential effects on DA uptake, we studied the effect of nicotine and lobeline in inhibiting [3H]DA uptake into striatal synaptosomes and synaptic vesicle preparations. Lobeline-induced alterations in DA presynaptic storage were also assessed.
Materials and Methods
Materials.
The following drugs and chemicals were used in this study: S(−)nicotine ditartrate, nomifensine maleate, mecamylamine hydrochloride, and GBR 12909 (Research Biochemicals, Inc., Natick, MA), tetrabenazine (Fluka Chemika-BioChemika, Ronkonkoma, NY), [3H]DA, specific activity 25.6 Ci/mmol (New England Nuclear, Boston, MA), DOPAC, DHBA, lobeline hemisulfate, pargyline hydrochloride, HEPES potassium tartrate, adenosine 5′-triphosphate magnesium salt (ATP-Mg++), l(+)tartaric acid and 1-octanesulfonic acid sodium salt (Sigma Chemical Co., St. Louis, MO), α-d-glucose and sucrose (Aldrich Chemical Company, Inc., Milwaukee, WI), ascorbic acid and ascorbic acid oxidase (AnalaR, BHD Ltd., Poole, U.K. and Boehringer Mannheim GmbH, Germany, respectively), glutaraldehyde, osmium tetroxide and copper grids (EMS Inc., Fort Washington, CA), eponate 12 (Ted Pella, Inc., Redding, CA), TS-2 tissue solubilizer (Research Products International, Mount Prospect, IL) and acetonitrile (HPLC grade) (EM Science, EM Industries, Cherry Hill, NJ). All other chemicals were purchased from Fisher Scientific (Pittsburgh, PA).
Subjects.
Male Sprague-Dawley rats (200–250 g) were obtained from Harlan Laboratories (Indianapolis, IN) and were housed two per cage with free access to food and water in the Division of Lab Animal Resources at the College of Pharmacy at the University of Kentucky. Experimental protocols involving the animals were in strict accordance with the NIH 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.
[3H]DA release assay.
The effects of lobeline and nicotine on [3H]overflow from rat striatal slices preloaded with [3H]DA were determined by using a previously published method (Dwoskin and Zahniser, 1986). Briefly, rat striatal slices (500 μm, 6–8 mg) were incubated in Krebs’ buffer (in mM: 118 NaCl, 4.7 KCl, 1.2 MgCl2, 1.0 NaH2PO4, 1.3 CaCl2, 11.1 α-d-glucose, 25 NaHCO3, 0.11l-ascorbic acid and 0.004 EDTA, pH 7.4, and saturated with 95% O2/5% CO2) in a metabolic shaker at 34°C for 30 min. Slices were incubated in fresh buffer containing 0.1 μM [3H]DA (6–8 slices/3 ml) for an additional 30 min. After rinsing, slices were transferred to a glass superfusion chamber maintained at 34°C and were superfused at 1 ml/min with oxygenated Krebs’ buffer containing pargyline (10 μM), a monoamine oxidase (MAO) inhibitor, and nomifensine (10 μM), a DA uptake inhibitor, to ensure that [3H]overflow primarily represented [3H]DA rather than [3H]DA metabolites (Cubeddu et al., 1979; Zumstein et al., 1981;Rapier et al., 1988). After 60 min of superfusion, two 5-min samples (5 ml) were collected to determine basal [3H]outflow.
For the nicotine or lobeline concentration-response studies, a single concentration of either nicotine (0.001–100 μM) or lobeline (0.01–100 μM) was added to the superfusion buffer of individual chambers after collection of the second basal sample, and the drug remained in the buffer for 60 min or until the end of experiment. Each chamber was exposed to only one concentration of nicotine or lobeline. The concentration-response relationship for each drug was determined using a repeated-measures design. In each experiment, one slice from the same rat was superfused in the absence of drug and served as control. To determine the calcium dependence of the effect of lobeline, concentration-response experiments were performed as described above; however, slices were superfused in the absence of CaCl2, and 0.5 mM EGTA was added to the superfusion buffer.
To determine the ability of mecamylamine to antagonize nicotine-evoked [3H]overflow, a repeated-measures design was also utilized. Individual slices were superfused with a single concentration (0.01–100 μM) of mecamylamine for 60 min, followed by 60 min of superfusion with nicotine (10 μM) in the presence of the various mecamylamine concentrations. One slice in each experiment was superfused in the absence of mecamylamine to determine the effect of nicotine alone. A control slice was superfused with buffer alone. To determine the ability of mecamylamine to antagonize lobeline-evoked [3H]overflow, a between-groups design was utilized. Slices were superfused for 60 min in the absence or presence of different concentrations (1–100 μM) of mecamylamine, a between-groups factor, followed by superfusion for 60 min with a range of concentrations (0.1–100 μM) of lobeline, a within-group factor.
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 counting (Packard model B1600 TR Scintillation Counter) with an efficiency of 59%.
Fractional release for each superfusate sample 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 was expressed as a percentage of total tritium in the tissue at the time of sample collection. Basal outflow was calculated from the average of the tritium collected in the two 5-min samples just before the addition of drug. Nicotine- or lobeline-evoked [3H]overflow was calculated by summing the increases in collected tritium that resulted from exposure to drug and subtracting the basal outflow for the equivalent period of drug exposure.
Endogenous DA release assay.
The effect of lobeline (0.1–100 μM) on endogenous DA and DOPAC overflow from striatal slices superfused in the absence of pargyline and nomifensine was determined by using a previously published method (Gerhardt et al., 1989) with minor modification. Slices were obtained and incubated for 30 min, followed by a second incubation for 30 min with fresh buffer containing 0.1 μM DA. Slices were then transferred to glass superfusion chambers and were superfused for 60 min with Krebs’ buffer. Two 1-min samples (1 ml) were collected to determine basal DA and DOPAC outflow. Subsequently, lobeline was added to the buffer and superfusate collected (15 consecutive 1-min samples followed by nine 1-min samples obtained every 5 min). At the end of the experiment, slice weight was determined, to normalize the amount of DA or DOPAC in superfusate per milligram wet slice weight. Tissue and superfusate samples were stored at −70°C for the determination of DA and DOPAC.
The concentration of endogenous DA and DOPAC in superfusate was determined via HPLC-EC. Superfusate samples were thawed, and ascorbic acid oxidase (2 μg in 10 μl) was added to 500 μl of superfusate. Then 50 μl of the resulting solution was injected onto the HPLC-EC system, which consisted of a Beckman Model 116 HPLC pump (Beckman, Fullerton, CA), a Beckman Model 504 autosampler, an ESA ODS ultrasphere C18 reverse-phase column (4.6 cm × 75 mm, 3-μM particle size; ESA, Bedford, MA) and an ESA 5100A coulometric electrochemical detector with a model 5011 detector cell (E1 = +0.05 V, E2 = +0.32 V). The eluent was 0.07 M citrate/0.1 M acetate buffer (pH 4) containing 50 mg/l disodium EDTA, 100 mg/l octylsulfonic acid–sodium salt and 7% methanol. All separations were performed at room temperature at a flow rate of 2 ml/min. Complete separation of DA and DOPAC and re-equilibration of the system required 5 min. Retention times of DA and DOPAC standards were used to identify relevant peaks. Peak heights were used to calculate detected amounts on the basis of standard curves. The detection limits were 1 and 2 pg/50 μl injected for DOPAC and DA, respectively.
Striatal DA and DOPAC content assay.
Striatal slices from superfusion experiments were assayed for DA and DOPAC content by a modification of a previously described method (Dubocovich and Zahniser, 1985). An aliquot (80 μl) of 0.1 M perchloric acid (pH 1.0) containing 0.29 μM DHBA (internal standard) was added to each striatal slice, and the mixture was sonicated with an Ultrasonic Processor (40-Watt Model, Sonics & Materials, Danbury, CT). The homogenate was centrifuged at 30,000 × g for 10 min at 4°C, and the supernatant was filtered (0.2-μm nylon membrane). An aliquot (50 μl) of the filtrate (1:1, 1:50, 1:100, 1:200 or 1:500 dilution with 0.1 M perchloric acid) was injected onto the above HPLC-EC system. The eluent was 6% acetonitrile, 10 μM EDTA, 1.4 mM 1-octane-sulfonic acid and 76 mM monobasic sodium phosphate (pH 3.1). All separations were performed at room temperature at a flow rate of 1 ml/min. Complete separation of DA and DOPAC and re-equilibration of the system required 9 min. The detection limits of DA and DOPAC were 0.2 and 0.05 pg/50 μl injected, respectively. Recovery of internal standard was routinely 75%.
[3H]DA uptake assay, striatal synaptosomal preparation.
The uptake of [3H]DA into striatal synaptosomes was determined by using a previously published method (Masserano et al., 1994) with minor modification. Striata from an individual rat were homogenized in 20 ml of cold 0.32 M sucrose containing 5 mM NaHCO3 (pH 7.4) with 16 up and down strokes of a Teflon pestle homogenizer (clearance approximately 0.003 inch). The homogenate was centrifuged at 2000 × g for 10 min at 4°C. The supernatant was centrifuged at 20,000 ×g for 15 min at 4°C. The pellet was resuspended in 2 ml of assay buffer (in mM: 125 NaCl, 5 KCl, 1.5 MgSO4, 1.25 CaCl2, 1.5 KH2PO4, 10 α-d-glucose, 25 HEPES, 0.1 EDTA, 0.1 pargyline and 0.1 ascorbic acid and saturated with 95% O2/5% CO2, pH 7.4). The final protein concentration was 400 μg/ml. The assay was performed in duplicate in a total volume of 500 μl. Aliquots (50 μl containing 20 μg of protein) were incubated with 50 μl of nicotine (final concentration, 0.001 nM–100 μM) or lobeline (final concentration, 0.01–1000 μM) in a metabolic shaker at 34°C for 10 min. Subsequently, a final DA ([3H]DA/unlabeled DA) concentration of 0.32 μM was added to each tube in a total volume of 66 μl, consisting of 16 μl of 0.61 μM [3H]DA and 50 μl of 3 μM DA. The incubation was continued for 10 min at 34°C. The reaction was terminated by the addition of 3 ml of ice-cold assay buffer. Samples were rapidly filtered through a Whatman GF/B filter using a Brandel cell harvester (model MP-43RS, Biochemical Research and Development Laboratories Inc., Gaithersburg, MD), and the filter was subsequently washed 3 times with 4 ml of ice-cold assay buffer containing 1 mM catechol. Filters had previously been soaked for 2 hr in the ice-cold assay buffer containing 1 mM catechol. Nonspecific uptake was determined in duplicate samples in the presence of 10 μM GBR 12909. Filters and 10 ml of scintillation cocktail were placed into scintillation vials, and radioactivity was determined by scintillation spectrometry.
[3H]DA uptake, striatal synaptic vesicle preparation.
The uptake of [3H]DA into striatal synaptic vesicles was determined by using a previously published method (Erickson et al., 1990). Striata from three rats (500 mg) were pooled and homogenized over a 2-min period in 14 ml of 0.32 M sucrose (pH 7.5) with 10 up and down strokes of a Teflon pestle (clearance approximately 0.009 inch). The homogenate was then centrifuged at 2000 × g for 10 min at 4°C, and the resulting supernatant was centrifuged at 10,000 × gfor 30 min at 4°C. Synaptosomes (buffy coat) were separated from the underlying mitochondria and cellular debris (reddish pellet) by gentle swirling in 2 ml of 0.32 M sucrose. The enriched synaptosome fraction (2.0 ml) was subjected to osmotic shock by the addition of 7 ml of distilled H2O and was homogenized with 5 up and down strokes of the Teflon pestle. The osmolarity was restored by the addition of 900 μl of 0.25 M HEPES and 900 μl of 1.0 M neutral potassium-tartrate buffer (pH 7.5) followed by a 20-min centrifugation (20,000 × g at 4°C). The supernatant was next centrifuged for 60 min (55,000 × g at 4°C). Then 1 ml of solution containing 10 mM MgSO4, 0.25 M HEPES and 1.0 M potassium-tartrate buffer was added to the supernatant, and the suspension was centrifuged (100,000 × g for 45 min at 4°C). Immediately before use, the final pellet was resuspended in the assay buffer (in mM: 25 HEPES, 100 potassium tartrate, 0.05 EGTA, 0.10 EDTA, 2 ATP-Mg++ and 1.7 ascorbic acid, pH 7.4). Aliquots (160 μl containing 8–10 μg protein) of the resuspension were incubated with 20 μl of drug (final concentrations: nicotine, 0.001–1000 μM; lobeline, 0.001–100 μM; tetrabenazine, 0.001–100 μM) and 20 μl of [3H]DA (final concentration 0.3 μM) for 8 min at 37°C in a total volume of 200 μl. The reaction was terminated by the addition of 2.5 ml of ice-cold assay buffer containing 2 mM MgSO4. Samples were rapidly filtered through Whatman GF/F filters using the Brandel cell harvester. The filters were then washed three times with 4 ml of ice-cold assay buffer containing 2 mM MgSO4. Filters had previously been soaked in 0.5% polyethylenimine (PEI) solution for 2 hr at 4°C. Nonspecific uptake was determined by incubation of duplicate samples at 0°C in the absence of drug. Filters were placed into scintillation vials, 10 ml of scintillation cocktail was added, and radioactivity was determined by scintillation spectrometry.
EM.
To confirm the purity of the isolated synaptic vesicles, vesicle pellets from rat striata were processed for EM. The pellet was fixed for 2 hr with 3.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.3). After a brief rinse in phosphate buffer, the pellet was postfixed for 2 hr in 1% osmium tetroxide in phosphate buffer. The pellet was then dehydrated five times in graded ethanol (50%, 70%, 80%, 90% and 100%) and embedded in Eponate 12 resin. Ultrathin (60–80-nm) sections were cut on an Ultracut E microtome (Reichert-Jung Inc., Wein, Austria) and collected on copper grids. The sections were then stained with saturated uranyl acetate in 70% ethanol and 0.04 M lead citrate. The grids were viewed with a Hitachi H-7000 transmission electron microscope (Hitachi, Tokyo, Japan).
Statistics.
Repeated-measures, one-way ANOVA was performed to analyze the results of the following experiments: the concentration effect of nicotine or lobeline on [3H]overflow, the concentration effect of lobeline on endogenous DA and DOPAC overflow, the ability of mecamylamine to antagonize nicotine (10 μM)-evoked [3H]overflow and the effect of lobeline on DA and DOPAC content in previously superfused striatal slices. Two-way ANOVAs were used to analyze the concentration effect of lobeline or nicotine on the time course of fractional [3H] release, the concentration effect of lobeline on the time course of endogenous DOPAC overflow, calcium dependence of lobeline-evoked [3H]overflow and the ability of mecamylamine to antagonize lobeline-evoked [3H]overflow. Inhibition of synaptosomal and vesicular [3H]DA uptake was analyzed by repeated-measures, one-way ANOVA and by an iterative nonlinear least-squares curve-fitting program (GraphPAD-PRIZM; GraphPAD, San Diego, CA) to obtain IC50values. Dunnett’s post-hoc test was used to compare treatment means to a single control mean. Also, Duncan’s New Multiple Range test or Fisher’s LSD post-hoc analysis was used to compare pairs of treatment means. Duncan’s New Multiple Range test was used when significant one-way ANOVAs were obtained or when significant main effects were obtained in the two-way ANOVAs. Fisher’s LSDpost-hoc analysis is a more conservative test, which takes into account error that cumulates during multiple comparisons of pairs of means. Fisher’s LSD analysis was used when the interaction term was significant in the two-way ANOVAs, specifically in thepost-hoc analysis of Drug × Time interactions. Statistical significance was reached when P < .05 (two-tailed, unless otherwise indicated).
Results
Effect of nicotine on superfused rat striatal slices preloaded with [3H]DA.
In a concentration-dependent manner, nicotine evoked an increase in the fractional release of tritium over the time course of the superfusion experiment (fig. 2, top panel). Repeated-measures, two-way ANOVA revealed a significant main effect of nicotine concentration (F (8,429) = 29.45, P < .0001) and a significant main effect of time (F (10,429) = 9.76, P < .0001), but the Concentration × Time interaction was not significant (F (80,429) = 1.22, P > .05). Fractional release peaked within 10 to 15 min after the addition of nicotine to the superfusion buffer. From 10 to 25 min after the addition of nicotine, fractional release was significantly increased above basal outflow, when the data were collapsed across nicotine concentration. At peak fractional release, the highest concentration of nicotine examined increased fractional release 2-fold above basal. Furthermore, when the data were collapsed across nicotine concentration, fractional release, from 30 to 45 min after nicotine addition, was not significantly different from basal, despite the presence of nicotine in the superfusion buffer throughout the superfusion period.
Presentation of the results as nicotine-evoked total [3H]overflow accentuates the concentration-dependent nature of the response to nicotine (fig. 2, bottom panel). Repeated-measures, one-way ANOVA revealed a significant nicotine-concentration effect (F (8,39) = 25.77, P < .0001). The lowest nicotine concentration that evoked a significant increase in [3H]overflow was 0.05 μM. A plateau in the concentration-response curve was not apparent over the concentration range examined. Higher concentrations of nicotine were not examined because of the extensive work of Westfall and collaborators (Westfall, 1974; Westfall et al., 1987) indicating that nicotine concentrations higher than 100 μM act to release DA from superfused rat striatal slices by a mechanism that is neither calcium-dependent nor nicotinic receptor-mediated.
Effect of lobeline on superfused rat striatal slices preloaded with [3H]DA.
Lobeline evoked a marked concentration-dependent increase in fractional release of tritium over the time course of the superfusion experiment (fig. 3). Repeated-measures, two-way ANOVA revealed a significant main effect of lobeline concentration (F (7,363) = 1057.13, P < .0001), a significant main effect of time (F (10,363) = 132.24, P < .0001) and a significant Concentration × Time interaction (F (70,363) = 44.85, P < .0001). Low concentrations (0.01–1 μM) of lobeline did not significantly increase fractional release during the entire superfusion period. Lobeline (3 μM) evoked a significant increase in fractional release 15 and 20 min after its addition to the buffer. Subsequently, fractional release returned toward basal, despite the continuous presence of lobeline in the buffer. Fractional release evoked by high concentrations (10–100 μM) of lobeline was significantly increased 10 min after the addition of lobeline to the buffer and remained significantly higher than basal until the end of the experiment.
Of note is the magnitude of the response to lobeline in comparison with that observed after superfusion with nicotine. Peak fractional release after superfusion with 30 and 100 μM lobeline was approximately 15% and 30%, respectively, of the total tritium present in the striatal slice (fig. 3, bottom panel). Furthermore, over the remainder of the superfusion period, fractional release in superfusate samples continued to be 10% to 20% of the total tritium in the slice. On the other hand, peak fractional release induced by the highest concentration (100 μM) of nicotine was only 2% of total tritium in the slice, and fractional release returned to basal during the course of the experiment (fig. 2, top panel). These results suggest the potential for depletion of DA storage pools after superfusion with lobeline at high concentrations.
Expression of the results as total [3H]overflow also revealed a concentration-dependent effect of lobeline and a marked increase in [3H]overflow evoked by high concentrations of lobeline (fig. 4). Repeated-measures, one-way ANOVA revealed a significant lobeline concentration effect (F (6,35) = 61.55, P < .0001). The lowest concentration of lobeline to evoke a significant increase in total [3H]overflow was 1 μM. As the lobeline concentration was increased, a significantly greater total [3H]overflow was evoked. Furthermore, a plateau in the concentration-response curve was not apparent over the concentration range examined.
The marked increase in fractional release and [3H]overflow in response to superfusion with high concentrations of lobeline (figs. 3 and 4) suggests that superfusion with lobeline may deplete striatal DA content. One-way ANOVA revealed a significant lobeline concentration effect on DA content (F (6,41) = 15.35, P < .0001) and DOPAC content (F (6,40) = 6.90, P < .0001). Superfusion with low concentrations (0.1–10 μM) of lobeline did not alter DA or DOPAC content (data not shown). When slices were superfused with high lobeline concentrations (30–100 μM), lobeline significantly depleted (42%–67%) DA content and increased (2-fold) DOPAC content compared with control (fig. 5).
Lobeline-induced [3H]overflow: lack of calcium dependence.
Previous studies (Westfall, 1974; Westfall et al., 1987) reported that nicotine (< 100 μM)-evoked [3H]overflow from rat striatal slices preloaded with [3H]DA was calcium-dependent. To determine whether lobeline-induced [3H]overflow was calcium-dependent, we determined the effect of lobeline in a calcium-free superfusion buffer containing 0.5 mM EGTA (table 1). Two-way ANOVA revealed a significant main effect of lobeline concentration (within-group factor, F (3,39) = 473.08, P < .001); however, the main effect of the inclusion of calcium in the buffer was not significant (between-groups factor, F (1,39)= 0.13, P > .05), and the interaction term also was not significant (F (3,39) = 1.64, P > .05). Thus the effect of lobeline on [3H]overflow was not altered after removal of calcium from the superfusion buffer.
Nicotine-evoked and lobeline-evoked [3H]overflow: mecamylamine antagonism.
In a concentration-dependent manner, mecamylamine significantly inhibited nicotine (10 μM)-evoked [3H]overflow from rat striatal slices preloaded with [3H]DA (table 2). Repeated-measures, one-way ANOVA revealed a significant mecamylamine concentration effect (F (5,38) = 4.46, P < .005). Concentrations of mecamylamine from 0.1 to 100 μM inhibited (57%–91%) the effect of nicotine in evoking [3H]overflow.
The time course of the effect of mecamylamine illustrates the pattern and extent of the inhibition of the nicotine-evoked increase in fractional release (fig. 6). Repeated-measures, two-way ANOVA revealed a significant main effect of mecamylamine concentration (F (6,599) = 19.59, P < .0001) and a significant main effect of time (F (11,599) = 4.98, P < .0001), but the Concentration × Time interaction was not significant (F (66,599) = 0.97, P > .05). When the data were collapsed across time, the lowest concentration of mecamylamine to produce a significant inhibition of nicotine’s effect was 0.01 μM. The time course illustrates the small but significant inhibition (36%) of nicotine’s effect produced by this low concentration of mecamylamine (fig. 6). Interestingly, the inhibitory effect of 0.01 μM mecamylamine was not detected when the results were expressed as total [3H]overflow (table 2). The maximal inhibitory effect of the highest concentration (100 μM) of mecamylamine is also illustrated in figure 6 for comparison.
The ability of mecamylamine (1–100 μM) to inhibit lobeline (0.1–100 μM)-evoked total [3H]overflow is shown in table3. Concentrations of mecamylamine that significantly inhibited nicotine-evoked [3H]overflow were utilized in this experiment. The effect of lobeline (0.1–100 μM) in the absence of mecamylamine represented control. Two-way ANOVA revealed a significant main effect of lobeline concentration (within-group factor,F (4,56) = 603.84, P < .0001); however, neither the main effect of mecamylamine concentration (between-groups factor, F (3,14) = 2.79, P > .05) nor the Lobeline × Mecamylamine interaction was significant (F (12,56) = 1.30, P > .05). Thus lobeline-evoked [3H]overflow was not inhibited by mecamylamine.
Effect of nicotine and lobeline on [3H]DA uptake into rat striatal synaptosomes and synaptic vesicles.
To determine whether modulation of DA uptake contributed to the increase in [3H]overflow evoked by nicotine or lobeline, we determined [3H]DA uptake into striatal synaptosomes and synaptic vesicles (fig. 7). Nicotine did not inhibit [3H]DA uptake into striatal synaptosomes over the concentration range (0.001 nM–100 μM) examined. Before determining the effect of nicotine on synaptic vesicular [3H]DA uptake, we determined the purity of the isolated synaptic vesicle preparation by electron microscopy of representative vesicle preparations (fig. 8). Plain spheroid or ellipsoid synaptic vesicle profiles of approximately 50 nm in diameter were the predominant membrane structures observed. Very few (≤ 1%) contaminating membrane fragments were present. The effect of nicotine on [3H]DA uptake into synaptic vesicles was analyzed by repeated-measures, one-way ANOVA, which revealed a significant nicotine concentration effect (F (9,28) = 3.30, P < .05). However, Dunnett’s post-hoc analysis revealed that significant inhibition of uptake occurred only at a very high concentration (1 mM) of nicotine (fig. 7).
Lobeline inhibited [3H]DA uptake into synaptosomes in a concentration-dependent manner (fig. 7). Repeated-measures, one-way ANOVA revealed a significant lobeline concentration effect (F (9,38) = 154.0, P < .0001). The lowest concentration of lobeline to produce a significant inhibition in the synaptosomal preparation was 30 μM. The IC50 value for lobeline to inhibit uptake into synaptosomes was 80 ± 12 μM. Moreover, in contrast to nicotine, lobeline potently inhibited [3H]DA uptake into synaptic vesicles in a concentration-dependent manner (F (8,26) = 28.60, P < .0001). The lowest concentration of lobeline to produce a significant inhibition in the synaptic vesicle preparation was 0.3 μM, and complete inhibition was obtained at 10 μM. The IC50 value for lobeline to inhibit vesicular uptake was 0.88 ± 0.001 μM, which was two orders of magnitude lower than that for lobeline-induced inhibition of synaptosomal [3H]DA uptake. Tetrabenazine (0.001–100 μM), a specific high-affinity inhibitor of the vesicular monoamine transporter, significantly inhibited striatal vesicular [3H]DA uptake in a concentration-dependent manner (F (9,28) = 23.78, P < .0001). The IC50 value for tetrabenazine to inhibit vesicular uptake was 77.7 ± 1.3 nM. Complete inhibition was obtained at 1 μM tetrabenazine. Thus lobeline was approximately one order of magnitude less potent than tetrabenazine in inhibiting vesicular [3H]DA uptake.
Effect of lobeline on DA and DOPAC overflow from superfused striatal slices.
To determine whether the increased cytosolic DA induced by lobeline was metabolized by MAO to DOPAC or the cytosolic DA escaped metabolism and was transported into the extracellular space, we determined the effect of lobeline on DA and DOPAC overflow in slices superfused in the absence of pargyline and nomifensine. At concentrations of 0.1 to 30 μM, lobeline did not increase DA overflow. At a high concentration of 100 μM, lobeline significantly increased DA overflow (1573 ± 979 pg/mg/60 min) compared with control (0 ± 0 pg/mg/60 min). However, a concentration effect of lobeline on DOPAC overflow from striatal slices was observed (fig. 9, top panel). Repeated-measures, one-way ANOVA revealed a significant concentration effect (F (5,33) = 88.59, P < .0001). The lowest concentration to evoke a significant increase in total DOPAC overflow was 1 μM, which similarly was the lowest concentration of lobeline to evoke a significant increase in [3H]overflow. A plateau in the concentration-response curve was not apparent over the concentration range examined (fig. 9, top panel).
Additionally, the pattern of the time course of lobeline-evoked DOPAC overflow (fig. 9, bottom panel) closely resembles that of the time course of lobeline-evoked fractional [3H] release (fig.3). Repeated-measures two-way ANOVA revealed a significant main effect of concentration (F (5,831) = 45.18, P < .0001), a significant main effect of time (F (25,831) = 669.3, P < .0001) and a significant Concentration × Time interaction (F (125,831) = 17.3, P < .0001). Low concentrations (0.1–1 μM) of lobeline did not significantly increase DOPAC overflow during the entire superfusion period. For lobeline concentrations of 10, 30 and 100 μM, DOPAC overflow was significantly increased after 10, 8, and 7 min, respectively, of superfusion with drug. Moreover, DOPAC overflow remained significantly higher than basal until the end of the experiment.
Repeated-measures, one-way ANOVA revealed a significant lobeline concentration effect on content of DA in superfused striatal slices (F (5,35) = 8.08, P < .0001). When slices were superfused with high concentrations (10–100 μM), lobeline significantly depleted DA content by 66% to 85% (fig.10). Thus lobeline depleted DA content in slices superfused either in the absence or in the presence of pargyline and nomifensine (figs. 10 and 5, respectively). However, a lower concentration (10 μM) of lobeline decreased DA content significantly more in the absence of the MAO inhibitor and the DA uptake inhibitor than in their presence. In contrast, lobeline did not significantly alter DOPAC content (repeated-measures, one-way ANOVA,F (5,35) = 1.11, P > .05). As expected, DOPAC content in control slices superfused in the absence of pargyline and nomifensine was greater (∼100-fold) than in control slices superfused with pargyline and nomifensine.
Discussion
Results from the present study demonstrate that, like nicotine, lobeline evokes [3H]overflow from rat striatal slices preloaded with [3H]DA in a concentration-dependent manner. However, in contrast to results with nicotine, lobeline-evoked [3H]overflow is calcium-independent and mecamylamine-insensitive. Although lobeline is thought to be a nicotinic agonist, the present results suggest that lobeline acts to evoke [3H]overflow from [3H]DA-preloaded striatal slices via a mechanism other than stimulation of nicotinic receptors. Moreover, in contrast to nicotine, lobeline potently inhibits striatal synaptosomal and vesicular [3H]DA uptake, leading to an increased concentration of cytosolic DA. Subsequently, the cytosolic DA is metabolized to DOPAC by MAO, and the DOPAC emerges from the presynaptic terminal, as indicated by the observed concentration-dependent increase in DOPAC overflow. Thus lobeline-induced inhibition of DA uptake and alteration of intracellular DA storage may contribute to the mechanism responsible for the lobeline-evoked increase in [3H]overflow from [3H]DA-preloaded striatal slices.
In agreement with reports of others, we found that nicotine evoked [3H]overflow from superfused rat striatal slices (Westfall, 1974; Giorguieff-Chesselet et al., 1979; Westfallet al., 1987; Harsing et al., 1992; Sacaanet al., 1995) and from rat or mouse striatal synaptosomes (Chesselet, 1984; Rowell et al., 1987; Rapier et al., 1988, 1990; Grady et al., 1992; Rowell and Hillebrand, 1992, 1994; Rowell, 1995) preloaded with [3H]DA. The nicotine concentration range (0.001–100 μM) chosen for the present study was based on extensive research demonstrating that at low concentrations (< 100 μM), the effect of nicotine was calcium-dependent and was antagonized by mecamylamine (i.e., nicotinic receptor-mediated), whereas at high concentrations (> 100 μM), a calcium-independent effect not antagonized by mecamylamine was observed (Giorguieff-Chesselet et al., 1979; Westfall et al., 1987; Rapier et al., 1988, 1990; Grady et al., 1992).
In previous studies utilizing the slice superfusion assay, nicotine was superfused for only short periods of time (3–10 min) (Giorguieff-Chesselet et al., 1979; Westfall et al., 1987; Harsing et al., 1992; Sacaan et al., 1995). Only one of these reports (Giorguieff-Chesseletet al., 1979) provided the time course of the effect of nicotine (1 μM), and in that study, [3H]- overflow remained elevated for the entire 10-min period of nicotine exposure. The present study illustrates a complete time course of exposure (over a 60-min superfusion period) to a low range of nicotine concentrations (0.01–100 μM), and it illustrates the time course and pattern of mecamylamine-induced inhibition of the effect of nicotine, indicative of nicotinic-receptor mediation. The time course illustrates that the peak effect of nicotine was reached within 10 to 15 min after the start of superfusion with drug. Despite continued superfusion with nicotine, the response returned to basal levels within 25 min, a result indicative of receptor desensitization. The present findings are of particular interest because in human smokers, a persistent nicotine blood level (0.1–1 μM) has been observed during the waking hours of each day (Benowitz et al., 1990).
Like nicotine, lobeline evoked [3H]overflow from [3H]DA-preloaded striatal slices in a concentration-dependent manner. However, as illustrated by the time course (fig. 3) and the concentration-response curve (fig. 4), the pattern and the magnitude of the effect of lobeline were different from nicotine. The peak effect occurred 10 to 20 min after the start of lobeline exposure, and, at least at low concentrations, the response returned to basal levels despite continued superfusion with lobeline. However, the response remained significantly above basal levels during superfusion with the higher lobeline concentrations. Moreover, the effect of lobeline on [3H]overflow was markedly increased (8–34-fold) compared with the effect of nicotine, particularly at the higher concentrations (10–100 μM) examined. Additionally, a depletion of striatal DA content was observed in the slices superfused with these high concentrations of lobeline, a result indicative of toxicity at least in vitro. Furthermore, in contrast to nicotine, the effect of lobeline was found in the present study to be calcium-independent and not to be inhibited by mecamylamine. Thus, despite the reported high affinity of lobeline for the [3H]nicotine binding site (see the Introduction), lobeline evokes [3H]overflow from rat striatal slices preloaded with [3H]DA by a mechanism other than stimulation of nicotinic receptors.
The present results further demonstrate that, in contrast to nicotine, lobeline potently inhibits [3H]DA uptake into striatal synaptosomes and vesicles. Significant inhibition of [3H]DA uptake into synaptic vesicles was observed at a low concentration of 0.3 μM lobeline, and the IC50 value for this effect was 0.88 μM. Additionally, at higher concentrations (≥ 30 μM), [3H]DA uptake into striatal synaptosomes was also significantly inhibited. The IC50 value for lobeline-induced inhibition of synaptosomal uptake was 80 μM,i.e., two orders of magnitude higher than that for inhibition of uptake into synaptic vesicles. The present results from the synaptosomal assay are in good agreement with a previous report of lobeline-induced inhibition of [3H]DA uptake into mouse striatal synaptosomes (Debler et al., 1988).
In the present study, nicotine inhibited vesicular [3H]DA uptake only at a very high concentration (∼ 1 mM), and no inhibition of synaptosomal [3H]DA uptake was observed. The failure of nicotine to inhibit DA uptake into striatal synaptosomes is in agreement with previous reports (Kramer et al., 1989;Izenwasser et al., 1991; Rowell and Hill, 1993). In the striatal chopped preparation, nicotine at very low concentrations (≥ 10 pM) has been reported to inhibit [3H]DA uptake by an indirect mechanism (Izenwasser et al., 1991); however, other investigators using the more intact striatal slice preparation were unable to observe any nicotine-induced inhibition of [3H]DA uptake (Rowell and Hill, 1993). Interestingly, [3H]DA uptake into β-NGF-treated PC12 cells transfected with the rat DA transporter cDNA was inhibited by nicotine (IC50 = 8 μM), and mecamylamine blocked nicotine’s effect (Yamashita et al., 1995), which suggests that nicotinic receptors may modulate DA uptake in these cells. More recently, nicotine (0.4 mg/kg) administered s.c. to rats was observed to increase the clearance of exogenously applied DA in an in vivo voltammetric study (Hart and Ksir, 1996), which suggests that nicotine induced an enhancement of DA clearance in striatum in vivo. Because nomifensine, a DA uptake inhibitor, was included in the superfusion buffer in the present study, the nicotine-evoked increase in [3H]overflow probably reflects a nicotine-induced increase in the release of DA rather than an inhibition of the DA transporter. Nevertheless, an indirect mechanism of action for nicotine at some other site, which modulates DA transport, as suggested by Izenwasser et al., (1991), cannot be ruled out by the results of the present study. Moreover, the results of the present study indicate that the synaptic vesicular DA transporter is significantly more sensitive to lobeline-induced inhibition than the plasma membrane DA transporter and that neither transport process is modulated to any great extent by nicotine. Because these two transporters are structurally and functionally different (see review, Brownstein and Hoffman, 1994), it is not surprising that they are differentially sensitive to inhibition by lobeline.
The lobeline-induced increase in DA concentration in the extracellular space (as reflected by an increase in [3H]overflow in superfusate in the presence of pargyline and nomifensine in the [3H]DA release assay) is consistent with the observed lobeline-induced inhibition of vesicular and synaptosomal [3H]DA uptake. Notably, the lowest concentration of lobeline to significantly evoke [3H]overflow in the superfusion assay was 1 μM, which is within the range of concentrations (0.3–100 μM) observed specifically to inhibit vesicular [3H]DA uptake. Higher concentrations (> 30 μM) of lobeline were required to detect the inhibition of synaptosomal [3H]DA uptake. The observation that the lobeline-induced [3H]overflow is not calcium-dependent suggests that the released DA originated from cytosolic rather than vesicular pools. Because lobeline is a very lipophilic compound (Barlow and Johnson, 1989; Reavill et al., 1990; Bhat et al., 1991), it could easily gain access to the vesicular transporter by passive entrance into the neuron and its vesicles. Lobeline-induced inhibition of vesicular DA uptake could occurvia two mechanisms: dissipation of the vesicle membrane proton gradient and/or interaction with a substrate site on the vesicular transporter. Because lobeline is a weak base, and as a result of the lower pH inside the vesicle, lobeline could accumulate in synaptic vesicles in its charged form (i.e., protonated). Once lobeline exceeded the buffering capacity within the vesicle, the vesicular pH gradient would be attenuated, with a resulting decrease in the energy available for DA uptake (Beers et al., 1986;Johnson, 1988). Subsequently, uncharged DA would diffuse out of the vesicles in accordance with the concentration gradient, such that DA concentrations in the cytosol would increase. Elevation of cytosolic DA would promote reverse transport and DA release from the presynaptic terminal into the extracellular space. The observation that high concentrations (30–100 μM) of lobeline markedly increased DA release under the conditions of the present study was further supported by the observed depletion of DA in the superfused striatal slices. Furthermore, neurotoxicity may result from the increased cytosolic DA, which could undergo auto-oxidation and enzymatic oxidative metabolism, leading to the increased formation of DOPAC, hydrogen peroxide, free radicals and active quinones (Graham et al., 1978; Slivka and Cohen, 1985). Thus the present results suggest that lobeline acts to redistribute intracellular DA pools within the presynaptic terminal, resulting in potential neurotoxicity.
To determine whether the lobeline-induced increase in cytosolic DA was metabolized by MAO to DOPAC or the DA escaped metabolism and was transported into the extracellular space, we determined the effect of lobeline on DA and DOPAC overflow in slices superfused in the absence of pargyline and nomifensine. Under these conditions, overflow of DA was detected only after superfusion with high concentrations (100 μM) of lobeline; however, lobeline (1–100 μM) produced a concentration-dependent, marked increase in DOPAC overflow in the absence of MAO inhibition. Thus the results suggest that lobeline exposure resulted in an increase in cytosolic DA, which was metabolized by MAO and detected as DOPAC in superfusate. The lowest concentration of lobeline to increase DOPAC overflow significantly was 1 μM, which is within the range of concentrations (0.3–100 μM) observed to inhibit vesicular DA uptake. The highest concentration (100 μM) examined probably increased the cytosolic DA concentration so much that it exceeded the capacity of MAO, and thus DA overflow was detected in superfusate. The increase in DA overflow in response to 100 μM lobeline may have come about via reversal of the synaptosomal DA transporter, because at this concentration of lobeline, [3H]DA uptake was inhibited by only 60%.
Furthermore, the observed decrease in striatal DA content in response to 10 to 100 μM lobeline is consistent with the marked increase in DOPAC overflow. A similar lobeline-induced depletion of striatal DA content was observed when slices were superfused in either the absence or the presence of pargyline and nomifensine. As expected, DOPAC content in control slices superfused in the absence of pargyline and nomifensine was greater (∼ 100-fold) than that in control slices superfused with pargyline and nomifensine in the buffer. Superimposed on this difference in striatal DOPAC content in control slices, high concentrations (30–100 μM) of lobeline increased DOPAC content in slices superfused with pargyline and nomifensine; however, no change in DOPAC content was observed when slices were superfused in the absence of the inhibitors. These results suggest that high concentrations of lobeline markedly increased cytosolic DA concentration, as reflected by an increase in DOPAC content.
The action of lobeline is reminiscent of that of amphetamine, a DA-releasing agent. Amphetamine is lipophilic, entering neurons by passive diffusion at moderate to high concentrations (Ross and Renyi, 1966; Fischer and Cho, 1979; Liang and Rutledge, 1982). At low concentrations, amphetamine enters neurons via the DA transporter, and as a result, DA is released into the extracellular space by carrier-mediated exchange diffusion (Fischer and Cho, 1979;Liang and Rutledge, 1982), a calcium-independent mechanism that is sensitive to DA uptake inhibitors (Hurd and Ungerstedt, 1989; Parker and Cubeddu, 1986a; Zaczek et al., 1991; Levi and Raiteri, 1993). Furthermore, amphetamine is a weak base that has been reported to interact with the vesicular substrate site (Schuldiner et al., 1993; Gonzalez et al., 1994), to enter synaptic vesicles and to dissipate the vesicular proton gradient, resulting in intracellular redistribution and subsequent release of neurotransmitter (Knepper et al., 1988; Sulzer and Rayport, 1990; Sulzeret al., 1995). In comparison with amphetamine, few studies have focused on the mechanism of action of lobeline; however, the present findings indicate many similarities in the action of these two drugs, even though lobeline has often been categorized as a nicotinic agonist (Decker et al., 1995).
On the other hand, differences between the action of lobeline and that of amphetamine are also apparent. First, amphetamine releases DA from presynaptic terminals, an action sensitive to DA transport inhibitors (Parker and Cubeddu, 1986a,b; Dwoskin et al., 1988). By contrast, as shown in the present study, lobeline releases DA per se only in the presence of MAO and DA transport inhibitors. Second, in the present study, lobeline was found to increase DOPAC overflow from rat striatal slices, whereas amphetamine has been reported to decrease DOPAC overflow under similar conditions (Parker and Cubeddu, 1986b; Dwoskin et al., 1988). One potential explanation for the latter difference may be that amphetamine, an excellent substrate for the synaptosomal DA transporter, rapidly depletes cytosolic DA pools, decreasing the concentration of DA available for intracellular metabolism by MAO. By contrast, lobeline potently inhibits vesicular DA uptake, increasing the cytosolic DA available for metabolism by MAO. Third, amphetamine has been reported to inhibit MAO activity (Mantle et al., 1976; Milleret al., 1980), whereas the results of the present study suggest that lobeline does not inhibit the activity of MAO. Thus the neurochemical effects of lobeline and amphetamine are similar, but not identical, and these differences may explain their differential effects in behavioral studies, e.g., on locomotor activity (Segal and Kuczenski, 1987; Stolerman et al., 1995).
In summary, in a concentration-dependent, calcium-independent and mecamylamine-insensitive manner, lobeline evoked [3H]overflow from rat striatal slices preloaded with [3H]DA. The lobeline-induced inhibition of vesicular DA transport and subsequent redistribution of presynaptic DA storage to increase cytosolic DA concentrations may be the mechanism by which lobeline acts. Clearly, lobeline acts by a mechanism different from that of nicotine, which may explain the reported differences between the behavioral effects of these two drugs and the differences in their ability to up-regulate nicotinic receptors after chronic administration. The use of lobeline as a long-term smoking cessation therapy should be carefully evaluated with respect to potential CNS neurotoxicity.
Acknowledgments
The authors thank Ms. Susan Buxton, Wendy Shaw and Mary Gale Engle for technical assistance and Mr. Larry Manzino for technical consultation.
Footnotes
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Send reprint requests to: Linda P. Dwoskin, Ph.D., College of Pharmacy, University of Kentucky, Rose Street, Lexington, Kentucky 40536-0082.
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↵1 This work was supported by a grant from the Tobacco and Health Research Institute, Lexington, Kentucky.
- Abbreviations:
- DA
- dopamine
- [3H]DA
- 3,4-ethyl-2-[N-3H]-dihydroxyphenylethylamine
- DHBA
- 3,4-dihydroxybenzylamine hydrobromide
- DHβE
- dihydro-β-erythroidine
- DOPAC
- dihydroxyphenylacetic acid
- EGTA
- ethylene glycol-bis(β-aminoethyl ether) N,N,N′,N′-tetraacetic acid
- EM
- electron microscopy
- GBR 12909
- 1-[2-[bis(4-fluorophenyl)methyl]ethyl]-4-[3-phenyl]piperazine dihydrochloride
- HPLC-EC
- high-pressure liquid chromatography with electrochemical detection
- MEC
- mecamylamine
- PEI
- polyethylenimine
- Received March 20, 1996.
- Accepted November 25, 1996.
- The American Society for Pharmacology and Experimental Therapeutics