Introduction

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Neuronal nicotinic acetylcholine receptors (nAChRs) are preferentially located presynaptically and primarily modulate synaptic activity by regulating neurotransmitter release (McGehee and Role, 1995; Wonnacott, 1997). nAChRs consist of two subunits, and , and combinations of nine  (2-10) and three  (2-4) subunits assembling as pentamers with a general stoichiometry of 2 and 3 (Anand et al., 1991). Hybridization cloning has revealed a surprising degree of subtype diversity among nAChRs (Deneris et al., 1991; Luetje et al., 1993; Chavez-Noriega et al., 1997). Results from immunoprecipitation studies indicate that more than two different subunits can assemble to form a functional receptor, greatly increasing the potential diversity of native nAChRs (Conroy et al., 1992; Forsayeth and Kobrin, 1997). Furthermore, individual neurons elaborate multiple nAChR subtypes (Poth et al., 1997), potentially increasing the complexity and making the elucidation of the function of specific receptor subtypes even more challenging. Thus, nAChR subtype diversity originates from differences in amino acid sequences of subunit proteins and from multiple combinations of subunits forming functional receptors. The exact subunit composition, stoichiometry, and arrangement of native nAChRs remain to be conclusively elucidated, as indicated herein by an asterisk after the subunit designation of native receptors (Lukas et al., 1999).

Heteromeric 42* nAChRs are likely the most abundant nAChR subtype in brain (Wada et al., 1989; Flores et al., 1992). However, 4 subunits may also form more complex combinations with 2, 3, and/or 4 subunits (Forsayeth and Kobrin, 1997). A second predominant nAChR, the 7* homomeric subtype, constitutes the major -bungarotoxin binding site in brain (Schoepfer et al., 1990). The predominance of nAChR subtypes does not necessarily reflect their functional importance. In this regard, the less prevalent 32* subtype has been suggested to mediate S()-nicotine-evoked dopamine (DA) release from presynaptic nerve terminals in striatum.

nAChRs are localized on DA cell bodies in the substantia nigra and DA terminals in the striatum (Wonnacott, 1997). Nicotine-evoked DA release is inhibited by the classical nAChR antagonists, mecamylamine or dihydroxy--erythroidine (DHE), indicating nAChR mediation of this effect (Grady et al., 1992; El-Bizri and Clarke, 1994; Rowell, 1995; Sacaan et al., 1995; Teng et al., 1997). The involvement of the 32* subtype in nicotine-evoked DA release has been suggested based on the sensitivity to the 32-selective antagonists, neuronal bungarotoxin (Schulz and Zigmond, 1989; Grady et al., 1992) and -conotoxin MII (Cartier et al., 1996; Kaiser and Wonnacott, 2000). The 32 selectivity of these antagonists has been indicated by their activity in recombinant cell-expression systems in which specific nAChR subtypes have been expressed (Luetje et al., 1990; Cartier et al., 1996). Importantly, -conotoxin MII only partially inhibited nicotine-evoked DA release (Kulak et al., 1997; Kaiser and Wonnacott, 2000), suggesting heterogeneity of presynaptic nAChRs on striatal DA terminals. The involvement of 4-, 2-, and 4-containing nAChRs in this response has also been suggested (Sharples et al., 2000). Results from studies utilizing 2 knockout mice also implicate 2-containing nAChR subtypes in the mediation of striatal DA release (Picciotto et al., 1998). Since rat substantial nigra neurons express mRNA for 3, 4, 5, 6, 7, 2, 3, and 4 subunits (Deneris et al., 1989; Wada et al., 1989; Dineley-Miller and Patrick, 1992; Charpantier et al., 1998; Arroyo-Jimenez et al., 1999), multiple nAChR subtypes may be involved in S()-nicotine-evoked DA release in striatum. These neurons express high levels of 6 and 3 mRNA (Deneris et al., 1989; Le Novere et al., 1996; Goldner et al., 1997; Charpantier et al., 1998), suggesting their likely candidacy for combination with 3 and 2 subunits in the mediation of S()-nicotine-evoked DA release in striatum.

Relatively little attention has focused on the development of nAChR subtype-selective antagonists (Dwoskin et al., 2000; Dwoskin and Crooks, 2001). Subtype-selective antagonists are needed to establish the role of specific nAChR subtypes in physiological function. Since S()-nicotine acts as an agonist at all nAChRs, modification of the S()-nicotine molecule by N-n-alkyl substitution was hypothesized to convert S()-nicotine into an antagonist, and with variation of the n-alkyl chain length, nAChR subtype selectivity may result. The present study determined the ability of N-n-alkylnicotinium analogs to inhibit S()-nicotine-evoked [³H] overflow from [³H]DA-preloaded striatal slices and the mechanism of inhibition involved.

	Experimental Procedures

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Materials. S()-Nicotine ditartrate, nomifensine maleate, and DHE were purchased from Sigma/RBI (Natick, MA). Pargyline hydrochloride was purchased from Sigma-Aldrich (St. Louis, MO). N-Methylnicotinium iodide (NMNI), N-n-butylnicotinium iodide (NnBNI), and N-n-octylnicotinium iodide (NONI) were prepared as described by Crooks et al. (1995). N-n-Hexylnicotinium iodide (NHxNI), N-n-heptylnicotinium iodide (NHpNI), N-n-nonylnicotinium iodide (NNNI), N-n-decylnicotinium iodide (NDNI), and N-n-dodecylnicotinium iodide (NDDNI) were prepared from S()-nicotine and the appropriate n-alkyl iodide using a modification of the general procedure described by Shibagaki et al. (1982). All compounds were fully characterized and determined to be free from S()-nicotine-utilizing spectroscopic (NMR and fast atom bombardment mass spectroscopy), chromatographic (silica gel), and combustion analysis procedures. Structures of the N-n-alkylnicotinium analogs are shown in Fig. 1. -D-Glucose was purchased from Aldrich Chemical Co. (Milwaukee, WI). Ascorbic acid (AnalaR grade) and TS-2 solubilizer were purchased from BDH Ltd. (Poole, Dorset, UK) and Research Products International (Mount Prospect, IL), respectively. [³H]DA (specific activity, 25.6 Ci/mmol) was obtained from PerkinElmer Life Sciences (Boston, MA). The remaining chemicals contained in the superfusion buffer were obtained from Fisher Scientific (Pittsburgh, PA).

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Structures of S()-nicotine and N-n-alkylnicotinium analogs. Note that the 2' S configuration of the nicotine molecule is preserved in all of the N-n-alkylnicotinium analogs. R = n-alkyl substituent.

Subjects. Male Sprague-Dawley rats (200-250 g) were obtained from Harlan Laboratories (Indianapolis, IN) and housed two per cage with free access to food and water in the Division of Lab Animal Resources in the College of Pharmacy, University of Kentucky. Experimental protocols involving the animals were in strict accordance with the 1996 National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at the University of Kentucky.

[³H]DA Release Assay. S()-Nicotine-evoked [³H] overflow from superfused rat striatal slices preloaded with [³H]DA was determined using a previously published method (Dwoskin and Zahniser, 1986). Briefly, coronal striatal slices (500 µm, 6-8 mg) were incubated for 30 min in Krebs' buffer (118 mM NaCl, 4.7 mM KCl, 1.2 mM MgCl₂, 1.0 mM NaH₂PO₄, 1.3 mM CaCl₂, 11.1 mM glucose, 25 mM NaHCO₃, 0.11 mM L-ascorbic acid, and 0.004 mM EDTA, pH 7.4, saturated with 95% O₂/5% CO₂) at 34°C in a metabolic shaker. Slices were then incubated for an additional 30 min in fresh buffer containing 0.1 µM [³H]DA. After rinsing, each slice was transferred to a superfusion chamber maintained at 34°C and was superfused (1 ml/min) with Krebs' buffer containing 10 µM nomifensine, a DA uptake inhibitor, and 10 µM pargyline, a monoamine oxidase inhibitor, such that [³H] overflow primarily represented [³H]DA rather than [³H] metabolites. After 60 min of superfusion, when basal outflow was stabilized, two 5-min samples (5 ml each) were collected to determine basal [³H] outflow.

Data Analysis. 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. Multiplying the value for fractional release by 100 transformed the sample data to a percentage of tissue [³H] content. Basal [³H] outflow was calculated from the average fractional release in the two 5-min samples just before addition of N-n-alkylnicotinium analog. Summation of superfusate [³H] as a percentage of sample [³H] above basal (12 consecutive samples collected during the 60-min drug-exposure period) was then used to define total [³H] overflow, i.e., superfusate [³H] as percentage of tissue [³H] content =  [(sample [³H] in superfusate above basal [³H] outflow  tissue [³H] content during the 5-min sample collection period) × 100]. Thus, S()-nicotine-, N-n-alkylnicotinium analog-, or DHE-evoked [³H] overflow was calculated by summing the increases in fractional release that resulted from exposure to drug, subtracting the basal [³H] outflow for an equivalent period of drug exposure, and dividing by tissue [³H] content at the time of sample collection.

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

Effect of S()-Nicotine on Superfused Rat Striatal Slices Preloaded with [³H]DA. Following superfusion with S()-nicotine, fractional release increased to a peak level between 10 and 15 min of superfusion and then diminished during the remainder of the period of superfusion, returning to basal levels of [³H]outflow (data not shown). S()-Nicotine evoked a significant increase in [³H] overflow from rat striatal slices preloaded with [³H]DA in a concentration-dependent manner (F_6,66 = 33.2, p < 0.0001), although a maximal effect was not evident at the highest concentration (100 µM) examined (Fig. 2, top panel). Since previous studies demonstrated that S()-nicotine-evoked [³H] overflow at concentrations of 100 µM is not mediated by nAChRs (Teng et al., 1997), data from 0.1 to 10 µM S()-nicotine concentrations were evaluated by an Eadie-Hofstee analysis, which revealed a curvilinear concentration-effect relationship (Fig. 2, bottom panel), suggesting that at least two components are involved in S()-nicotine-evoked [³H] overflow from [³H]DA-preloaded striatal slices. The data were fitted to a two-component hyperbolic function, which allowed the estimation of maximal response and EC₅₀ values for apparent high- and low-affinity components of S()-nicotine-evoked [³H] overflow. High- and low-affinity components with EC₅₀ values of 100 nM and 360 µM, respectively, were revealed, suggesting activation of two different nAChR subtypes. Maximal effect for the high- and low-affinity components of S()-nicotine-evoked [³H] overflow was obtained at values of 0.53 and 50.2% tissue [³H] content, respectively.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   S()-Nicotine increases [3H] overflow from superfused rat striatal slices preloaded with [3H]DA. Concentration dependence of S()-nicotine-evoked [3H] overflow (top). S()-Nicotine was added to the buffer containing nomifensine (10 µM) and pargyline (10 µM) during a 60-min superfusion period. [3H] overflow was not detected from slices superfused under buffer control conditions [i.e., in the absence of S()-nicotine]. Eadie-Hofstee plot of [3H] overflow evoked by 0.1 to 10 µM S()-nicotine (bottom). Linear curve fits are Eadie-Hofstee representations of the two components of S()-nicotine-evoked [3H] overflow, derived from the hyperbolic fit to the nontransformed data presented in the inset. Data are expressed as mean ± S.E.M. total [3H] overflow (top) and as total [3H] overflow  [S()-nicotine] (bottom). CON, buffer control. , p < 0.05, , p < 0.01, different from control; Dunnett's post hoc comparisons. n = 8 to 10 rats.

DHE: Intrinsic Activity and Inhibition of S()-Nicotine-Evoked [³H] Overflow. DHE evoked a significant increase in [³H] overflow (F_5,35 = 13.0, p < 0.0001). Superfusion with a wide range of DHE concentrations (0.01-10 µM) alone did not evoke [³H] overflow (Fig. 3); however, superfusion with a high concentration of DHE (100 µM) resulted in an increase in [³H] overflow to a value of 1.68 ± 0.40% tissue [³H] content. In a concentration-dependent manner, DHE (0.01-10 µM) significantly inhibited S()-nicotine (10 µM)-evoked [³H] overflow compared with the S()-nicotine control (F_5,29 = 12.5, p < 0.0001; Fig. 4). The IC₅₀ value for DHE was 1.58 µM (95% confidence interval, 0.36, 6.94 µM). The highest concentration (10 µM) of DHE, which alone produced no intrinsic activity, was found to inhibit ~80% of the response to S()-nicotine (10 µM).

View larger version (28K): [in this window] [in a new window]   Fig. 3.   Novel N-n-alkylnicotinium analogs evoke [3H] overflow from [3H]DA-preloaded rat striatal slices in a concentration-dependent manner; comparison with DHE. Superfusion buffer contained nomifensine (10 µM) and pargyline (10 µM) from the start of superfusion. N-n-Alkylnicotinium analog or DHE was added to the buffer following 60 min of superfusion, and superfusion continued for 60 min with analog or DHE before addition of S()-nicotine (10 µM) to the buffer. Intrinsic activity was determined during the first 60-min period of superfusion with analog or DHE. In each experiment, one striatal slice was superfused in the absence of drug and served as the buffer control. [3H] overflow was not detected from slices superfused under buffer control conditions (i.e., in the absence of analog or DHE). Numbers in parentheses next to each analog abbreviation indicate number of carbons in n-alkyl chain for that analog. Data are expressed as mean ± S.E.M. total [3H] overflow (percentage of tissue). CON, buffer control. n = 3 to 10 rats/compound.

View larger version (28K): [in this window] [in a new window]   Fig. 4.   In a concentration-dependent manner, N-n-alkylnicotinium analogs inhibit S()-nicotine-evoked [3H] overflow from superfused rat striatal slices; comparison with DHE. Superfusion buffer contained nomifensine (10 µM) and pargyline (10 µM) from the start of superfusion. N-n-Alkylnicotinium analog or DHE was added to the buffer following 60 min of superfusion, and superfusion continued for 60 min with analog or DHE before addition of S()-nicotine (10 µM) to the buffer. Slices were superfused with analog plus S()-nicotine for an additional 60 min. In each experiment, one striatal slice was superfused with 10 µM S()-nicotine in the absence of N-n-alkylnicotinium analog or DHE and served as the S()-nicotine control. An additional striatal slice was superfused in the absence of either drug and served as the buffer control. [3H] overflow was not detected in superfusate from the buffer control slices (not shown). Data are expressed as mean ± S.E.M. total [3H] overflow (top) and as a percentage of [3H] overflow in the S()-nicotine control condition (bottom). Numbers in parentheses next to each analog abbreviation indicate number of carbons in the n-alkyl chain for that analog (bottom). n = 3 to 10 rats/compound.

N-n-Alkylnicotinium Analog-Evoked [³H] Overflow from Striatal Slices Preloaded with [³H]DA. Intrinsic activity of the N-n-alkylnicotinium analogs was assessed during the 60-min period of superfusion with each analog prior to addition of S()-nicotine to the superfusion buffer. Analogs with an n-alkyl chain length of 6 carbons or greater evoked significant amounts of [³H] overflow, with the exception of NNNI, which did not evoke [³H] overflow [NHxNI (C₆), F_6,35 = 2.85, p < 0.05; NHpNI (C₇), F_6,24 = 85.3, p < 0.0001; NONI (C₈), F_6,33 = 79.9, p < 0.0001; NDNI (C₁₀), F_6,49 = 564, p < 0.0001; and NDDNI (C₁₂), F_6,27 = 78.8, p < 0.0001] (Fig. 3). The amount of [³H] overflow was generally related to carbon chain length, such that, as the n-alkyl chain length increased, the concentration of analog that elicited intrinsic activity decreased; i.e., NDNI (C₁₀) evoked significant [³H] overflow at 1 µM, and NDDNI (C₁₂) at 10 µM, whereas NONI (C₈), NHpNI (C₇), and NHxNI (C₆) evoked [³H] overflow at 100 µM (Fig. 3).

N-n-Alkylnicotinium Analog-Induced Inhibition of S()-Nicotine-Evoked [³H] Overflow from Striatal Slices Preloaded with [³H]DA. The ability of the N-n-alkylnicotinium analogs to inhibit S()-nicotine-evoked [³H] overflow is shown in Fig. 4; IC₅₀ values derived from the nonlinear sigmoid curve fits to these data are presented in Table 1. Analog concentrations that were observed to evoke [³H] overflow (i.e., intrinsic activity) were not evaluated for inhibition of the effect of S()-nicotine. NDNI (C₁₀ analog) did not exhibit significant inhibitory activity (F_3,29 = 0.638, p = 0.597). One-way ANOVA revealed significant concentration-dependent inhibition for most other analogs in this series [NMNI (C₁), F_3,15 = 5.02, p < 0.05; NPNI (C₃), F_6,38 = 4.33, p < 0.005; NnBNI (C₄), F_3,19 = 8.83, p < 0.005; NHxNI (C₆), F_5,20 = 3.81, p < 0.05; NHpNI (C₇), F_5,24 = 26.3, p < 0.0001; NONI (C₈), F_5,27 = 10.5, p < 0.0001; NNNI (C₉), F_5,23 = 4.58, p < 0.01; and NDDNI (C₁₂), F_4,18 = 9.00, p < 0.001]. A 4000-fold range of IC₅₀ values was observed among the analogs (Table 1). NPNI was the least potent of the N-n-alkylnicotinium analogs with an IC₅₀ value of 37.4 µM. NnBNI and NHxNI exhibited IC₅₀ values of 9 and 3 µM, respectively, whereas NHpNI, NONI, and NNNI had IC₅₀ values in the range of 0.2 to 0.8 µM. NDDNI, the C₁₂ analog, had the highest potency with an IC₅₀ value of 9 nM. In comparison, DHE exhibited intermediate potency (IC₅₀ = 1.58 µM) relative to these N-n-alkylnicotinium analogs (Fig. 4 and Table 1). The overall rank order of potency for inhibition of S()-nicotine-evoked [³H] overflow was NDDNI NNNI = NONI = NHpNI > DHE > NHxNI = NnBNI > NPNI = NMNI > NDNI.

View this table: [in this window] [in a new window]   TABLE 1 Concentration (IC50) of N-n-alkylnicotinium analog required to reduce S()-nicotine (10 µM)-evoked [3H] overflow from [3H]DA-preloaded rat striatal slices by 50%: comparison with DHE IC50 values and 95% confidence interval values were derived from fixed slope sigmoid fits to the percent control transformed data illustrated in Fig. 4, bottom panel.

View larger version (32K): [in this window] [in a new window]   Fig. 5.   Time course of NDDNI-induced inhibition of S()-nicotine-evoked fractional release. NDDNI (1 nM-1 µM) was added to the superfusion buffer of individual chambers 60 min prior to the addition of S()-nicotine (10 µM) to the buffer. Superfusion continued in the presence of NDDNI and S()-nicotine, and superfusion samples were collected at 5-min intervals. The vertical arrow indicates addition of S()-nicotine to the buffer. Data are expressed as mean ± S.E.M. fractional release as a percentage of basal [3H] outflow as a function of time of superfusion (min); n = 6 rats.

View larger version (21K): [in this window] [in a new window]   Fig. 6.   Potency to inhibit S()-nicotine-evoked [3H] overflow from superfused rat striatal slices preloaded with [3H]DA varies linearly with n-alkyl chain length. Log IC50 values for N-n-alkylnicotinium analog inhibition of S()-nicotine-evoked [3H] overflow were derived from nonlinear sigmoid curve fits of the mean percent S()-nicotine control data illustrated in Fig. 4 (bottom). NDNI produced no inhibition of the effect of S()-nicotine, such that its IC50 value was not included in the linear regression analysis (r2 = 0.940). n = 3 to 10 rats/analog.

Mechanism of N-n-Alkylnicotinium Inhibition. The competitive versus noncompetitive nature of NONI inhibition of the high-affinity component of S()-nicotine-evoked [³H] overflow was determined by Schild analysis. NONI was considered to be a representative inhibitor from the N-n-alkylnicotinium series. The S()-nicotine concentration-response relationship was determined in the absence and presence of three NONI concentrations (0.1, 1, and 10 µM) (Fig. 7, top panel). Inclusion of NONI (1 and 10 µM) in the buffer produced rightward shifts in the concentration-response curves for S()-nicotine relative to the response curves observed for the control condition (in the absence of NONI) or in the presence of 0.1 µM NONI. Eadie-Hofstee transformation of these data (Fig. 7, bottom panel) was also plotted (minus control and 100 µM S()-nicotine data) to better visualize shifts in the S()-nicotine concentration-response relationship in the presence of NONI. NONI produced a left- and downward shift in the overall S()-nicotine concentration-response profiles, suggesting that both high- and low-affinity components of S()-nicotine-evoked [³H] overflow were inhibited by inclusion of NONI in the assay buffer.

View larger version (26K): [in this window] [in a new window]   Fig. 7.   NONI (C8 analog) competitively inhibits S()-nicotine-evoked [3H] overflow from superfused rat striatal slices. Superfusion buffer contained nomifensine (10 µM) and pargyline (10 µM) from the start of superfusion. After 60 min of superfusion, slices were superfused in the absence or presence of NONI (0.1, 1, or 10 µM) for a 60-min period prior to the addition of S()-nicotine (1 nM-100 µM) to the buffer, and superfusion continued for an additional 60 min. In each experiment examining the effect of a single concentration of NONI, an independent set of striatal slices was superfused with 1 nM to 100 µM S()-nicotine in the absence of NONI, serving as the S()-nicotine control condition. Since the S()-nicotine controls [i.e., concentration response for S()-nicotine in the absence of NONI] were not significantly different (F2,91 = 0.4032, p = 0.669) among the series of experiments, these data were pooled for graphical presentation. An additional striatal slice in each experiment was superfused in the absence of either drug and served as the buffer control (CON). Data are expressed as mean ± S.E.M. total [3H] overflow during the 60-min period of exposure to S()-nicotine in the absence and presence of NONI as a function of S()-nicotine concentration (top). Schild regression plot of the high-affinity component of S()-nicotine-evoked [3H] overflow is presented in the inset (top). Data are also presented in the form of the Eadie-Hofstee plot of total [3H] overflow  S()-nicotine concentration (bottom), derived from the hyperbolic fit to nontransformed data presented in the inset. Data from the highest concentration (100 µM) of S()-nicotine tested were not included in these plots. n = 3 to 8 rats/NONI concentration.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

The current study demonstrates that S()-nicotine evokes [³H] overflow from [³H]DA-preloaded rat striatal slices in a time- and concentration-dependent manner. Previous studies have suggested the involvement of non-nicotinic receptor-mediated DA release at high S()-nicotine concentrations (~100 µM); whereas concentrations of ~10 µM nicotine evoke DA release that is completely inhibited by mecamylamine and DHE, indicating that this latter response is via a nicotinic receptor-mediated mechanism (Westfall, 1974; Grady et al., 1992; Teng et al., 1997). The current study provides a more detailed analysis of the response to 0.1 to 10 µM S()-nicotine using Eadie-Hofstee analysis, which revealed a biphasic curve, suggesting the contribution of more than one process underlying this response. Fitting a hyperbolic function to the data provided two components: a high-affinity (EC₅₀ = 100 nM), low-capacity (maximal overflow = 0.53% tissue [³H]) component, and a low-affinity (EC₅₀ = 360 µM), high-capacity (maximal overflow = 50% tissue [³H]) component. The EC₅₀ value obtained for the high-affinity component is comparable with that reported by others (480 nM-5 µM) using rat and mouse striatal synaptosomal and slice preparations (Grady et al., 1992; Teng et al., 1997; Wonnacott et al., 2000). These results suggest that S()-nicotine interacts with either two conformational forms of a single subtype of nAChR, or with two different nAChR subtypes, to evoke DA release. The involvement of more than one nAChR subtype in nicotine-evoked DA release has been proposed by others (Kulak et al., 1997; Kaiser et al., 1998; Sharples et al., 2000). Specifically, the Conus magus peptide, -CTX MII, only inhibits ~50% of nicotine-evoked [³H]DA release from rat striatal synaptosomes (Kulak et al., 1997; Kaiser et al., 1998). -CTX MII has been shown to be a potent and selective antagonist of acetylcholine-evoked currents in Xenopus laevis oocytes expressing the 32 receptor subtype (Cartier et al., 1996), implicating this subtype as at least one of the possible 2-containing presynaptic nAChRs mediating S()-nicotine-stimulated DA release. Recent work by Sharples et al. (2000) indicates that the 42* nAChR may mediate the -CTX MII-insensitive component of nicotinic agonist-evoked [³H]DA release. The identity of other nAChR subtypes located on the dopaminergic presynaptic terminals, which mediate this effect of S()-nicotine (such as those containing the 6 subunit), remains speculative.

Evidence indicates that high micromolar concentrations of S()-nicotine also activate 7* homomeric nAChRs to evoke [³H]DA release via an indirect mechanism, i.e., direct stimulation of the release of glutamate, which in turn indirectly stimulates DA release (McGehee et al., 1995; Gray et al., 1996). Furthermore, glutamate receptor antagonists have been shown to inhibit ~20 to 50% of nicotine-evoked [³H]DA release from striatal slices but not from striatal synaptosomes (Wonnacott et al., 2000), indicating that local circuitry within the slice is sufficient to reveal this indirect action of nicotine on dopaminergic neurotransmission. These results further suggest that the low-affinity component observed in the present assay may not be due entirely to nonspecific factors but also to activation of 7* homomeric nAChRs. The present study, however, focused on the high-affinity component of S()-nicotine-evoked striatal [³H]DA release for characterization of the mechanism of inhibition produced by these novel N-n-alkylnicotinium analogs. As previously discussed, the 32* subtype appears to be a strong candidate for mediation of the inhibition of the high-affinity component of S()-nicotine-evoked [³H]DA release from rat striatal slices.

An important contribution of the current research is the emergence of a novel class of nAChR antagonists based upon a specific structural modification of the S()-nicotine molecule. This series of N-n-alkylnicotinium analogs was evaluated for intrinsic activity and for inhibition of S()-nicotine-evoked [³H] overflow from [³H]DA-preloaded striatal slices. At relatively high concentrations (10-100 µM), analogs with n-alkyl chain lengths greater than 4 carbons exhibited intrinsic activity in the DA release assay. The concentration of analog that produced intrinsic activity was dependent upon n-alkyl chain length, i.e., the greater the number of carbons, the lower the concentration at which [³H] overflow was evoked. Analogs with n-alkyl chain length of 6 to 8 carbon atoms evoked [³H] overflow at 100 µM concentration, whereas analogs with n-alkyl chains of 10 and 12 carbon atoms evoked [³H] overflow at 10 µM. This effect of the N-n-alkylnicotinium analogs is similar to that observed for high concentrations (100 µM) of DHE (Crooks et al., 1995; present study). Thus, due to intrinsic activity in this assay, the use of high concentrations of either N-n-alkylnicotinium analogs or DHE as nAChR antagonists is not justified, since this intrinsic activity compromises any mechanistic interpretation derived. Furthermore, DA release evoked by high concentrations of either NONI or NDNI was not inhibited by DHE (data not shown), indicating that analog-evoked DA release is not via a nicotinic receptor-mediated mechanism.

The current structure-activity relationships (SARs) reveal that quaternization of the pyridine-N atom of S()-nicotine results in analogs that inhibit S()-nicotine-evoked [³H]DA release, and that analog inhibitory potency varies linearly with n-alkyl chain length. The greater the number of carbon atoms in the n-alkyl chain, the greater the potency (i.e., the lower the IC₅₀ value) for inhibition of S()-nicotine-evoked [³H] overflow. Low-potency antagonists (IC₅₀ >10 µM) possessed n-alkyl groups of 1 to 4 carbons. NDDNI, the C₁₂ N-n-alkyl analog, had the highest potency (IC₅₀ value = 9 nM). Thus, a relatively long, N-n-alkyl chain appears to be an important determinant of antagonist potency in this series of N-quaternized S()-nicotine analogs. The relatively simple, linear relationship revealed an orderly progression in potency from C₁ (NMNI) to C₁₂ (NDDNI). Although regression analysis revealed a linear relationship between N-n-alkyl chain length and potency to inhibit S()-nicotine-evoked [³H] overflow, the N-n-decyl analog, NDNI, unexpectedly did not exhibit inhibitory activity at concentrations devoid of intrinsic activity. This observation indicates that NDNI, unlike all the other analogs in its class, does not inhibit with the nAChR subtype(s) mediating S()-nicotine-evoked DA release. We hypothesize that the N-decyl moiety may exist in solution in a unique conformation compared with analogs with either shorter or longer n-alkyl chain lengths, which may explain its unusual profile. Of further note, the more potent analogs produced 80 to 100% maximal inhibition of the response to S()-nicotine. In comparison, DHE had an intermediate potency relative to the N-n-alkylnicotinium analogs. Thus, N-n-alkylnicotinium analogs constitute a new class of nicotinic receptor antagonist, and the C₁₂ analog, NDDNI, was at least two orders of magnitude more potent than the classical nAChR antagonist, DHE, as an inhibitor of nAChR(s) mediating S()-nicotine-evoked DA release.

The C₈ analog, NONI, was chosen as a representative compound for determining the mechanism of N-n-alkylnicotinium-induced inhibition. S()-Nicotine concentration-response curves were obtained in the absence and presence of three concentrations of NONI. NONI produced a rightward shift in the concentration response curve for S()-nicotine, a left- and downward shift in the Eadie-Hofstee analysis, and the Schild regression was linear with a slope near unity. The pA₂ derived from the x-intercept of the Schild regression was 7.096, providing a respective K_i value for NONI of 80.2 nM. The latter K_i value from the Schild analysis was 8-fold lower than the IC₅₀ value obtained for NONI inhibition of [³H]DA release evoked by 10 µM S()-nicotine. The apparent discrepancy in these values is likely due to the restriction of the Schild analysis to the high-affinity component of S()-nicotine-evoked [³H]DA release, to isolate a subpopulation of nAChR subtype involved. Importantly, the K_i value obtained from the Schild analysis is three orders of magnitude lower than its K_i value for the high-affinity S()-[³H]nicotine binding site (K_i value = ~80 µM; L. H. Wilkins Jr., V. P. Grinevich, J. T. Ayers, P. A. Crooks, and L. P. Dwoskin, manuscript submitted for publication). Thus, these findings indicate that NONI interacts in a competitive manner with the nAChR, mediating the high-affinity component of S()-nicotine-evoked [³H]DA release.

Compared with agonist molecules, it has been proposed that antagonists, which are generally large molecules, dock onto the agonist-binding site but extend beyond the region of agonist binding (Sheridan et al., 1986). The additional structural bulk associated with the antagonist molecules is believed to prevent the receptor protein from achieving the open-channel form (Sheridan et al., 1986). In this respect, the active N-n-alkylnicotinium analogs are of significantly larger molecular weight than S()-nicotine, and the sterically bulky N-n-alkyl chain may interact within a hydrophobic cavity extending outside the normal volume for agonist binding to the receptor. The N-n-alkylnicotinium analogs have been proposed to interact with the 32* nAChR in the unprotonated form, leading to a reversal in the role of the pharmacophoric nitrogen-containing moieties (Crooks et al., 1995). That is, the quaternary pyridinium center of the antagonist molecule interacts with the binding site that normally accommodates the protonated pyrrolidine nitrogen in the agonist-binding mode, and the unprotonated pyrrolidine nitrogen of the antagonist molecule substitutes for the pyridine N of the agonist at the hydrogen-bonding site of the nAChR. Alternatively, these N-n-alkylnicotinium molecules may interact with the S()-nicotine binding site in a manner allowing free positioning of the n-alkyl chain into the receptor ion channel, thereby sterically blocking ion flux through the channel.

In summary, the current research establishes a new class of nAChR antagonist, resulting from structural modification of the S()-nicotine molecule. Members of this nAChR antagonist class exhibit potent and competitive inhibition of the nAChR subtype mediating S()-nicotine-evoked DA release from dopaminergic nerve terminals in striatum. In this novel analog series, the structure-activity relationship reveals that analogs with N-n-alkyl chains ranging from C₇ to C₁₂ were the most potent antagonists of the nAChRs mediating S()-nicotine-evoked DA release. Importantly, the C₁₀ analog, NDNI, was unique within this series, since it did not inhibit S()-nicotine-evoked DA release. Furthermore, NDNI was most potent in inhibiting the high-affinity S()-[³H]nicotine binding site in rat brain (42*; L. H. Wilkins, V. P. Grinevich, J. T. Ayers, P. A. Crooks, and L. B. Dwoskin, manuscript submitted for publication). Currently, drug discovery is focused on nAChRs as novel targets for the development of therapeutic agents for a wide variety of central nervous system diseases. Such novel N-n-alkylnicotinium antagonists may be useful tools in establishing a role for these receptor subtypes in physiological function and for unraveling the complexities of the action of specific nAChR subtypes in physiological function.