Introduction

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Cigarette smoking is the number one health problem accounting for more illness and deaths in the United States than any other factor (Surgeon General's Report, 1988). Dependence liability for the major tobacco alkaloid, nicotine, stems from nicotine's intrinsic reinforcing properties, suggested to be the result of activation of DA pathways in brain (Fibiger and Phillips, 1987; Corrigall et al., 1992, 1994; Balfour and Benwell, 1993). Nicotinic receptors are found in high density in cell body and terminal areas of the nigrostriatal DA pathway (Clarke et al., 1984; Clarke et al., 1985a; Reavill et al., 1988). A reduction in the number of nicotinic receptors was observed in striatum after 6-hydroxydopamine treatment, suggesting localization on DA presynaptic terminals in striatum (Schwartz et al., 1984; Clarke and Pert, 1985). Furthermore, systemic administration or iontophoretic application of nicotine stimulates cell firing of substantia nigra neurons in electrophysiological studies (Lichtensteiger et al., 1982; Clarke et al., 1985b).

Nicotine facilitates DA release from striatal nerve terminals in in vivo studies using microdialysis in striatum (Imperato et al., 1986; Toth et al., 1992), and in in vitro superfusion studies using striatal slices (Arqueros et al., 1978; Giorguieff-Chesselet et al., 1979; Westfall, 1974; Westfall et al., 1987; Izenwasser et al., 1991; Harsing et al., 1992; Schulz et al., 1993, Sacaan et al., 1995) and synaptosomes (Takano et al., 1983; Chesselet, 1984; Rowell et al., 1987; Rapier et al., 1988, 1990; Grady et al., 1992; Rowell and Hillebrand, 1994; El-Bizri and Clarke, 1994; Rowell, 1995). Concentrations (0.1-1 µM) of nicotine, which correspond to plasma levels in moderate smokers (Russell et al., 1980; Kogen et al., 1981; Benowitz et al., 1990; Henningfield et al., 1993), evoked DA release in the latter in vitro studies. Moreover, nicotine-evoked striatal DA release was Ca⁺⁺-dependent, stereoselective and inhibited by MEC or DHE (Westfall et al., 1987; Rapier et al., 1988, 1990; Grady et al., 1992; El-Bizri and Clarke, 1994; Sacaan et al., 1995). MEC is a centrally active, noncompetitive nicotinic receptor antagonist, which blocks the open ion channel of the nicotinic receptor more effectively than the closed channel (Varanda et al., 1985; Loiacono et al., 1993; Peng et al., 1994). DHE is a selective, competitive nicotinic receptor antagonist, which displaces nicotine from its binding site (Reavill et al., 1988; Grady et al., 1992), and inhibits nicotine's electrophysiological effects (Vidal and Changeux, 1989; Alkondon and Albuquerque, 1991; Mulle et al., 1991).

In contrast to the plethora of studies investigating the neuropharmacological effects of nicotine, few studies have investigated the effects of nornicotine, a major tobacco alkaloid (Bush et al., 1993) and minor peripheral nicotine metabolite (Kyerematen et al., 1988; Zhang et al., 1990; Kyerematen and Vesell, 1991; Curvall and Vala, 1993; Benowitz et al., 1994). Of note, both nornicotine enantiomers are present in tobacco products (Kisaki and Tamaki, 1961). Although nornicotine is a minor peripheral metabolite of nicotine in various animal species (Cundy and Crooks, 1984; Curvall and Vala, 1993), high levels of nornicotine have been found in rat brain after s.c. [2-¹⁴C]nicotine administration, suggesting local formation in brain via N-demethylation of nicotine (Crooks et al., 1995, 1997). Moreover, at 4 hr after peripheral nicotine administration, the nornicotine concentration in brain was nearly equal to that of nicotine (Crooks et al., 1997). Because nornicotine has a significantly longer plasma half life (7.2-8.5 hr) than does nicotine (0.9-1.4 hr) (Kyerematen et al., 1990), nornicotine may also have a longer CNS residence time compared to nicotine, and nornicotine may accumulate in brain after repeated nicotine administration.

The structure of nornicotine suggests that it may have significant nicotinic receptor agonist properties. Both nornicotine enantiomers displace [³H]S(-)nicotine binding from its high affinity sites in rat brain membranes (Reavill et al., 1988; Copeland et al., 1991; Zhang and Nordberg, 1993). S(-)Nornicotine evokes a concentration- and Ca⁺⁺-dependent increase in endogenous DA release from rat striatal slices (Dwoskin et al., 1993). Taken together, these results suggest that nornicotine acts at neuronal nicotinic receptors and contributes to the cental nervous system effects of nicotine and tobacco smoking.

The purpose of our study was to determine if nornicotine evokes [³H]overflow from rat striatal slices preloaded with [³H]DA in a stereoselective manner and if the S(-)nornicotine-evoked DA release was inhibited by mecamylamine and DHE, providing additional evidence for a nicotinic receptor-mediated mechanism.

	Methods

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Materials. S(-)Nicotine ditartrate, nomifensine maleate, mecamylamine HCl and dihydro--erythroidine HBr were purchased from Research Biochemicals, Inc. (Natick, MA). S(-)Nornicotine and R(+)nornicotine were synthesized as perchlorate salts (P. A. Crooks and A. Ravard, unpublished methods). [³H]DA (3,4-ethyl-2[N-³H]dihydroxyphenylethylamine; specific activity, 25.6 Ci/mmol) was purchased from New England Nuclear (Boston, MA). Ascorbic acid, -D-glucose, and pargyline hydrochloride were purchased from AnalaR (BHD Ltd., Poole, U.K.), Aldrich Chemical Company, Inc. (Milwaukee, WI) and Sigma Chemical Co. (St. Louis, MO), respectively. TS-2 Tissue solubilizer was purchased from Research Products International (Mount Prospect, IL). 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, 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 assays. Drug effects on [³H]overflow from rat striatal (caudate-putamen) slices preloaded with [³H]DA was determined using a previously published method (Dwoskin and Zahniser, 1986). Briefly, rat striatal slices (500 µm, 6-8 mg) were incubated for 30 min in Krebs' buffer (in mM; 118 NaCl, 4.7 KCl, 1.2 MgCl₂, 1.0 NaH₂PO₄, 1.3 CaCl₂, 11.1 glucose, 25 NaHCO₃, 0.11 L-ascorbic acid and 0.004 ethylenediaminetetraacetic acid, pH 7.4, saturated with 95% O₂/5% CO₂, at 34°C). Slices were then incubated for an additional 30 min in buffer containing 0.1 µM [³H]DA. Each slice was transferred to a superfusion chamber and superfused (1 ml/min) with Krebs' buffer containing nomifensine (10 µM), a DA uptake inhibitor, and pargyline (10 µM), a monoamine oxidase inhibitor, to insure that the [³H]overflow primarily represented [³H]DA, rather than [³H]metabolites (Cubeddu et al., 1979; Zumstein et al., 1981; Rapier et al., 1988). When basal outflow was stabilized after 60 min superfusion, two 5-min (5 ml) samples were collected to determine basal [³H]outflow followed by superfusion with different concentrations of drugs. For all the experiments, slices from a given rat were randomly assigned to all drug concentrations. Each chamber containing one slice was exposed to only one concentration of either S(-)nicotine, S(-)nornicotine or R(+)nornicotine. The design of the superfusion chamber incorporated a bubble trap, which trapped air inadvertently introduced into the tubing when changing solutions. Additionally, the superfusion pump was turned off momentarily while tubing was switched to drug or control solution, such that manipulations in control and drug conditions were identical.

Statistics. A repeated-measures two-way ANOVA was performed to analyze the concentration-dependent effect of S(-)nicotine and the time course of the S(-)nicotine-induced increase in fractional release. For one-way and two-way ANOVAs analyzing total [³H]overflow, the data were log transformed before statistical analysis to be in accordance with the assumption of homogeneity of variance. Repeated-measures one-way ANOVA was used to analyze the concentration-dependency of S(-)nicotine-evoked [³H]overflow. Although the data from complete concentration range (0.01 µM-3 mM) of S(-)nicotine were analyzed statistically, only the statistical analyses for the data from the low concentrations (0.01-100 µM) are provided. This is because of the reported involvement of a nonnicotinic receptor-mediated mechanism in the response to the higher concentrations (>100 µM) (Westfall, 1974; Westfall et al., 1987; Grady et al., 1992) and because the significance of the effect of the low concentrations (<100 µM) of S(-)nicotine was obscured by the more robust effect of the high concentrations. The effect of S(-)nornicotine was analyzed similarly as was the effect of S(-)nicotine. Total-[³H] remaining in the slices after superfusion with either S(-)nicotine or S(-)nornicotine was analyzed by repeated-measures, one-way ANOVA. Studies determining the ability of MEC or DHE to antagonize the effect of S(-)nicotine were analyzed by repeated-measures, one-way ANOVA. Two-way ANOVA was performed to analyze the ability of MEC or DHE (between-subject factors) to inhibit different concentrations (within-subject factor) of S(-)nornicotine-evoked [³H]overflow. Three-way ANOVA was performed to analyze nornicotine's stereoselectivity (between-subject factor), concentration dependency (within-subject factor) and time course of effect (within-subject factor). An iterative nonlinear least-squares curve-fitting program (GraphPAD-PRIZM; GraphPAD, San Diego, CA) was used to obtain EC₅₀ values for MEC-sensitive or DHE-sensitive S(-)nornicotine evoked [³H]overflow, and the EC₅₀ values were compared by Students' t-test. A protected version of Fisher's LSD test (i.e., only preplanned comparisons were considered to limit the overall type-1 error rate) was used for post hoc analysis. Results were considered statistically significant when P < .05 (two-tailed).

	Results

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Effect of S(-)nicotine on superfused rat striatal slices preloaded with [³H]DA. S(-)Nicotine evoked an increase in [³H]overflow from rat striatal slices preloaded with [³H]DA in a concentration-dependent manner (fig. 1). However, a plateau in the concentration-response curve was not observed, even at concentrations up to 3 mM. A significant main effect of concentration (1-100 µM) (F_(5,684) = 88.87, P < .0001), a significant main effect of time (F_(13,684) = 11.07, P < .0001), and a significant concentration x time interaction (F_(65,684) = 2.53, P < .0001) were found. Basal [³H]outflow under buffer control conditions was stable over the course of the experiment (i.e., no significant differences were found between the first two superfusate samples and later samples collected during the course of superfusion). Because the rate of basal outflow for the buffer control condition was stable over the course of the experiment, S(-)nicotine-evoked fractional release was compared statistically to both the predrug basal outflow (with-in slice comparison) and to the no-drug buffer control condition (between-slice comparison). Fractional release peaked 10 to 15 min after S(-)nicotine addition to the buffer, and subsequently decreased toward basal, despite the presence of S(-)nicotine throughout the superfusion period. When the data were expressed as total [³H]overflow, the lowest concentration of S(-)nicotine to produce a significant [F_(5,49) = 27.11, P < .0001] increase in [³H]overflow was 0.1 µM, although no significant increase in fractional release was observed at this concentration.

View larger version (31K): [in this window] [in a new window]   Fig. 1.   In a concentration-dependent manner, S(-)nicotine increased total [3H]overflow from rat striatal slices preloaded with [3H]DA. Results for the time course (top) or the concentration-response (bottom) are presented as a composite of two series of experiments with an overlap in concentrations (see "Methods"). Top panel illustrates the time course of the response to 60 min superfusion with S(-)nicotine (0.1-100 µM). S(-)Nicotine was added to the buffer after the collection of the second 5-min sample (as indicated by the arrow). In each experiment, a slice was superfused in the absence of drug and this constituted the control condition. Data are expressed as mean ± S.E.M. fractional release. *P < .05, different from basal fractional release, and different from fractional release from control slices at the same time point; $P < .05, different from fractional release from control slices at the same time point; Fisher's LSD post hoc comparisons. Bottom panel illustrates S(-)nicotine-evoked total [3H]overflow for the complete range of concentrations (0.01 µM-3 mM). The inset illustrates the concentration-response over the low concentration range. [3H]Overflow was not detected from slices superfused under control conditions. Data are expressed as mean ± S.E.M. total [3H]overflow. #P < .01, ##P < .001, different from control; Fisher's LSD post hoc comparisons. n = 5-12 rats.

Effect of S(-)nornicotine on superfused rat striatal slices preloaded with [³H]DA. S(-)Nornicotine evoked an increase in [³H]overflow in a concentration-dependent manner (fig. 2). A plateau in the concentration-response curve was not observed even at concentrations up to 3 mM. A significant main effect of concentration (0.1-100 µM) [F_(4,433) = 78.05, P < .0001], a significant main effect of time [F_(13,433) = 10.59, P < .0001], and a significant concentration x time interaction [F_(52,433) = 2.53, P < .0001] were found. Because the rate of basal [³H]outflow for buffer control condition was stable over the course of the experiment, S(-)nornicotine-evoked fractional release was compared statistically to both the predrug basal outflow (with-in slice comparison) and the no-drug buffer control condition (between-slice comparison). Fractional release peaked 10 to 15 min after S(-)nornicotine addition to the buffer, and subsequently decreased toward basal, despite the presence of S(-)nornicotine throughout the superfusion period. When the data were expressed as total [³H]overflow, the lowest concentration of S(-)nornicotine to produce a significant [F_(4,36) = 60.23, P < .0001] increase in [³H]overflow was 0.1 µM, although no significant increase in fractional release was observed at this concentration. Total [³H] remaining in the slices after superfusion with the high concentrations (1 and 3 mM) of S(-)nornicotine was decreased significantly (30 and 53%, respectively) compared to control slices [F_(7,57) = 3.10, P < .01, data not shown]. Superfusion with lower concentrations (<1 mM) did not result in a decrease in [³H] remaining in the tissue. Thus, the decreased fractional release after prolonged superfusion with low concentrations (<1 mM) of S(-)nornicotine is not due to the depletion of DA striatal content.

View larger version (30K): [in this window] [in a new window]   Fig. 2.   In a concentration-dependent manner, S(-)nornicotine increased total [3H]overflow from rat striatal slices preloaded with [3H]DA. Top panel illustrates the time-course of the response to 60 min superfusion with S(-)nornicotine (0.1-100 µM). In each experiment, a slice was superfused in the absence of drug and this constituted the control condition. Data are expressed as mean ± S.E.M. fractional release. *P < .05, different from basal fractional release and from fractional release from the control slice at the same time point; $P < .05, different from fractional release from control slice at the same time point; Fisher's LSD post hoc comparisons. Bottom panel illustrates the concentration-dependence of S(-)nornicotine-evoked total [3H]overflow for the complete range of concentrations (0.1 µM-3 mM) examined. Data are expressed as mean ± S.E.M. total [3H]overflow. The inset illustrates the concentration-response over the low concentration range. [3H]Overflow was not detected from slices superfused under control conditions. #P < .001, different from control; Fisher's LSD post hoc comparisons; n = 8 rats.

MEC and DHE antagonism of S(-)nicotine- and S(-)nornicotine-evoked [³H]overflow from rat striatal slices preloaded with [³H]DA. Superfusion with MEC (0.01-100 µM) alone did not alter [³H]overflow (table 1). In a concentration-dependent manner, MEC significantly [F_(5,38) = 8.56, P < .001] inhibited S(-)nicotine (10 µM)-evoked [³H]overflow compared to the S(-)nicotine control (fig. 3). The highest concentration of MEC examined robustly inhibited (91%) the effect of S(-)nicotine, and was chosen to study the inhibition of S(-)nornicotine-evoked [³H]overflow. MEC inhibited [³H]overflow evoked by S(-)nornicotine (0.1 µM-70 µM) (fig. 4). A significant main effect of concentration [F_(8,121) = 36.10, P < .0001], a significant main effect of MEC [F_(1,121) = 26.29, P < .0005] and a significant interaction [F_(8,121) = 2.31, P < .05] were found. Thus, the effect of S(-)nornicotine to evoke [³H]overflow was MEC-sensitive. Interestingly, the effect of S(-)nornicotine at concentrations  70 µM was robustly inhibited (60-80%) by MEC (fig. 4); however, the effect of high concentrations (100 µM) was inhibited marginally (3-20%, data not shown). Thus, high S(-)nornicotine concentrations evoked [³H]overflow that was not MEC sensitive.

View this table: [in this window] [in a new window]   TABLE 1 Effect of MEC and DHE on [3H]overflow from rat striatal slices preloaded with [3H]DA

View larger version (18K): [in this window] [in a new window]   Fig. 3.   MEC and DHE inhibited S(-)nicotine-evoked [3H]overflow from rat striatal slices in a concentration-dependent manner. MEC (0.01-100 µM) and DHE (0.01-10 µM) inhibition of S(-)nicotine (10 µM)-evoked [3H]overflow is illustrated in the top and bottom panels, respectively. Slices were superfused with MEC or DHE for 60 min before the addition of S(-)nicotine to the buffer. Superfusion continued for 60 min with MEC or DHE plus S(-)nicotine in the buffer. In each experiment, a slice was superfused in the absence of drug, and overflow was not observed (i.e., tritium in superfusate over the duration of superfusion was not different from basal outflow; data not shown). Another slice was superfused with 10 µM S(-)nicotine and served as the S(-)nicotine control. Data are expressed as mean ± S.E.M. total [3H]overflow. *P < .05, **P < .005, different from S(-)nicotine control; Fisher's LSD post hoc comparisons. n = 8 rats for MEC; n = 5 rats for DHE experiments.

View larger version (23K): [in this window] [in a new window]   Fig. 4.   MEC and DHE inhibition of S(-)nornicotine-evoked [3H]overflow from rat striatal slices preloaded with [3H]DA. Results for MEC or DHE inhibition of the effect of S(-)nornicotine are presented each as a composite of two series of experiments with an overlap in S(-)nornicotine concentrations (see "Methods"). S(-)Nornicotine- (0.1-100 µM) evoked total [3H]overflow in the absence (control) and presence of MEC (100 µM) or DHE (10 µM) is illustrated in the top and bottom panel, respectively. Slices superfused with MEC or DHE for 60 min before the addition of S(-)nornicotine to the buffer for 60 min; and subsequently, slices were superfused with MEC plus S(-)nornicotine or DHE plus S(-)nornicotine in the buffer. Data are presented as mean ± S.E.M. total [3H]overflow. MEC, mecamylamine; norNic: S(-)nornicotine; *P < .05, different from S(-)nornicotine control; Fisher's LSD post hoc comparisons. n = 5-12 rats for MEC experiments; n = 5-10 rats for DHE experiments.

View larger version (18K): [in this window] [in a new window]   Fig. 5.   MEC-sensitive and DHE-sensitive [3H]overflow evoked by S(-)nornicotine. MEC-sensitive (top panel) or DHE-sensitive (bottom panel) S(-)nornicotine-evoked [3H]overflow was determined by transforming the data illustrated in figure 4. The transformation was performed by subtracting S(-)nornicotine-evoked [3H]overflow in the presence of respective antagonist from that in the absence of antagonist for each individual experiment. The means ± S.E.M. of the differential total [3H]overflow are illustrated. n = 5-12 rats for MEC; n = 5-10 rats for DHE experiments.

Stereoselective effect of nornicotine. Both enantiomers of nornicotine increased fractional release (data not shown) and [³H]overflow (fig. 6) in a concentration-dependent manner. Three-way ANOVA revealed significant main effects of enantiomer [F_(1,1605) = 95.73, P < .0001], concentration [F_(5,1605) = 88.97, P < .0001], and time [F_(14,1605) = 19.88, P < .0001]. Also, a significant enantiomer x concentration interaction [F_(5,1605) = 4.17, P < .001], and a significant concentration x time interaction [F_(70,1605) = 3.51, P < .001] were found; however the enantiomer x time interaction [F_(14,1605) = 1.08, P > .05] and the three-way interaction [F_(70,1605) = .52, P > .05] were not significant. Thus, when the data were collapsed across time, significant stereoselectivity was observed at concentrations from 1 to 100 µM. Expressing the data as [³H]overflow clearly illustrates the concentration-dependence, the stereoselectivity and the tendency for a greater response of S(-)enantiomer compared to the R(+)enantiomer as the concentration was increased.

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Nornicotine evoked a stereoselective and concentration-dependent increase in total [3H]overflow from rat striatal slices preloaded with [3H]DA. Data are expressed as mean ± S.E.M. total [3H]overflow. *P < .0001, different from the R(+) enantiomer at same concentration; Fisher's LSD post hoc comparisons. n = 10 rats/enantiomer.

	Discussion

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Our study demonstrates that S(-)nornicotine evokes [³H]overflow from rat striatal slices preloaded with [³H]DA in a concentration-dependent manner, with a magnitude of response similar to that for S(-)nicotine. Nornicotine stereoselectively evoked an increase in [³H]overflow, and moreover, the effect of low concentrations (<100 µM) of S(-)nornicotine were antagonized by the nicotinic receptor antagonists, MEC and DHE. Taken together with our previous findings demonstrating that S(-)nornicotine-evoked DA release is Ca⁺⁺ dependent (Dwoskin et al., 1993), the results suggest that S(-)nornicotine at low concentrations evokes [³H]overflow from rat striatal slices preloaded with [³H]DA via stimulation of nicotinic receptors.

In our study, S(-)nicotine evoked [³H]overflow from the [³H]DA-preloaded rat striatal slices in a concentration-dependent manner, in agreement with others using rat striatal slices (Westfall et al., 1987; Izenwasser et al., 1991; Sacaan et al., 1995) and rat or mouse striatal synaptosomes (Rowell et al., 1987; Rapier et al., 1988, 1990; Grady et al., 1992, 1994; El-Bizri and Clarke, 1994; Rowell and Hillebrand, 1994; Rowell, 1995). In most of the previous studies, striatal tissue was exposed to S(-)nicotine for only short periods (either a pulse stimulus or for 3-10 min). Only one study using rat striatal slices provided the time course of the effect of nicotine at a single concentration (1 µM), and [³H]overflow peaked at 2.5 min and remained at this elevated level for the entire 10-min period of exposure (Giorguieff-Chesselet et al., 1979). In studies using rodent striatal synaptosomes preloaded with [³H]DA, time courses illustrate that nicotine exposure (0.3-5 µM, pulse-10 min duration) produced a rapid increase in [³H]overflow which peaked at 1 to 2 min, and subsequently required 10 min to return to basal levels (Rapier et al., 1988; Grady et al., 1994; Rowell and Hillebrand, 1994; Rowell, 1995). Our study extends the previous work and provides the complete time course of the effect of S(-)nicotine (1-100 µM). Under the conditions of our study, a peak effect was observed at 10 to 15 min and by 30 to 60 min of superfusion with the low concentrations (<100 µM), response returned to basal. Differences in the timing and pattern of response may be due to differences in one or more parameters, i.e., species, tissue preparation, drug-exposure duration and superfusion flow rate. The slower and more prolonged response in assays using striatal slices may in a large part be due to the time required for diffusion of drug, because of the presence of significant barriers to drug permeation as a result of intact cellular connection, compared to the synaptosomal preparation.

In our study, the return of fractional [³H]release toward basal levels despite the continued presence of S(-)nicotine in the superfusion buffer is indicative of receptor desensitization, and was not the result of depletion of striatal [³H]DA content. Receptor desensitization is additionally supported by the observation that S(-)nicotine was able to release [³H]DA during a period when fractional release returned to basal after a prior S(-)nicotine exposure (Dwoskin et al., 1995). Although returning toward basal levels, the response to higher concentrations of S(-)nicotine was still significantly different from basal at the end of the superfusion period. Our findings are of particular interest in light of the observation that throughout the waking hours of the day, human smokers titrate their blood concentration (0.1-1.0 µM) of S(-)nicotine to an intrinsically preferred level (Benowitz et al., 1990). Thus, the latter results suggest that neuronal nicotinic receptors in the brain of a moderate smoker are more likely exposed to a continuous concentration of S(-)nicotine, as well as to intermittent exposures to high concentrations coincident with tobacco smoking.

Few studies have examined the effects of nicotine metabolites on DA release in vitro, and metabolite-induced effects on DA release in vivo have not been investigated to date. Racemic nornicotine has been shown to evoke a concentration-dependent increase in [³H]overflow from [³H]DA-preloaded mouse striatal synaptosomes (Grady et al., 1992). Enantiomerically pure S(-)nornicotine (0.1-100 µM, 15 min exposure) evokes a concentration-dependent and Ca⁺⁺-dependent increase in endogenous DA release from rat striatal slices (Dwoskin et al., 1993). In the present study, the effect of a wider range of S(-)nornicotine concentrations (0.1 µM-3 mM) on [³H]overflow was determined. The concentration response curve for S(-)nornicotine appears very similar to that for S(-)nicotine, and the pattern of the time course indicates receptor desensitization during prolonged S(-)nornicotine exposure.

In a recent study, the concentration-response curve for S(-)nicotine-evoked [³H]overflow from [³H]DA-preloaded rat striatal slices was observed to reach a plateau between 10 and 100 µM (EC₅₀ = 3.7 µM; Sacaan et al., 1995). Receptor desensitization, nonspecific effects at high concentrations and release of neurotransmitters that inhibit DA release may have contributed to the plateau. Izenwasser et al. (1991), using rat striatal minces, also observed a plateau, but at much lower concentrations (0.1 µM) than observed by Sacaan et al. (1995). In our study, the S(-)nicotine concentration-response curve did not reach a plateau, in agreement with a previous study that used rat striatal slices, a similar nicotine-concentration range and a shorter (5-min) drug-exposure period (Westfall et al., 1987). The reason for the inconsistencies in the observation of a plateau is not known. Variations in experimental conditions are evident, including slice thickness, superfusion chamber volume, buffer flow rate, duration of exposure and superfusate collection. However, a plateau was also not observed in the present studies with a 2-min nicotine exposure period (data not shown). Therefore, duration of exposure is not the factor responsible for differences in the pattern of DA release. The experimental condition responsible for the discrepancy in the results may be the duration of superfusate collection. In the Izenwasser et al. (1991) and Sacaan et al. (1995) studies, striatal slices were exposed to nicotine for 3-10 min and samples were collected for 10 to 30 min; time courses were not provided. The plateau in the response observed in the latter studies may have resulted from a truncation of the full effect of drug exposure, because the collection period may not have included the complete response.

In studies using rodent striatal synaptosomes, a plateau in the concentration-response curve at concentrations between 1 to 100 µM has been observed by some investigators (Rapier et al., 1988; El-Bizri and Clarke, 1994; Rowell et al., 1987; Rowell, 1995), but not by others (Grady et al., 1992). However, when the data were presented as peak response, a plateau was observed for S(-)nicotine (EC₅₀ = 0.33 µM; Grady et al., 1994). In the Grady et al. (1992) study, a maximal response (EC₅₀ = 0.48 µM) was defined as one that was insensitive to MEC inhibition, indicating that the full concentration-response represented both specific and nonspecific effects. Thus, the nicotinic receptor-mediated portion of the response can be determined by sensitivity to nicotinic-receptor antagonists. Good agreement in nicotine EC₅₀ values has been obtained either when a plateau was observed or when the maximal response was defined by antagonist sensitivity.

Because a plateau in the S(-)nornicotine concentration-response curve was not observed in our study, nicotinic receptor mediation was assessed by sensitivity to nicotinic antagonists. MEC has been reported to be a noncompetitive inhibitor of the NMDA receptor, acting at the MK-801 site within the channel (Reynolds and Miller, 1988; Snell and Johnson, 1989; Court et al., 1990). To verify that the effect of S(-)nornicotine was mediated by nicotinic receptors, competitive nicotinic receptor inhibition with DHE (Vidal and Changeux, 1989; Alkondon and Albuquerque, 1991; Mulle et al., 1991) was studied. MEC and DHE robustly inhibited nicotine-evoked [³H]overflow from [³H]DA-preloaded striatal slices, in good agreement with previous studies (El-Bizri and Clark, 1994; Sacaan et al., 1995). Moreover, MEC and DHE also effectively inhibited [³H]overflow evoked by low concentrations of S(-)nornicotine, but not by high concentrations ( 100 µM). Based on visual inspection of the S(-)nornicotine concentration-response curves in the absence and presence of the antagonists, the overall depression of the S(-)nornicotine concentration-response curves in the presence of MEC and the shift to the right of the concentration-response curve in the presence of DHE are consistent with noncompetitive and competitive antagonism, respectively. MEC-sensitive and DHE-sensitive concentration-response curves revealed maximal responses of S(-)nornicotine, and EC₅₀ values were determined. Interestingly, S(-)nornicotine has a similar potency to release DA from striatum compared to S(-)nicotine. Thus, the results suggest that the effect of S(-)nornicotine at concentrations < 100 µM is the result of stimulation of nicotinic receptors. High concentrations of S(-)nornicotine may release DA via a nonselective mechanism that is insensitive to inhibition by MEC and DHE, or by a nicotinic receptor that is not sensitive to MEC or DHE. Regardless, it is unlikely that such high concentrations of nornicotine are pharmacologically relevant or are present in the human smoker's brain.

In our study, the effect of S(-)nornicotine was significantly greater than the effect of R(+)nornicotine at concentrations of 1, 10 and 100 µM, consistent with an interpretation of a receptor-mediated effect. The observations that nornicotine-evoked [³H]overflow was stereoselective at 100 µM, but not sensitive to inhibition by nicotinic-receptor antagonists, further suggests that this response is mediated by a nonnicotinic receptor or by a nicotinic receptor that is not sensitive to MEC or DHE. Interestingly, nornicotine competes with [³H]nicotine for its binding site in rat brain membranes; however, stereoselective displacement is not observed (Reavill et al., 1988; Copeland et al., 1991; Zhang and Nordberg, 1993).

Concentrations (0.1 µM) of nornicotine, within the low range found to release DA in our study (i.e., pharmacologically relevant), have been detected in rat brain 4 hr after administration (s.c.) of radiolabeled-nicotine (0.54 mg/kg), and the presence of nornicotine in brain was suggested to result at least in part from nicotine metabolism in the CNS (Crooks et al., 1995, 1997). The dose of nicotine administered is moderate relative to reported studies examining the behavioral effects and in vivo neurochemical effects of nicotine in rats. These low concentrations of nornicotine in the CNS after peripherial nicotine administration may be pharmacologically relevant with regard to brain concentrations after tobacco smoking and peripheral nicotine administration in man. The nicotine dose administered to rats in the studues of Crooks et al. (1995, 1997) resulted in plasma nicotine concentrations somewhat higher than daytime levels of plasma nicotine found in habitual smokers; however, one objective of the latter studies was to determine an acute dose of peripheral nicotine that would give rise to pharmacologically relevant levels of nornicotine in the CNS. Nevertheless, the results are relevant to tobacco smoking, because nornicotine has a higher polarity and water solubility than nicotine, and thus, nornicotine is predicted to efflux from the CNS compartment much more slowly than nicotine. Therefore, there is potential for the accumulation of nornicotine in the CNS during chronic nicotine exposure, and this is clearly of relevance with regard to habitual smokers considering that nornicotine is an active nicotine metabolite. In addition, another factor to consider is that nornicotine is an alkaloid in cigarette tobacco which contributes to the total amount of nornicotine in the CNS (i.e., the amount of nornicotine present in the CNS that is not the result of N-demethylation of nicotine). The concentration of nornicotine in the smoker's brain is not known, however, the concentration of nornicotine in smoker's urine is minor (1-10%) relative to nicotine (Kyerematen et al., 1990; Zhang et al., 1990; Benowitz et al., 1994). Thus, in smokers, brain nornicotine most likely results from both the alkaloidal source (tobacco) and from metabolism of nicotine in the periphery and/or in the CNS. Although nornicotine is reported to be a major tobacco alkaloid (Bush et al., 1993), nornicotine yield and bioavailability during tobacco smoking have not been determined.

In summary, S(-)nornicotine evokes DA release from rat striatal slices in a concentration-dependent manner and desensitizes nicotinic receptors. Under these conditions, the effect of nornicotine is stereoselective and antagonized by MEC and DHE, suggesting an action at nicotinic receptors. In conclusion, our results suggest that nornicotine may contribute to the neuropharmacological effects of nicotine and tobacco usage, and further study of nornicotine pharmacology is necessary.