Introduction

Top Abstract Introduction Materials & Methods Results Discussion References

Lobeline (-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; Olin et 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).

View larger version (10K): [in this window] [in a new window]   Fig. 1.   The structure of S()nicotine and of 2S, 6R, 8S()lobeline.

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 (Geller et al., 1971), most subsequent studies failed to replicate these original findings (Schechter and Rosecrans, 1972; Reavill et 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 acting via 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 (K_d = 1-7 nM) (Lippiello and Fernandes, 1986; Reavill et 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 (K_i = 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; Bhat et al., 1991, 1994; Marks et al., 1992; Sanderson et 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; Rowell et al., 1987; Rapier et al., 1988, 1990; Grady et 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

Top Abstract Introduction Materials & Methods Results Discussion References

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), -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 -D-glucose, 25 NaHCO₃, 0.11 L-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.

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.

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 -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 × g for 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 IC₅₀ values. 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 LSD post-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 the post-hoc analysis of Drug × Time interactions. Statistical significance was reached when P < .05 (two-tailed, unless otherwise indicated).

	Results

Top Abstract Introduction Materials & Methods Results Discussion References

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.

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Time course of nicotine-evoked fractional release (top panel) and concentration dependence of nicotine-evoked total [3H]overflow (bottom panel) from rat striatal slices preloaded with [3H]DA. Nicotine was added to the superfusion buffer after the second sample (as indicated by the arrow) and remained in the buffer until the end of the experiment. Pargyline (10 µM) and nomifensine (10 µM) were included in the superfusion buffer from the start of superfusion. Data in the top panel are presented as means ± S.E. fractional release, which represents the tritium in the sample as a percentage of the total tritium in the slice at the time of sample collection. Data in the bottom panel are presented as mean ± S.E. total [3H]overflow, which was calculated by summing the increases in collected tritium resulting from exposure to drug and subtracting the basal outflow for the equivalent period of drug exposure. § P < .05, different from basal (5-10 min), when fractional release was collapsed across nicotine concentration; * P < .05, different from 0 to 0.01 µM and 1 to 100 µM; ** P < .05, different from 0 to 0.1 µM and 100 µM; *** P < .05, different from 0 to 10 µM; Duncan's New Multiple Range test. n = 4 to 9 rats.

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.

View larger version (33K): [in this window] [in a new window]   Fig. 3.   Time course of lobeline-evoked fractional release from rat striatal slices preloaded with [3H]DA. Lobeline was added to the superfusion buffer after collection of the second sample (as indicated by the arrow) and remained in the buffer until the end of the experiment. Pargyline (10 µM) and nomifensine (10 µM) were included in the superfusion buffer from the start of superfusion. Data are presented as mean ± S.E. fractional release. The top panel illustrates the time course of the fractional release evoked by low concentrations (0.01-3 µM) of lobeline, and the bottom panel shows that evoked by high concentrations (3-100 µM). * P < .05, different from basal outflow;  P < .05, different from the peak responses at 25 min for 0.01 to 3 µM and 30 to 300 µM; § P < .05, different from the peak responses of 0.01 to 10 µM and 100 µM; # P < .05, different from the peak responses of 0.01 to 30 µM; Fisher's LSD post-hoc test. n = 6 rats.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Concentration dependence of lobeline-evoked total [3H]overflow from rat striatal slices preloaded with [3H]DA. Data are presented as mean ± S.E. total [3H]overflow. The inset illustrates total [3H] overflow evoked by the lower concentrations (0.01-1 µM) of lobeline. Control slices that were superfused with buffer in the absence of lobeline did not evoke [3H]overflow (i.e., fractional release was not different from basal during the course of superfusion). * P < .05, different from control and each of the other lobeline concentrations; Duncan's New Multiple Range test. n = 6 rats.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   DA and DOPAC content in rat striatal slices superfused with high concentrations (30-100 µM) of lobeline. DA and DOPAC content in control and lobeline-exposed slices were determined after superfusion with buffer containing pargyline (10 µM) and nomifensine (10 µM). Data are presented as mean ± S.E. ng/mg protein. * P < .05, different from control; ** P < .001, different from control; Dunnett's post-hoc test. n = 8 rats.

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.

View larger version (29K): [in this window] [in a new window]   Fig. 6.   Time course of the effect of mecamylamine to inhibit nicotine (10 µM)-evoked fractional release of tritium from [3H]DA-preloaded striatal slices. For clarity of graphical presentation, we show only significant effects of the lowest and highest concentrations (0.01 and 100 µM, respectively) of mecamylamine. Data are presented as mean ± S.E. fractional release as percentage of basal outflow. Experiments were performed as described in table 2. The time course begins at the time of nicotine (10 µM) addition to the superfusion buffer containing mecamylamine. The control represents fractional release in the absence of either mecamylamine or nicotine in the superfusion buffer. Duncan's New Multiple Range test revealed a significant inhibitory effect of 0.01 µM mecamylamine when the data were collapsed across time of superfusion. n = 8 rats.

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).

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Effects of nicotine (0.01-1000 µM) and lobeline (0.01-1000 µM) on rat striatal synaptosomal and synaptic vesicular [3H]DA uptake.  nicotine, synaptosomal [3H]DA uptake;  nicotine, vesicular [3H]DA uptake;  lobeline, synaptosomal [3H]DA uptake;  lobeline, vesicular [3H]DA uptake. Data are presented as mean ± S.E. percentage of total [3H]DA uptake. Total [3H]DA uptake for synaptosomes and vesicles was 109 ± 9.80 pmol/min/mg and 168 ± 8.96 pmol/min/mg, respectively. Nonspecific [3H]DA uptake in synaptosomal experiments as determined by incubation with 10 µM GBR 12909 and in vesicular experiments as determined by incubation at 0°C was 2% and 20% of total [3H]DA uptake, respectively. Experiments examining the effect of nicotine on synaptosomal uptake included a low concentration range (0.001-1 nM), but no effect was observed, and for clarity of graphical presentation, these results are not illustrated. * P < .05, different from total [3H]DA uptake; Dunnett's post-hoc test. n = 3 to 6 rats.

View larger version (180K): [in this window] [in a new window]   Fig. 8.   An electron micrograph of purified synaptic vesicles from rat striatum. Magnification of the micrograph was ×105.

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).

View larger version (25K): [in this window] [in a new window]   Fig. 9.   Concentration dependence of lobeline-evoked total DOPAC overflow from striatal slices (top panel) and the time course of effect (bottom panel). Data in the top panel are presented as mean ± S.E. total DOPAC overflow and are expressed as picograms of DOPAC released per milligram wet weight over the 60-min period of superfusion with lobeline. In the time course, data are expressed as mean ± S.E. picograms of DOPAC in superfusate per milligram wet weight per minute. Lobeline was added to the superfusion buffer after the second sample (as indicated by the arrow in the bottom panel) and remained in the buffer until the end of the experiment. These experiments were performed in the absence of pargyline and nomifensine in the superfusion buffer. Top panel: * P < .05, different from control and each of the other lobeline concentrations; Duncan's New Multiple Range test. Bottom panel: * P < .05, different from the corresponding basal values (1-2 min) for 10 to 100 µM;  different from the peak responses at 22 min for 0.01 to 1 µM and 30 to 100 µM; $ P < .05, different from the peak responses of 0.1 to 10 µM and 100 µM; # P < .05, different from the peak response of 0.1 to 30 µM; Fisher's LSD post-hoc test. n = 6 rats.

View larger version (19K): [in this window] [in a new window]   Fig. 10.   DA and DOPAC content in rat striatal slices after superfusion with lobeline (0.1-100 µM). DA content (top panel) and DOPAC content (bottom panel) in control and lobeline-exposed slices were determined after superfusion in the absence of pargyline and nomifensine. Data are presented as mean ± S.E. ng/mg protein. * P < .05, different from control; Dunnett's post-hoc test. n = 6 rats.

	Discussion

Top Abstract Introduction Materials & Methods Results Discussion References

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; Westfall et al., 1987; Harsing et al., 1992; Sacaan et 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-Chesselet et 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 -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 occur via 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; Sulzer et 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 sl