Introduction

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Neuronal nicotinic acetylcholine receptors (nAChRs) are members of a ligand-gated ion channel family of receptors, consisting of transmembrane proteins of pentameric structure with potentially diverse composition (Anand et al., 1991). S-()-Nicotine activates all the known subtypes of nAChRs, although with varying affinities (Parker et al., 1998). Heteromeric nAChRs exist as combinations of  and  subunits; however, the exact subunit composition, stoichiometry, and arrangement of the subunits of native nAChRs have not been elucidated conclusively (Lukas et al., 1999).

Great functional diversity is suggested by the identification of 12 genes encoding 2-10 and 2-4 subunits, and from results of in situ hybridization studies revealing discrete, but overlapping, CNS distribution of mRNAs encoding these subunits (Wada et al., 1989; Dineley-Miller and Patrick, 1992; Séguéla et al., 1993; Le Novère et al., 1996). In addition to heteromeric nAChRs, homomeric nAChRs also are present in the CNS and are believed to consist of 7, 8, or 9 subunits; the 7* nAChR is one of the most abundant nAChR subtypes in brain (Wada et al., 1989; Flores et al., 1992). The 7* nAChR subtype is sensitive to inhibition by -bungarotoxin and methyllycaconitine (MLA) (Schoepfer et al., 1990; Orr-Urtreger et al., 1997; Davies et al., 1999); [³H]MLA has been reported to be a useful radioligand for probing this nAChR subtype (Davies et al., 1999).

The 42* subtype is also a predominant nAChR in the CNS and is probed by high- affinity S-()-[³H]nicotine binding. Binding of S-()-[³H]nicotine to nAChRs in homogenates of rodent brain is reversible and stereospecific, and represents a single class of high-affinity sites located at the interface of the / subunits (Lippiello et al., 1987; Reavill et al., 1988; Zhang and Nordberg, 1993). Greater than 90% of high-affinity S-()-[³H]nicotine binding sites are immunoprecipitated with anti-2 antibody (Whiting and Lindstrom, 1987; Flores et al., 1992). Furthermore, mice lacking the 2 subunit do not exhibit high-affinity S-()-[³H]nicotine binding (Zoli et al., 1998). Taken together, these results strongly suggest that 42* is the nAChR subtype probed by S-()-[³H]nicotine binding in brain.

nAChR subunit composition is an important factor that determines the relative affinity of nAChR antagonists at the S-()-[³H]nicotine binding site (Harvey and Luetje, 1996; Harvey et al., 1996; Chavez-Noriega et al., 1997). The relative inhibitory potency of the classic nAChR antagonist, dihydro--erythroidine (DHE), at multiple recombinant nAChRs has provided insight into the contribution of  and  subunits to antagonist sensitivity. DHE inhibition of agonist-induced currents in Xenopus oocytes afforded the following rank order of sensitivity of expressed rat nAChRs: 44 > 42 = 32 > 22 > 24 34 (Harvey and Luetje, 1996; Harvey et al., 1996). Similarly, the rank order for DHE inhibition of expressed human nAChR was 44 > 42 > 22 = 32 = 24 34 (Chavez-Noriega et al., 1997). As such, both  and  subunit N-terminal binding domains are important for sensitivity to DHE (Harvey and Luetje, 1996; Harvey et al., 1996). With respect to native receptors in rat brain, [³H]DHE competitively binds to nAChRs with high affinity (K_d ~10 nM; Williams and Robinson, 1984). Taken together, these results indicate that DHE binds to agonist recognition sites on 42* native nAChR receptors.

Recently, exciting developments in drug discovery have indicated that nAChR agonists may be useful for the treatment of cognitive dysfunction, neurodegeneration, and other CNS diseases (Lloyd and Williams, 2000; Glennon and Dukat, 2000). However, relatively little attention has focused on the development of nAChR antagonists as drug candidates (Dwoskin et al., 2000; Dwoskin and Crooks, 2001). Our previous research has discovered a new class of nAChR antagonists resulting from N-n-alkylation of the S-()-nicotine molecule (Dwoskin et al., 1999). These S-()-nicotine analogs exhibit potent and competitive inhibition of the nAChR subtype (362* subtype) mediating S-()-nicotine-evoked dopamine (DA) release from dopaminergic nerve terminals in striatum (Wilkins et al., 2002). Structure-activity relationships (SARs) reveal that analogs with N-n-alkyl chains ranging from C₇ to C₁₂ were the most potent antagonists of native 362* nAChRs. The C₁₀ analog, N-n-decylnicotinium iodide (NDNI), was unique in this respect, since it did not inhibit S-()-nicotine-evoked DA release. To determine the nAChR-subtype selectivity of this new class of N-n-alkylnicotinium antagonists, the current study evaluated the ability of these antagonists to inhibit high-affinity S-()-[³H]nicotine binding to rat striatal membranes (42* subtype) and to inhibit [³H]MLA binding to rat whole brain membranes (7* subtype). Analog affinity was compared with that of the classic antagonist, DHE, and the mechanism of interaction of two N-n-alkylnicotinium analogs, NDNI and N-n-octylnicotinium iodide (NONI), with 42* nAChRs was also determined.

	Materials and Methods

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Materials. DHE, S-()-nicotine di-d-tartrate, HEPES, Tris[hydroxymethyl]aminomethane hydrochloride (Trizma HCl), Tris[hydroxymethyl]aminomethane base (Trizma), and polyethylenimine (PEI) were purchased from Sigma/RBI (Natick, MA). R-(+)-Nicotine was purchased from Toronto Research Chemicals (Toronto, ON, Canada). S-()-[³H]Nicotine (S-()[N-methyl-³H]; specific activity, 80 Ci/mmol and (±)-[³H]methyllycaconitine ([1,4(S),6,14,16]-20-ethyl-1,6,14,16-tetramethoxy-4[[[2-([3-³H]methyl-2,5-dioxo-1-pyrrolidinyl)benzoyl]oxy]methyl]aconitane-7,8-diol); specific activity, 25.4 Ci/mmol; [³H]MLA) were purchased from PerkinElmer Life Sciences (Boston, MA) and Tocris Cookson Ltd. (Bristol, U.K.), respectively. Scintillation cocktail 3a70B was purchased from Research Products International Corp. (Mt. Prospect, IL). Remaining chemicals used in the buffers were obtained from Fisher Scientific (Pittsburgh, PA). N-Methylnicotinium iodide (NMNI), N-n-propylnicotinium iodide (NPNI), N-n-butylnicotinium iodide (NnBNI), and NONI were prepared as described by Crooks et al. (1995). N-Ethylnicotinium iodide (NENI), N-n-pentylnicotinium iodide (NPeNI), N-n-hexylnicotinium iodide (NHxNI), N-n-heptylnicotinium iodide (NHpNI), N-n-nonylnicotinium iodide (NNNI), NDNI, N-n-undecylnicotinium iodide (NUNI), and N-n-dodecylnicotinium iodide (NDDNI) were prepared from S-()-nicotine and the appropriate n-alkyl iodide using the general procedure described by Rui et al. (2002). All compounds were fully characterized by elemental analysis and determined to be free from S-()-nicotine utilizing spectroscopic (¹H and ¹³C nuclear magnetic resonance, and fast atom bombardment mass spectroscopy), thin layer chromatographic (silica gel), and combustion analysis procedures. Structures of the N-n-alkylnicotinium analogs are shown in Fig. 1.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Structures of S-()-nicotine, N-n-alkylnicotinium analogs, methyllycaconitine (MLA) and dihydro--erythroidine (DHE). Note that the 2' S configuration of the S-()-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 were housed two per cage with free access to food and water in the Division of Laboratory Animal Resources at the College of Pharmacy, University of Kentucky. Experimental protocols involving animals were in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at the University of Kentucky.

S-()-[³H]Nicotine Saturation Binding. For each experiment, striata from two to four rats were homogenized using a Tekmar Polytron in 10 volumes of ice-cold modified Krebs-HEPES buffer (20 mM HEPES, 118 mM NaCl, 4.8 mM KCl, 2.5 mM CaCl₂, 1.2 mM MgSO₄, pH 7.5). Homogenates were incubated (5 min at 37°C) and centrifuged (29,000g for 20 min at 4°C). Tissue pellets were resuspended in 10 volumes of ice-cold Milli-Q water (Millipore Corp., Bedford, MA), incubated (5 min at 37°C), and centrifuged (29,000g for 20 min at 4°C). The tissue pellets were again resuspended in 10 volumes of ice-cold 10% Krebs-HEPES buffer, then incubated and centrifuged as described. Final tissue pellets were stored at 70°C in fresh 10% Krebs-HEPES buffer until use. Upon assay, pellets were resuspended in 10% Krebs-HEPES buffer, incubated, and centrifuged as previously described. Final pellets were resuspended in 2.0 ml of ice-cold Milli-Q water, and the amount of protein (~200 µg of protein/100 µl of membrane suspension) was determined (Bradford, 1976).

Inhibition of S-()-[³H]Nicotine Binding. Striatal membranes were prepared as previously described. Inhibition of specific S-()-[³H]nicotine binding by synthetic N-n-alkylnicotinium analogs was assessed using a previously described method (Crooks et al., 1995). Briefly, assays were performed in triplicate in a final volume of 200 µl of Krebs-HEPES buffer containing 250 mM Tris buffer (pH 7.5, 4°C). Reactions were initiated by the addition of 100 µl of membrane suspension to tubes containing 50 µl of Krebs-HEPES buffer or one of at least seven concentrations (0.1 nM-1 mM, final concentration) of S-()-nicotine, R-(+)-nicotine, DHE or N-n-alkylnicotinium analog and 50 µl of S-()-[³H]nicotine (3 nM, final concentration). Nonspecific binding was determined in triplicate in the presence of 10 µM S-()-nicotine. Following incubation (90 min at 4°C), reactions were terminated by dilution of samples with ice-cold Krebs-HEPES buffer followed by immediate filtration through Whatman GF/B glass fiber filters (presoaked in 0.5% PEI) using the cell harvester. Filters were processed and radioactivity was determined as previously described.

Inhibition of [³H]MLA Binding. Whole rat brain (minus cortex, striatum, and cerebellum) was homogenized in 20 volumes of ice-cold hypotonic buffer (2 mM HEPES, 14.4 mM NaCl, 0.15 mM KCl, 0.2 mM CaCl₂, and 0.1 mM MgSO₄, pH 7.5). Homogenates were incubated at 37°C for 10 min and centrifuged (25,000g for 15 min at 4°C). Pellets were washed three times by resuspension in 20 volumes of the same buffer, followed by centrifugation using the above parameters. Final pellets were resuspended in incubation buffer to provide ~150 µg of protein/100 µl membrane suspension. Binding assays were performed in duplicate, in a final vol of 250 µl of incubation buffer, containing 20 mM HEPES, 144 mM NaCl, 1.5 mM KCl, 2 mM CaCl₂, 1 mM MgSO₄, and 0.05% bovine serum albumin, pH 7.5. Assays were initiated by the addition of 100 µl of membrane suspension to 150 µl of sample containing 2.5 nM [³H]MLA and one of at least six concentrations (30 nM-100 µM, final concentration) of analog, and incubated for 2 h at room temperature. Nonspecific binding was determined in the presence of 10 µM MLA. Assays were terminated by dilution with 3 ml of ice-cold incubation buffer followed by immediate filtration through Schleicher and Schuell (Keene, NH) #32 glass fiber filters (presoaked with 0.5% PEI) using the cell harvester. Filters were processed as described above in the S-()-[³H]nicotine binding assay.

Mechanism of Analog Inhibition of S-()-[³H]Nicotine Binding. Striatal membrane homogenates were prepared as previously described. Saturation of specific S-()-[³H]nicotine binding was determined in the absence and presence of three concentrations of NDNI or NONI. Reactions were initiated by addition of 100 µl of membrane suspension to tubes containing 50 µl S-()-[³H]nicotine (0.625-20 nM, final concentration) and 50 µl of Krebs-HEPES buffer (control, i.e., absence of analog) or one of three concentrations of NDNI or NONI. Concentrations of NDNI (0.6, 2, and 6 µM) and NONI (0.66, 2.2, and 22 µM) were chosen based on results obtained from the inhibition isotherms. Nonspecific binding at each S-()-[³H]nicotine concentration was determined in duplicate in the presence of 10 µM S-()-nicotine. Following incubation (90 min at 4°C), reactions were terminated and filters processed as previously described.

Analysis of S-()-[³H]Nicotine Binding Data. For S-()-[³H]nicotine saturation binding isotherms, specific S-()-[³H]nicotine binding was expressed as femtomoles per milligram of protein and plotted as a function of S-()-[³H]nicotine concentration. Data were fitted by one- and two-site hyperbolic functions using weighted (1/Y² minimized), nonlinear least-squares regression, since the nonweighted fit minimizing Y² was not unique. One-site binding was modeled using the equation, Y = (B_max · X)/(K_d + X), where Y = specific S-()-[³H]nicotine binding, X = S-()-[³H]nicotine concentration, B_max = maximum binding density, and K_d = the dissociation binding constant. Two-site binding was modeled using the equation, Y = (B_max1 · X)/(K_d1 + X) + (B_max2 · X)/(K_d2 + X), where B_max1 and B_max2 = maximum binding density for sites 1 and 2, respectively, and K_d1 and K_d2 = dissociation binding constants for sites 1 and 2, respectively. Fits were compared using the F statistic, and the one-site model was chosen unless the two-site model provided a significantly (p < 0.05) better fit.

(log EC₅₀X) · n

Analyses of Analog Binding Inhibition Data. For N-n-alkylnicotinium analog inhibition of S-()-[³H]nicotine binding, data were expressed as specific S-()-[³H]nicotine bound as a percentage of control and plotted as a function of log analog concentration. Data were fit by a variable slope model, using nonlinear least-squares regression, such that Y = Bt + ((Tp  Bt)/(1 + 10)^{(log IC₅₀X) · n})), where Y = specific S-()-[³H]nicotine binding, X = log [S-()-[³H]nicotine], Bt and Tp = minimum and maximum specific S-()-[³H]nicotine binding densities, respectively, log IC₅₀ = log[compound], which decreased S-()-[³H]nicotine receptor occupancy by 50%, and n = the pseudo-Hill coefficient. Analog affinity constants (K_i values) were calculated using the equation, K_i = IC₅₀/(1 + concentration of S-()-[³H]nicotine/K_d), where IC₅₀ = the concentration of analog inhibiting S-()-[³H]nicotine binding by 50% and K_d = the S-()-[³H]nicotine dissociation constant determined from initial saturation binding experiments (Cheng and Prusoff, 1973).

X  log IC₅₀

Correlation of N-n-Alkylnicotinium Inhibition of S-()-[³H]Nicotine Binding to Striatal Membranes and S-()-Nicotine-Evoked [³H]DA Overflow from Superfused Striatal Slices. To evaluate the nAChR subtype selectivity of this novel series of analogs, log IC₅₀ values for each analog to inhibit S-()-nicotine-evoked [³H]DA overflow, which were obtained from a previous report (Wilkins et al., 2002), were plotted as a function of the log IC₅₀ values derived from S-()-[³H]nicotine binding inhibition curves (Fig. 2). Pearson's analysis of these data was used to detect the presence of a significant correlation between these nAChR sites.

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Saturation binding of S-()-[3H]nicotine to rat striatal membranes. Binding curves showing specific () and nonspecific () S-()-[3H]nicotine binding. Inset, Scatchard plot of specific S-()-[3H]nicotine binding. Data represent the mean ± S.E.M. of at least four independent observations.

	Results

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S-()-[³H]Nicotine Saturation Binding. Binding of S-()-[³H]nicotine to rat striatal membranes was saturable and specific (Fig. 2). Nonspecific binding was ~12% of total binding at a S-()-nicotine concentration approximating the K_d. A one-site hyperbolic model provided the best fit to the data, suggesting that equilibrium binding of S-()-[³H]nicotine represents interaction with a single population of binding sites. Values for affinity (K_d, 95% confidence interval) and maximum density (B_max ± S.E.M.) were 1.94 (1.36, 2.51) nM and 97.6 (±4.7) fmol/mg protein, respectively. Variable slope sigmoid fit (R² = 0.995) of specific S-()-[³H]nicotine binding as a function of log S-()-[³H]nicotine concentration provided similar estimates for K_d and B_max of 1.56 (0.92, 2.64) nM and 89.8 (±5.9) fmol/mg protein, respectively, with a Hill coefficient of 0.963 (r² = 1). Linear regression (r² = 0.948) of the Scatchard-transformed data provided similar estimates of K_d and B_max values of 1.94 (1.31, 2.56) nM and 98.1 (±5.2) fmol/mg protein (Fig. 2, inset).

N-n-Alkylnicotinium Analog Inhibition of Specific S-()-[³H]Nicotine Binding. Inhibition curves for N-n-alkylnicotinium analogs as well as the reference compounds S-()-nicotine, R-(+)-nicotine, and DHE are shown in Fig. 3. Comparisons were made between simple- and variable-slope sigmoid curve fits to the data for each compound and were found not different, with exceptions of DHE (F_1,5 = 18.5, p < 0.05) and S-()-nicotine (F_1,6 = 28.9, p < 0.05), suggesting a more complex interaction of these two compounds with S-()-[³H]nicotine binding sites. Inhibition parameters and slope factors are provided in Table 1. Similar to the reference compounds, the N-n-alkylnicotinium analogs completely inhibited S-()-[³H]nicotine binding. S-()-Nicotine had the highest affinity (K_i = 0.8 nM) of the compounds tested. R-(+)-Nicotine had an affinity (K_i = 49 nM) ~60-fold lower than that of S-()-nicotine, indicating a high degree of enantioselectivity for nicotine at high-affinity S-()-[³H]nicotine binding sites. Analog affinities varied across a 230-fold range (K_i values ranged from 93 nM to 20 µM; Table 1). From the series, the C₁₀ analog NDNI exhibited the greatest affinity, with a K_i value of 93 nM, which was not different from either the C₁₂ analog NDDNI (K_i = 140 nM) or DHE (K_i = 143 nM). The remaining synthetic analogs exhibited lower affinities, with an overall rank order of the compounds tested: S-()-nicotine > R-(+)-nicotine > NDNI = NDDNI = DHE > NHxNI > NNNI = NENI = NHpNI > NMNI > NnBNI > NONI = NPNI (Table 1).

View larger version (37K): [in this window] [in a new window]   Fig. 3.   Inhibition of specific S-()-[3H]nicotine binding to striatal membranes by N-n-alkylnicotinium analogs: comparison with DHE, S-()-nicotine, and R-(+)-nicotine. S-()-[3H]Nicotine binding is expressed as a percentage of binding in the absence of analog (control, CON). Curves are variable-slope sigmoid fits to mean data. For visual clarity, standard errors are not illustrated; however, variability obtained is indicated in Table 1. Symbols in legend for S-()-nicotine, R-(+)-nicotine, and DHE are connected by dashed rather than solid lines. Legend (top to bottom) also provides rank order of affinity from highest to lowest. Numbers inside parentheses indicate n-alkyl chain length. Data represent the mean of n = 4-6 independent observations from triplicate measurements at each concentration.

View this table: [in this window] [in a new window]   TABLE 1 Inhibition of specific S-()-[3H]nicotine binding to rat striatal membranes by novel N-n-alkylnicotinium analogs: comparison with reference compounds DHE, S-()-nicotine, and R-(+)-nicotine IC50 values were derived from variable-slope, sigmoid curve fits to the mean data from four to six independent observations. Ki values were derived from IC50 values using the Cheng-Prusoff equation.

N-n-Alkylnicotinium Analog Inhibition of [³H]MLA Binding. Results from full concentration-response curves for the analogs to inhibit [³H]MLA binding revealed that only four analogs (NENI, NHxNI, NHpNI, and NONI) at the highest concentration of 100 µM inhibited binding by greater than 50% of total specific binding (Table 2). Based on these results, K_i values were obtained for NENI, NHxNI, NHpNI, and NONI from one-site competition models of the data (Table 2). None of the analogs in this series exhibited high affinity for [³H]MLA binding sites.

Analog Affinity for S-()-[³H]Nicotine Binding Sites as a Function of n-Alkyl Chain Length. A plot of the log transform of the S-()-[³H]nicotine binding inhibition constant (log K_i) as a function of n-alkyl chain length for each analog is presented in Fig. 4. Linear regression of analog affinity (K_i) for S-()-[³H]nicotine binding sites by n-alkyl chain length was performed with NONI (C₈ analog) included and excluded from the analysis, because the K_i value for NONI appeared to deviate from the linear trend. With the log K_i value for NONI included in the analysis, the linear model did not provide a good fit to the data (r² = 0.338), and the slope of the regression line was not significantly different from a value of zero (F_1,8 = 7.08, p < 0.078). When the log K_i value for NONI was excluded, the linear model provided a significantly better fit (r² = 0.560), with a significantly non-zero slope (-0.156; F_1,7 = 8.91, p < 0.05). Therefore, with the exception of NONI, analog affinity varied in a linear fashion with n-alkyl chain length, i.e., affinity increased with increasing chain length.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Linear relationship of N-n-alkylnicotinium analog affinity for S-()-[3H]nicotine binding sites and n-alkyl chain length. When the log Ki value for NONI was excluded from the analysis, the linear model provided a significantly better fit (r2 = 0.560) than when NONI was included. Thus, analog affinity for S-()-[3H]nicotine binding sites in rat striatum varies in a linear fashion with n-alkyl chain length. Each point represents the log Ki value derived from nonlinear curve fitting of the inhibition data shown in Fig. 3. Symbols representing each analog are as indicated in Fig. 3.

Mechanism of N-n-Alkylnicotinium Analog-Induced Inhibition of S-()-[³H]Nicotine Binding. Within the series of analogs examined, the C₁₀ and C₈ analogs, NDNI and NONI, respectively, represent extremes in affinity for S-()-[³H]nicotine binding sites. The mechanism by which NDNI and NONI interact with high-affinity S-()-[³H]nicotine binding sites was determined by assessing shifts in the S-()-[³H]nicotine concentration-binding curves in the presence of three concentrations of NDNI or NONI relative to control (in the absence of analog). NDNI and NONI concentrations were chosen based on K_i values determined in the previous inhibition studies (Table 2). S-()-[³H]Nicotine saturation binding isotherms were observed to be parallel in the absence and presence of analog, as evidenced by the better fit of the data to the simple slope sigmoid model compared with the variable slope model (Table 3).

View this table: [in this window] [in a new window]   TABLE 3 Specific S-()-[3H]nicotine binding affinity (Kd) and maximum densities (Bmax) in the absence and presence of the N-n-alkylnicotinium analogs NDNI and NONI Parameters were determined from studies using rat striatal membranes. Kd and Bmax values were derived from linear regression analysis of the Scatchard-transformed data from saturation binding isotherms. F-statistics were calculated for comparison of simple- vs. variable-slope sigmoid curve fits.

View larger version (25K): [in this window] [in a new window]   Fig. 5.   Scatchard transforms of saturation S-()-[3H]nicotine binding isotherms in the absence and presence of three concentrations of NDNI (top panel) and NONI (bottom panel). Concentrations of NDNI (0.6, 2 and 6 µM) and NONI (0.66, 2.2 and 22 µM) were chosen based on Ki values obtained from inhibition curves shown in Fig. 2. Each point represents the mean values for n = 4 - 5 independent observations from duplicate measurements at each concentration of S-()-[3H]nicotine. Values for Kd and Bmax derived from these linear Scatchard analyses are shown in Table 3.

View larger version (22K): [in this window] [in a new window]   Fig. 6.   S-()-[3H]Nicotine affinity (log Kd) as a function of NDNI or NONI concentration. Kd values were derived from nonlinear fits to the saturation binding isotherms in the absence and presence of three concentrations of NDNI or NONI. The simple model of competitive interaction provided the best fit to data sets for each analog.

Correlation of N-n-Alkylnicotinium Inhibition of S-()-[³H]Nicotine Binding to Striatal Membranes and S-()-Nicotine-Evoked [³H]DA Overflow from Superfused Striatal Slices. To evaluate the nAChR subtype selectivity of this series of analogs, data from both the present S-()-[³H]nicotine binding assays and previously reported S-()-nicotine-evoked [³H]DA overflow assays (Wilkins et al., 2002) were analyzed by correlation analysis, and the results are shown in Fig. 7. NDNI did not inhibit S-()-nicotine-evoked [³H]DA overflow and, as such, the highest concentration (100 µM) examined in the current study was included in the correlation analysis. Correlation of IC₅₀ values for inhibition of S-()-nicotine-evoked [³H]DA overflow and of IC₅₀ values for inhibition of S-()-[³H]nicotine binding was not significant (Pearson r = 0.255, p > 0.05). Another correlation analysis was performed subsequently, in which the data for NDNI and NONI were excluded as outliers. Upon exclusion of NDNI and NONI, a significant correlation was obtained (Pearson r = 0.855, p < 0.05). Therefore, the lack of correlation observed when data for NDNI and NONI were included in the analysis suggests that inhibition of S-()-nicotine-evoked [³H]DA overflow is not well correlated with inhibition of S-()-[³H]nicotine binding. This interpretation is supported by the observations that NDNI did not inhibit S-()-nicotine-evoked [³H]DA overflow but potently inhibited S-()-[³H]nicotine binding. Also, NONI did not inhibit S-()-[³H]nicotine binding but potently inhibited nicotine-evoked [³H]DA overflow. Interestingly, when the correlation analysis was performed excluding NDNI and NONI, a relationship was revealed suggesting that the remaining analogs interact with common nAChR sites.

View larger version (26K): [in this window] [in a new window]   Fig. 7.   Lack of correlation of N-n-alkylnicotinium analog inhibition of S-()-nicotine-evoked [3H]DA overflow from superfused striatal slices with inhibition of S-()-[3H]nicotine binding to striatal membranes. Each point represents the log IC50 value derived from nonlinear curve fitting of the inhibition data for S-()-[3H]nicotine binding (Fig. 3) and for the S-()-nicotine-evoked [3H]DA overflow (taken from Wilkins et al., 2002). NDNI did not inhibit S-()-nicotine-evoked [3H]DA overflow, and as such, the highest concentration (100 µM) examined was used in the Pearson's correlation analysis. Symbols representing each analog are as indicated in Fig. 3.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The current study demonstrates that N-n-alkylnicotinium analogs with n-alkyl chain lengths from 1 to 12 carbons, which have been previously shown to act as antagonists at 362* nAChRs mediating S-()-nicotine-evoked [³H]DA overflow (Wilkins et al., 2002), have varying affinity for S-()-[³H]nicotine binding sites in striatum. A linear relationship was observed such that with increasing n-alkyl chain length, affinity for the 42* nAChR subtype increased. The most potent analog was NDNI (C₁₀ analog; K_i value = 90 nM). In contrast, the analogs did not exhibit high affinity for [³H]MLA binding sites, indicating low affinity for 7* nAChRs. Furthermore, the present study demonstrates that the interaction of these analogs with high affinity S-()-[³H]nicotine binding sites on the 42* subtype is via a competitive mechanism. Importantly, NDNI did not inhibit S-()-nicotine-evoked [³H]DA overflow from superfused striatal slices and, thus, appears to be selective for the 42* subtype. In contrast, NONI (C₈ analog) had low affinity for S-()-[³H]nicotine binding sites but potently inhibited S-()-nicotine-evoked [³H]DA overflow (IC₅₀ = 0.62 µM), demonstrating selectivity for 362* nAChRs mediating S-()-nicotine-evoked [³H]DA overflow.

Affinity and maximum binding density estimates (1.94 nM and 97.6 fmol/mg protein, respectively) obtained from S-()-[³H]nicotine saturation binding analysis were modeled best by a one-site hyperbolic function. Linear Scatchard transformation and Hill coefficient of 0.963 indicated an interaction with a single class of binding sites. Current parameter estimates were within the range of reported values, using either striatal or whole brain membranes (K_d = 0.4-14 nM; B_max = 55-200 fmol/mg protein; Lippiello et al., 1987; Martino-Barrows and Kellar, 1987; Reavill et al., 1988). Variability in K_d and B_max estimates may be attributed to methodological differences in assays employed (e.g., inclusion of protease or cholinesterase inhibitors in tissue preparation buffers, variation in Mg²⁺ and Ca²⁺ concentrations in binding buffers). Validation of current assay conditions was provided by comparable K_i values for S-()-nicotine, R-(+)-nicotine, and DHE (K_i = 0.8, 50, and 140 nM, respectively), as reported by others (Martino-Barrows and Kellar, 1987; Reavill et al., 1988). Thus, the present assay reliably measured interaction with high-affinity S-()-[³H]nicotine binding sites in striatum.

Inhibition of specific S-()-[³H]nicotine binding by S-()-nicotine, R-(+)-nicotine, DHE, and each N-n-alkylnicotinium analog was modeled using a variable-slope sigmoid equation. With the exception of S-()-nicotine and DHE, slope factors derived were near unity for R-(+)-nicotine and each of the analogs examined, indicating a simple competitive interaction with high-affinity S-()-[³H]nicotine binding sites. In contrast, DHE and S-()-nicotine exhibited shallow slopes (n < 1), suggesting that these compounds recognize either more than one agonist binding site on a single 42* receptor, or more than one conformational state of the 42* nAChR, or more than one nAChR subtype. Interestingly, multiple binding sites for [³H]DHE in rat cortical membranes have been detected (Williams and Robinson, 1984), suggesting that DHE distinguishes more than one binding site or more than one state of the 42* nAChR. In the latter study, a pseudo-Hill coefficient of 0.9 for S-()-nicotine inhibition of [³H]DHE binding was observed, suggesting that S-()-nicotine interacts with one of multiple [³H]DHE sites.

N-Alkylation of the S-()-nicotine molecule converts this potent agonist into analogs that selectively and competitively interact with 42* nAChRs. N-n-Alkylnicotinium analogs exhibited affinities for S-()-[³H]nicotine binding sites across an ~200-fold concentration range, from ~90 nM (NDNI) to ~20 µM (NONI). The relationship between analog affinity for S-()-[³H]nicotine binding sites was a linear function of n-alkyl chain length, with the exception of NONI. These N-n-alkylnicotinium analogs are larger molecules than S-()-nicotine, and those with longer chain lengths (C₉, C₁₀, and C₁₂) were more potent inhibitors of S-()-[³H]nicotine binding than those with shorter chain lengths (C₁-C₇). The higher affinity of the longer n-alkyl chain analogs may reflect a stronger association with agonist binding sites on 42* nAChRs, due to increased lipophilic interaction of the carbon chain with a hydrophobic amino acid-rich region of the protein near the binding pocket. As such, this lipophilic interaction may stabilize the analog-receptor complex, thereby increasing the affinity for the agonist recognition site by long-chain analogs.

The mechanism of N-n-alkylnicotinium analog interaction with S-()-[³H]nicotine binding sites was determined using two analogs exhibiting extreme K_i values (K_i = 90 nM and 20 µM for NDNI and NONI, respectively). NDNI and NONI differ structurally by only two methylene units in the alkyl chain length. Scatchard analyses of S-()-[³H]nicotine saturation binding in the absence and presence of NDNI or NONI demonstrated that affinity of S-()-[³H]nicotine for its binding sites decreased in the presence of increasing concentrations of analog, with no change in B_max value. The competitive interaction of NDNI and NONI with high affinity S-()[³H]nicotine binding sites was assessed further using the Lew and Angus analysis, which alleviates concern regarding variability in the estimate of B_max. The simple, one-site model best fit the data, corroborating the interpretation of a competitive interaction of NDNI and NONI with high-affinity S-()-[³H]nicotine binding sites. These results suggest that NDNI and NONI competitively interact with either specific amino acid residues directly involved in S-()-[³H]nicotine binding or with nearby residues allowing for steric hindrance of the interaction of S-()-[³H]nicotine with its high-affinity binding site.

To evaluate nAChR subtype selectivity of the N-n-alkylnicotinium analogs, correlation analysis of data from both the present S-()-[³H]nicotine binding assays and previously reported S-()-nicotine-evoked [³H]DA overflow assays (Wilkins et al., 2002) revealed that NDNI and NONI stand out as selective analogs for 42* and 362* nAChR subtypes, respectively. NDNI exhibited high affinity for S-()-[³H]nicotine binding sites but did not inhibit S-()-nicotine-evoked [³H]DA overflow. On the other hand, NONI was a potent inhibitor of S-()-nicotine-evoked [³H]DA overflow but exhibited low affinity for S-()-[³H]nicotine binding sites. Thus, NDNI and NONI appear to be excellent lead compounds for probing agonist recognition sites on 42* and 362* nAChRs, respectively.

The 362* subtype has been suggested to mediate S-()-nicotine-evoked DA release primarily based on sensitivity to the 362-selective antagonists, neuronal bungarotoxin (Schulz and Zigmond, 1989; Grady et al., 1992) and -conotoxin MII (Cartier et al., 1996). The 362 selectivity of these antagonists is indicated by activity in recombinant systems expressing specific nAChR subtypes (Luetje et al., 1990; Cartier et al., 1996) and by results from nAChR knockout mice studies (Champtiaux et al., 2002; Picciotto and Corrigall, 2002). Importantly, -conotoxin MII only partially inhibited nicotine-evoked DA release (Kulak et al., 1997), suggesting the potential involvement of other nAChR subtypes in this response, such as 4-, 2-, and 4-containing nAChRs (Picciotto et al., 1998; Sharples et al., 2000). Rat substantia nigra neurons express mRNA for 3, 4, 5, 6, 7, 2, 3, and 4 subunits (Wada et al., 1989; Dineley-Miller and Patrick, 1992; Charpantier et al., 1998). Inasmuch as high levels of 6 and 3 mRNAs are expressed in substantia nigra DA neurons (Le Novère et al., 1996; Goldner et al., 1997; Charpantier et al., 1998), their potential combination with 3 and 2 subunits in the mediation of S-()-nicotine-evoked DA release in striatum is probable, but has not been established conclusively.

The current results show that N-n-alkylnicotinium analogs interact with both 42* and 362* nAChR subtypes, but not with the 7* nAChR subtype. As was observed in the interaction of these analogs with 362* (Crooks et al., 1995; Wilkins et al., 2002), the current SAR reveals that an increase in affinity at 42* nAChRs is dependent on increasing n-alkyl chain length. Whereas the current receptor binding assays do not provide information as to whether these N-n-alkylnicotinium analogs function as agonists or antagonists at 42* receptors, recently, these analogs have been observed to inhibit S-()-nicotine-evoked ⁸⁶Rb⁺ efflux from preloaded rat thalamic synaptosomes, a functional assay for 42* receptors, suggesting an antagonist mechanism of action (L. H. Wilkins, D. K. Miller, J. T. Ayers, P. A. Crooks and L. P. Dwoskin, manuscript submitted for publication). The results suggest that the binding site on the 42* subtype that normally accommodates S-()-nicotine also accommodates these charged, more sterically bulky molecules, perhaps in a unique binding mode. As such, the unprotonated form of these analogs was proposed previously to interact with the 362* subtype, in a manner in which the roles of the pharmacophoric nitrogen-containing moieties are reversed (Crooks et al., 1995; Wilkins et al., 2002). Moreover, comparison of the SAR of the analogs at both the 42* and 362* nAChR subtypes reveals that the selectivity of the subtype interaction cannot be explained simply by lipophilicity alone. Thus, the relative lack of interaction of NONI with 42* and the lack of interaction of NDNI with 362* suggest that each of these molecules exists in a unique molecular conformation that is recognized by one subtype but is not compatible with the other. Based on our current knowledge, it is likely that the  subunit plays a critical role in this surprising selective recognition profile of NDNI and NONI.

In summary, a series of N-n-alkylnicotinium analogs exhibited a wide range of affinity for S-()-[³H]nicotine binding sites representing 42* nAChRs in striatum. When the n-alkyl substituent ranged from C₁ to C₁₂, a linear relationship between n-alkyl chain length and analog affinity was found, with the exception of the C₈ analog, NONI. The ability of NONI to potently inhibit S-()-nicotine-evoked [³H]DA overflow from superfused striatal slices, combined with its low affinity for S-()-[³H]nicotine and [³H]MLA binding sites, suggests selectivity for the 362* nAChR subtype. The C₁₀ analog, NDNI, exhibited the highest affinity for the 42* subtype; however, this analog did not interact with either 362* or 7* subtypes. Selectivity for the 42* subtype combined with competitive interaction with S-()-nicotine binding sites indicates that NDNI is an excellent candidate for studying the structural topography of agonist recognition sites on 42* nAChRs, for establishing the antagonist pharmacophore for this subtype, and for defining its role in physiological function and pathological disease states.