Introduction

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NAChR belong to the family of ligand-gated ion channels (c.f., Sargent, 1993). NAChR are pentameric in structure and are proposed to be composed of two  and three  subunits (Cooper et al., 1991). At least 10 different gene products (2-9 and 2-4) for NAChR subunits have been identified by molecular cloning techniques (Sargent, 1993; Elgoyhen et al., 1994; Schoepfer et al., 1990). The distribution of the various subunits in rat brain, as revealed by in situ hybridization and immunoprecipitation, showed distinct expression patterns for different subunits from the widely distributed 2 subunit as compared to the highly restricted 4 subunit (Wada et al., 1989; Duvoisin et al., 1989). Pair-wise expression of  and  subunits in Xenopus oocytes revealed different pharmacological profiles for the various subunit combinations, suggesting different physiological roles and various receptor subtypes (Luetje and Patrick, 1991; Gerzanich et al., 1995).

Decrements in the numbers of NAChR have been demonstrated in various neurodegenerative diseases such as AD and PD (Whitehouse et al., 1988; Aubert et al., 1992; Lange et al., 1993). Administration of ()-nicotine to AD patients was beneficial in ameliorating the attention and information processing deficits seen in these patients (Sahakian and Coull, 1994; Newhouse et al., 1988). Demonstration of the potential beneficial effects of nicotine injection in the management of PD patients dates back to 1926 (Moll, 1926). Since then, sporadic reports have shown beneficial effects of nicotine in ameliorating PD symptoms (Marshall and Schnieden, 1966; Zdonczyk et al., 1988; Fagerstrom et al., 1994).

The clinical use of nicotine as a therapeutic agent is severely limited by its cardiovascular and neuromuscular side effects (Benowitz, 1986). This is thought to result from its nonselective activation of all subtypes of NAChR. A subtype selective NAChR agonist could therefore prove to be of greater therapeutic value in PD and AD. In addition, subtype selective probes are likely to be useful in defining the physiological role(s) of NAChR subtypes in vivo. Such selective pharmacological tools are starting to emerge. GTS-21 has been recently described to display selectivity for 7 (Hunter et al., 1994; de Fiebre et al., 1995). In addition, ABT-418 has recently been described as an 42 selective agonist (Arneic et al., 1994). We describe the novel NAChR agonist SIB-1765F ([±]-5-ethynyl-3-(1-methyl-2-pyrrolidinyl)pyridine fumarate (fig. 1) that shows a pharmacological profile consistent with it being a subtype selective NAChR agonist.

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Structures of SIB-1765F and ()-nicotine.

	Materials and Methods

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1-[7,8-³H] Norepinephrine ([³H]-NE, 40 Ci/mmol) was purchased from Amersham (Arlington Heights, IL); 3,4-[7-³H] dihydroxyphenylethylamine ([³H]-DA, 20 Ci/mmol), [³H]-Cytisine (38.2 Ci/mmol), [³H]-quinuclidinyl benzilate (QNB; 84.5 Ci/mmol) and [¹²⁵I]--bungarotoxin (BTX; 12-14 mCi/mg), [³H]-di-2-toluoyl guanidine (DTG; 35.2 Ci/mmol), [³H]-pentazocine (31.6 Ci/mmol) were purchased from NEN (Boston, MA). (+)-[³H]-Epibatidine (51.3 Ci/mmol) was purchased from Amersham Inc. (Arlington Heights, IL). ()-Nicotine hydrogen tartrate, mecamylamine HCl, dopamine HCl, dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), d-tubocurarine (d-TC), tacrine, acetylcholinesterase and butyrylcholinesterase were purchased from Sigma Chemical Co. (St. Louis, MO). Dihydro--erythroidine (DHE), nomifensine and bupropion were purchased from Research Biochemicals Inc. (RBI, Natick, MA). All other reagents were of highest quality commercially available. SIB-1765F was synthesized by the SIBIA Department of Medicinal Chemistry as per methods previously published (Cosford et al., 1996). Cell lines expressing human NAChR subunits were supplied by the Molecular Neurobiology Lab at SIBIA (Stauderman et al., 1995).

Animals

Male Sprague-Dawley rats (250-300 g) purchased from Harlan (San Diego, CA) were used throughout the study. The rats were maintained in temperature (22-24°C) and humidity (50-55%) controlled quarters for at least 2 days before use on a 12-hr light-dark cycle. All the experiments using experimental animals in the present investigation were conducted as per institution- (NIH and SIBIA Neurosciences Inc. Animal Care and Use Committee) approved guidelines.

Radioligand Binding Assays

The assays for binding of [³H]-nicotine and [³H]-cytisine, [³H]-QNB and [¹²⁵I]-BTX to rat cortical membranes were conducted as per methods described by Flynn and Mash (1986), Yamamura and Snyder (1974) and Marks et al., (1986), respectively. The assays for binding of [³H]-DTG to rat cortical membranes and [³H]-pentazocine to guinea pig brain cortical membranes were performed as per methods described by Weber et al. (1986). In all assays, an assay buffer (50 mM Tris, pH 7.4 at 25°C) containing in mM; NaCl, 120; KCl, 5; CaCl₂, 2 and MgCl₂, 1 was used. Reactions were terminated by rapid filtration using a Brandell Cell Harvester (Brandel Instruments, Gaithersburg, MD). Nonspecific binding to NAChR, MAChR and BTX-sensitive NAChR was defined by nicotine (10 µM), atropine (1 µM) or BTX (1 µM), respectively. Haloperidol (10 µM) was used to define nonspecific binding to sigma receptors. For NAChR binding assays, the assay buffer also included atropine (1 µM) to block muscarinic sites. Affinities of SIB-1765F to various human NAChR subunit combinations were determined by its ability to displace the binding of (+)-[³H]-epibatidine to human embryonic kidney (HEK293) cells stably expressing human NAChR subunit combinations 42 and 44. Membranes were prepared by scraping cells from 10-cm culture dishes in culture medium and centrifuging for 10 min at 2000 × g. Pellets were resuspended in assay buffer, homogenized and centrifuged for 15 min at 2000 × g. The pellet was frozen at 20°C until use. The assay conditions were as follows: cell membranes (6-100 mg protein), (+)-[³H]-epibatidine (200 pM); incubation time, 2 hr on ice and nonspecific binding was defined by ()-nicotine (10 µM) or (±)-epibatidine (10 nM). The assay was terminated by rapid filtration on GF/C filters soaked in 0.5% polyethylineimine for at least 2 hr. In addition, the selectivity of SIB-1765F was evaluated in ligand binding assays for several neurotransmitters, neuropeptides and lipid mediators (table 1) by Pan Labs Inc. (Bothell, WA) as per validated protocols described in the Pan Labs product literature.

Measurement of Cytosolic Ca⁺⁺ Concentration by the Plate-Based Fluo-3 Assay

Cells stably transfected with expression plasmids encoding human 42 (h42) or h44 NAChR subunits were plated at 2 × 10⁵ cells/well on a 96-well poly-L-lysine-coated microtiter plate. Twenty-four hours later, the cells were washed with 200 µl of HBS (in mM: NaCl, 125; KCl, 5; MgCl₂, 0.62; CaCl₂, 1.8; glucose, 20; HEPES, 20; pH 7.4) and incubated with 30 µl of 20 mM fluo-3-acetoxymethyl ester (fluo-3/AM; Molecular Probes, Inc., Eugene, OR) for 2 hr at 20°C in the dark. The fluo-3/AM stock solution was prepared in HBS containing 2% DMSO and 0.2% pluronic F127. Residual unloaded dye was washed with 200 µl HBS, and subsequently, 180 µl HBS were added to each well. The fluorescence measurements were carried out using a plate-reading fluorimeter (Cambridge Technology, Inc., Watertown, MA). First, the basal fluorescence (F_b) was determined before drug addition. Next, 20 µl of drug solution were added to each well and the fluorescence was recorded for 60 sec at 0.33-sec intervals to determine the peak response (F_p). The maximal fluorescence (F_max) was determined after lysing the cells with Triton X-100 (final concentration of 0.20%). To record the minimum fluorescence (F_min), MnCl₂ was then added to a final concentration of 10 mM. All fluorescence determinations were performed in quadruplicate. The peak and basal cytosolic Ca⁺⁺ concentrations [Ca⁺⁺]_i were calculated according to Grynkiewicz et al. (1985).

Neurotransmitter Release Assays

Superfusion release assays in the various brain areas were conducted as previously described (Sacaan et al., 1995). Briefly, rats were decapitated and the brain rapidly dissected on ice. The specific areas of interest were cross chopped (300 µm) in a McIlwain tissue chopper and incubated in the presence of either 60 nM [³H]-DA or 50 nM [³H]-NE for 30 min at 37°C in Krebs buffer containing (in mM: sodium chloride, 119.5; potassium chloride, 3.3; calcium chloride, 1.3; potassium dihydrogen phosphate, 1.2; magnesium sulfate, 1.2; EDTA, 0.03 and glucose 11.0; pargyline, 0.01) that was continuously gassed with 95% to 5% O₂/CO₂ mixture. Tissue was then transferred to chambers and was continuously superfused with oxygenated buffer. Slices were exposed to test compounds for 3-min intervals and antagonists were applied 9 min before and during the application of test compounds. The fractional efflux of tritium was estimated as the amount of radioactivity in the superfusate fraction relative to the total amount in the tissue, multiplied by 100.

Measurement of Cholinesterase Activity

The potential inhibitory effects of SIB-1765F on cholinesterase activity were evaluated in vitro on human serum butyrylcholinesterase, and human erythrocyte acetylcholinesterase and ex vivo on rat plasma butyrylcholinesterase. Ex vivo analysis of rat plasma butyrylcholinesterase was determined as follows. Six male Sprague-Dawley rats were used for the determination of plasma butyrylcholinesterase. Before compound injection, rats were anesthetized (isofluorane) and approximately 0.5 ml of blood was collected into heparin-containing tubes by retroorbital sinus puncture to establish a base-line plasma cholinesterase level. After a 10- to 15-min recovery from anesthesia, animals were given SIB-1765F (20 mg/kg, s.c.). Fifteen min later, animals were killed by decapitation and trunk blood was collected into heparin containing tubes. The tubes were centrifuged for 10 min at 1000 × g and plasma samples were collected for cholinesterase analysis.

The effects of SIB-1765F on human erythrocyte acetylcholinesterase and human serum butyrylcholinesterase were determined on enzymes purchased from Sigma and used without further purification. Analysis of all cholinesterase activity followed the method of Ellman et al. (1961) as described below, with minor modifications to the buffer conditions.

Nonspecific cholinesterase inhibition was determined by adding 5 µl of test compound (dissolved in DMSO) and 5 µl (3 mU) of cholinesterases (Lin-Trol, Sigma) to 250 µl of BTC reagent (Sigma) in a 96-well plate. BTC reagent contains 5 mM butyrylthiocholine iodide and 0.25 mM 5,5-dithiobis-(2-nitrobenzoic acid) buffered to pH 7.2. Acetylcholinesterase inhibition was assayed by adding 5 µl of test compound (dissolved in DMSO) and 125 µl (6 mU) of human erythrocyte acetylcholinesterase (Sigma) to 125 µl of a buffer containing in mM: Tris, 100 pH 7.4; NaCl, 240; KCl, 10; CaCl₂, 4; MgCl₂, 2; acetylthiocholine, 10 and DTNB, 0.5. Butyrylcholinesterase inhibition was determined by adding 5 µl of test compound (dissolved in DMSO) and 125 µl (6 mU) of human serum butyrylcholinesterase (Sigma) to 125 µl of 2× BTC reagent. Ex vivo rat plasma butyrylcholinesterase inhibition was determined using 230 µl of BTC reagent, and 20 µl of rat plasma. Enzymatic activity and reaction rates were determined by recording the increase in the absorbance at 415 nm on a 96-well plate reader (Bio-Rad Laboratories, model 3350, Hercules, CA), using the Bio-Rad kinetic analysis software package.

[³H]-DA Uptake

[³H]-DA uptake in rat striatal synaptosomes was conducted as described by Izenwasser et al. (1991). Briefly, 100 µl of crude P₂ synaptosomal fraction were preincubated in the presence of varying concentrations of test compounds (SIB-1765F, ()-nicotine, bupropion or nomifensine) for 10 min before the addition of [³H]-DA (final concentration of 10 nM) and the incubation was continued for an additional 5 min in a 37°C shaking water bath. The assay was terminated using a Brandel Cell harvestor and GF/C fiber glass filters (Brandel; Gaithersburg, MD). Samples were counted in a liquid scintillation counter after the addition of 5 ml scintillant.

In Vivo Microdialysis

Stereotaxic surgery. Male Sprague-Dawley rats (200-250 g, Harlan, San Diego, CA) were anesthetized with 2% isofluorane in air and placed in a Kopf rodent stereotaxic apparatus (David Kopf Instruments, Tujunga, CA). The guide cannula (21-gauge; Plastics One, Roanoke VA) was introduced into the left hemisphere through a burr hole in the skull. Implantation coordinates, according to the stereotaxic atlas of Paxinos and Watson (1986), were AP = +2.7 mm, L = +1.4 mm and V = 5.5 mm for the nucleus accumbens and AP = +0.7 mm, L = +2.3 mm and V = 4.0 mm for the striatum. Three small screws were placed into the skull and secured with dental acrylic cement. The incisions were closed with wound clips and animals were placed in bedded plastic cages while recovering from anesthesia and housed separately until tested. Animals were allowed to recuperate from surgery for at least 3 days before the start of experiments.

Microdialysis. Loop-type microdialysis probes, of a 2- and 3-mm lengths (for the nucleus accumbens and striatum, respectively), and 6000 molecular weight cut-off dialysis membrane (ESA, Chelmsford, MA) were used in these studies. Microdialysis probes were inserted in the brain of the animals that were previously implanted with stainless steel guide cannulae under brief isofluorane anesthesia. The dialysis probe, on insertion, extended 2 or 3 mm below the guide cannulae. Experiments were started immediately after probe insertion. Animals were perfused at a constant rate of 1.0 µl/min with artificial CSF, consisting of in mM: NaCl, 145; KCl, 2.7; MgCl₂, 1.2; and CaCl₂, 1.2. Twenty-min fractions were collected for HPLC analysis. After the establishment of a stable baseline for dopamine levels, which takes approximately 4 hr after the implantation of the probe, SIB-1765F was injected s.c. at a dose of 25 mg/kg. Dopamine release under these conditions is known to be tetrodotoxin sensitive and calcium dependent, suggesting a neuronal origin (data not shown).

Dopamine turnover. In time-course experiments, rats (n = 6-10) were killed at selected times (15, 30, 60, 120 and 180 min) after s.c. administration of ()-nicotine (0.4 mg/kg), (±)-epibatidine (3 µg/kg) or SIB-1765F (40 mg/kg) or saline. In dose-response experiments, rats were killed 30 min after s.c. administration of various doses of ()-nicotine (0.05, 0.1, 0.2 and 0.4 mg/kg), (±)-epibatidine (0.1, 0.2, 1 and 3 µg/kg) or SIB-1765F (5, 10, 20 and 40 mg/kg) or saline. For experiments with antagonists, rats were pretreated with mecamylamine at a dose of 3 mg/kg 15 min before the injection of agonist and killed 30 min later. Striatal and olfactory tubercle samples were collected and stored at 70°C until analyzed. Tissue levels of biogenic amines and metabolites were measured by HPLC analysis as per protocols described earlier (Rao et al., 1996) and the levels are expressed as nmol/mg protein (mean ± S.E.M., n = 6-10). All doses refer to free bases, unless otherwise stated.

Data analysis. Data were analyzed using the software Sigma Stat (Jandel, San Rafael, CA) with the Student's t-test, Mann-Whitney U-test or one-way analysis of variance followed by appropriate post hoc tests as detailed in the figure legends. Significance was defined as a P < .05 in all cases. IC₅₀, EC₅₀ and Hill slopes were determined by nonlinear regression analysis, while allowing the top and bottom values to float, using the software Prism (GraphPad, San Diego, CA). K_i values were calculated from IC₅₀ values using the equation of Cheng and Prusoff (1973).

	Results

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Ligand binding. SIB-1765F displaced [³H]-cytisine binding to rat cortical membranes with an K_i of 7.5 ± 1.4 nM. The corresponding value for ()-nicotine was 6.9 ± 1.8 nM (fig. 2A). In contrast, SIB-1765F was far less potent than ()-nicotine in displacing [¹²⁵I]--BTX binding (50% displacement at 100 µM; fig. 2B). SIB-1765F displaced the binding of the muscarinic ligand, [³H]-QNB (fig. 2C) and the sigma ligands, [³H]-pentazocine and [³H]-DTG (table 1), in the range of 5 to 10 µM. The hill slopes for these assays ranged from 0.85 ± 0.1 to 1.1 ± 0.15. In contrast, SIB-1765F did not have any appreciable affinity for several neuropeptide, neurotransmitter and lipid mediator receptors (table 1). SIB-1765F and ()-nicotine displayed similar properties in displacing the binding of (±)-[³H]-epibatidine to HEK293 cells stably expressing h42 NAChR subunits (7.5 ± 2.7 and 4.4 ± 0.8 nM, respectively, P > .05), whereas SIB-1765F showed a nearly 20-fold lower affinity for h44 receptor than did ()-nicotine (235 ± 44 nM vs. 14.3 ± 3.2 nM, respectively, P < .05) (fig. 3).

View larger version (10K): [in this window] [in a new window]   Fig. 2.   Displacement of [3H]-cytisine, [125I]--Btx and [3H]-QNB binding to rat cortical membranes by SIB-1765F and ()-nicotine. Graph depicts a representative experiment performed in triplicate and repeated three to seven times with similar results.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Displacement of [3H]-epibatidine binding to HEK293 cell membranes stably expressing h42 (A) and h44 (B) subunits by ()-nicotine and SIB-1765F. Graph depicts a representative experiment performed in triplicate and repeated five to six times with similar results.

Intracellular calcium. ()-Nicotine and SIB-1765F increased [Ca⁺⁺]_i in HEK293 cells stably expressing h42 or h44 NAChR subunits in a concentration-dependent manner, but had no effect on host HEK293 cells (data not shown). The EC₅₀ values for ()-nicotine and SIB-1765F revealed that the latter was approximately 10-fold more potent at activating h42 containing cells compared to h44 containing cells (table 2). In contrast, ()-nicotine displayed a modest 3-fold potency difference. In addition, SIB-1765F was approximately 20-fold less potent in the RD cells (EC₅₀ = 50 ± 11 µM) that express the neuromuscular nicotinic ACh receptor subtypes (Bencherif and Lukas, 1991) as compared to its activity at h42 cell line.

View this table: [in this window] [in a new window]   TABLE 2 Effect of SIB-1765F on [3H]-epibatidine binding and [Ca+2]i measurement in HEK293 cells stably expressing h42 and h44

Cholinesterase inhibition. SIB-1765F and ()-nicotine did not inhibit human butyrylcholinesterase or human acetylcholinesterase although the prototypical cholinesterase inhibitors tacrine and eserine markedly inhibited the activity of cholinesterase with submicromolar IC₅₀ values (table 3). In addition, ex vivo analysis of rat plasma samples taken 15 min after s.c. injection of SIB-1765F (20 mg/kg) did not show any inhibition of plasma cholinesterase activity. In the same experiment, the cholinesterase inhibitor eserine, at a dose of 0.2 mg/kg, s.c., produced approximately 45% inhibition of serum cholinesterase activity.

View this table: [in this window] [in a new window]   TABLE 3 Effect of ()-nicotine and SIB-1765F on in vitro human cholinesterases activitya

Inhibition of [³H]-DA uptake. SIB-1765F and ()-nicotine did not affect uptake of [³H]-DA into rat striatal crude synaptosomes although the reference compounds, nomifensine and bupropion, inhibited uptake of [³H]-DA with submicromolar IC₅₀ values (table 4).

View this table: [in this window] [in a new window]   TABLE 4 Effect of ()-nicotine and SIB-1765F on [3H]-DA uptake in rat striatal synaptosomesa

Release of [³H]-DA from rat striatum and olfactory tubercles. SIB-1765F produced a concentration-dependent increase in [³H]-DA efflux from rat striatal and olfactory tubercle slices (fig. 4, A and B) with EC₅₀ values 99.6 ± 12.2 and 39.6 ± 9.5 µM, respectively. Data are expressed as % of a maximally effective concentration of ()-nicotine (10 µM) that was run in parallel with SIB-1765F. The maximal response produced by SIB-1765F in the striatum was significantly different from that produced by a maximally effective concentration of ()-nicotine (F 5,18) = 13.5, P < .0001; Dunnett's t test, q = 2.414; P < .05). In the olfactory tubercles, SIB-1765F effect was not significantly different from that produced by ()-nicotine; (P > .05). [³H]-DA release evoked by SIB-1765F was antagonized by Mec (3 µM; P < .05) and DHE (100 µM; P < .05), but not by d-TC (100 µM) (fig. 5; P > .05). Superfusion with antagonists alone did not alter basal neurotransmitter release (data not shown).

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Stimulation of [3H]-DA release from rat striatal slices (A) and rat olfactory tubercle slices (B). Data were expressed as % of an EC100 concentration of ()-nicotine (10 µM) which was run in parallel and was fitted using the program PRISM (Graph Pad) and represent the mean ± S.E.M. of four (striatum) and three (olfactory tubercles) experiments performed in triplicate.

View larger version (28K): [in this window] [in a new window]   Fig. 5.   Effect of NAChR antagonists Mec, d-TC and DHE on SIB-1765F-induced [3H]-DA release. Mec and DHE produced a statistically significant inhibition of SIB-1765F-induced [3H]-DA release from striatal and olfactory tubercle slices although d-TC did not produce a statistically significant inhibition. Columns represent means ± S.E.M. of three independent experiments performed in duplicate. * P < .05 against control, Student's t test.

Release of [³H]-NE from rat hippocampus, prefrontal cortex and thalamus. To investigate the selectivity of SIB-1765F-induced neurotransmitter release, its effects on the release of [³H]-NE from rat hippocampal, prefrontal cortex and thalamic slices were investigated. These studies showed marked regional selectivity of the effect of SIB-1765F on [³H]-NE release. In the hippocampus, SIB-1765F at the highest concentration tested (1 mM) elicited [³H]-NE release that was only 27% of the response of an EC₈₀ concentration of ()-nicotine (fig. 6). Coapplication of SIB-1765F with nicotine did not result in attenuation of ()-nicotine's response (data not shown). In the prefrontal cortex and the thalamus, SIB-1765F was nearly equiefficacious with ()-nicotine (85 and 72%, respectively, of the maximally effective concentration of ()-nicotine) (fig. 6). The effects of SIB-1765F on [³H]-NE release in the prefrontal cortex were significantly attenuated by the removal of extracellular calcium or by Mec (3 µM) (fig. 7).

View larger version (27K): [in this window] [in a new window]   Fig. 6.   Effects of SIB-1765F on [3H]-NE release from rat hippocampal, prefrontal cortex and thalamic slices. Data are expressed as % of the EC80 of ()-nicotine in the hippocampus and the EC100 of ()-nicotine in the prefrontal cortex and the thalamus. Columns represent the mean ± S.E.M. of three different experiments performed in duplicate.

View larger version (15K): [in this window] [in a new window]   Fig. 7.   Effect of Mec and 0 mM extracellular calcium on SIB-1765F-induced [3H]-NE release in rat PFC slices. Both Mec and 0 mM extracellular calcium significantly inhibited the effects of SIB-1765F. Columns with bars represent the mean ± S.E.M. of three to six experiments performed in triplicate. * P < .05 against the respective SIB-1765F alone, one-way ANOVA followed by Dunnett's test.

In vivo microdialysis. SIB-1765F (25 mg/kg s.c.) produced an increase in the extracellular levels of DA in both the striatum and the nucleus accumbens perfusates (183 ± 34 and 920 ± 440% of baseline, respectively; P < .05, Mann-Whitney U test) (fig. 8). Further analysis of the data in the nucleus accumbens indicated that rats segregated into low responders with a peak increase 182 ± 20% of baseline (n = 4) and high responders with a peak increase of 1689 ± 700% of baseline (n = 4). Gross anatomical examination of the probe placement did not reveal any difference between the two groups.

View larger version (16K): [in this window] [in a new window]   Fig. 8.   Effect of SIB-1765F (25 mg/kg, s.c.) on DA release measured by in vivo microdialysis in the striatum and the nucleus accumbens. Animals were implanted with dialysis probes and perfused with artificial CSF at a rate of 1 µl/min. Twenty-µl samples were analyzed by HPLC with electrochemical detection. Data represent the mean ± S.E.M. of 11 rats in the striatum and 8 rats in the nucleus accumbens. * Statistically significant from baseline P < .05, Mann-Whitney U test.

DA turnover. The effects of behaviorally active doses of SIB-1765F (Menzaghi et al., 1995) were studied on striatal and olfactory tubercle DA turnover and compared with those of ()-nicotine and (±)-epibatidine. None of the three treatments affected the steady-state DA levels (data not shown). Changes in HVA closely followed those of DOPAC and are not reported. ()-Nicotine (0.05-0.4 mg/kg, base) and SIB-1765F (5-40 mg/kg, salt) increased the levels of DOPAC, an index of nerve terminal DA metabolism (Sharman, 1981), in striatum and olfactory tubercles in a dose-dependent manner (fig. 9, A and B). (±)-Epibatidine (0.2-3 µg/kg) also significantly increased the levels of DOPAC in the striatum and the olfactory tubercles; however, the effect was not dose dependent (fig. 10A). In addition, the effect of epibatidine did not appear to be stereoselective (fig. 10B).

View larger version (20K): [in this window] [in a new window]   Fig. 9.   Effects of ()-nicotine (A) and SIB-1765F (B) on rat striatal and olfactory tubercles DOPAC levels expressed as a percentage of response from saline-treated animals. Values represent the mean ± S.E.M. (n = 6-10 rats/dose). * P < .05 vs. saline controls, one-way ANOVA followed by Dunnett's test.

View larger version (35K): [in this window] [in a new window]   Fig. 10.   Effects of (±)-epibatidine and its isomers on rat striatal and olfactory tubercles DOPAC levels expressed as a percentage of response from saline-treated animals. Values represent the mean ± S.E.M. (n = 6-10 rats/dose). * P < .05 vs. saline controls, one-way ANOVA followed by Dunnett's test.

View larger version (23K): [in this window] [in a new window]   Fig. 11.   Time related effects of ()-nicotine, SIB-1765F and (±)-epibatidine on rat striatal (A) and olfactory tubercles (B) DOPAC levels expressed as a percentage of response from saline-treated animals. Values represent the mean ± S.E.M. (n = 6-10 rats/dose). *, ** or # P < .05 vs. saline controls, one-way ANOVA followed by Dunnett's test. # SIB-1765F; * ()-nicotine and ** (±)-epibatidine.

View larger version (47K): [in this window] [in a new window]   Fig. 12.   Pretreatment with mecamylamine (3 mg/kg; 15 min) attenuates increases in striatal (A) and olfactory tubercles (B) DOPAC levels in response to s.c. injections of ()-nicotine, SIB-1765F or (±)-epibatidine. DOPAC levels are expressed as a percentage of response from saline-treated animals. Rats were given agonists 15 min after mecamylamine and killed 30 min later. Values represent the mean ± S.E.M. (n = 6-10 rats/dose). * P < .05 vs. saline controls (Dunnett's test); ** P < .05 vs. agonist alone (Neuman-Keul's test).

	Discussion

Top Abstract Introduction Materials & Methods Results Discussion References

Despite great strides in the molecular characterization of NAChR, our understanding of the physiological role of these receptors is hampered in part by the limited availability of selective ligands. The prototypical NAChR agonist, ()-nicotine, has a multitude of pharmacological actions as well as dose limiting side effects that hinder its use in in vivo studies in humans and laboratory animals. There is a need for subtype-selective NAChR ligands with a favorable tolerability profile to both assess the therapeutic potential of such ligands and to delineate the roles of different NAChR subtype in normal physiology. Recently, ligands such as (±)-epibatidine (Badio and Daly, 1994), GTS-21 (Hunter et al., 1994) and ABT-418 (Arneric et al., 1994, 1995) became available to probe the function of NAChR. Although (±)-epibatidine is a desirable ligand due to its high affinity and selectivity (Badio and Daly, 1994; Houghtling et al., 1994, 1995), the in vivo toxicity of this ligand limits its use as a pharmacological probe and therapeutic agent (Rupniak et al., 1994; Rao et al., 1996). GTS-21 and ABT-418 have been proposed as 7- and 42-selective NAChR agonists, respectively (Hunter et al., 1994; de Fiebre et al., 1995; Arneric et al., 1994, 1995). As part of our efforts to develop subtype-selective NAChR agonists with therapeutic potential, we have characterized SIB-1765F, a compound with a novel pharmacological profile and NAChR subtype-selectivity.

The profile of SIB-1765F is quite distinct from ()-nicotine. SIB-1765F binds to NAChRs with nanomolar affinity both in [³H]-nicotine and [³H]-cytisine binding assays. The high affinity NAChR binding sites labelled by [³H]-cytisine in the rat brain have been proposed to represent a major proportion of 42-containing NAChR (Flores et al., 1992). The ability of SIB-1765F to displace the binding of [³H]-cytisine in rat brain membranes suggests an interaction with 42-containing NAChR. In contrast to nicotine, SIB-1765F exhibited a much lower affinity for -BgT-sensitive NAChR. Ligand binding and calcium flux studies using HEK293 cells stably expressing human recombinant NAChR further support NAChR subtype selectivity of SIB-1765F over nicotine. Unlike ()-nicotine, which does not discriminate between NAChR containing h42 and h44 subunits, SIB-1765F exhibits a greater selectivity for h42 as demonstrated by its ability to displace [³H]-epibatidine binding and to increase [Ca⁺⁺]_i with higher potency in h42-containing cells than in h44-containing cells.

Extensive ligand binding characterization of SIB-1765F also demonstrates selectivity of this compound for NAChR over receptors for several neurotransmitters, neuropeptides and lipid mediators (see table 1). In the ligand-gated ion channel superfamily of receptors, (e.g., GABA_A, 5-HT₃ and NAChR), SIB-1765F exhibits more than 10,000-fold selectivity for NAChR. Within the cholinergic receptor family, i.e., NAChR and muscarinic cholinergic receptors that use the same endogenous neurotransmitter ACh, SIB-1765F exhibits more than 6000-fold selectivity for NAChR as defined by the displacement of [³H]-nicotine and [³H]-QNB binding. The only other ligand binding site at which SIB-1765F exhibits micromolar affinity is the sigma "receptor," a site with unknown function (Walker et al., 1990).

SIB-1765F did not show appreciable inhibition of human acetyl and human and rat butyrylcholinesterase activity up to millimolar concentrations. In addition, s.c. administration of SIB-1765F at behaviorally active doses (Menzaghi et al., 1995) did not show appreciable inhibition of rat plasma cholinesterase. Because many of the behavioral effects of SIB-1765F were observed with brain levels of SIB-1765F in the range of 1 to 10 µM (Rao et al., unpublished results), activation of NAChR through a mechanism that increases endogenous ACh levels via inhibition of AChE is unlikely.

NAChR have been proposed to regulate neurotransmitter release in vivo and in vitro through presynaptic mechanisms (Balfour, 1982; Wonnacott et al., 1990). The abilities of nicotinic ligands to regulate release of DA or NE is well documented (Arqueros et al., 1978; Westfall, 1974, 1987; El-Bizri and Clarke, 1994; Giorguieff et al., 1977; Marks et al., 1986; Sacaan et al., 1995, 1996). The pharmacology of NAChR that regulate striatal DA release is distinct from those regulating hippocampal NE release, reflective of receptor heterogeneity and indicative of different NAChR subtypes mediating neurotransmitter release (Sacaan et al., 1995, 1996; Clarke and Reubin, 1996). Therefore, the effects of SIB-1765F were examined on [³H]-DA release from rat striatal and olfactory tubercle slices and [³H]-NE release from rat hippocampal, thalamic and PFC slices with a view to elucidating differential selectivity for SIB-1765F. SIB-1765F had a greater efficacy than ()-nicotine at releasing [³H]-DA from rat striatal slices, although it was less potent than ()-nicotine [EC₅₀ values of 99.6 ± 12 µM for SIB-1765F and 3.9 ± 0.6 µM for nicotine (Sacaan et al., 1995), respectively]. Similar observations were made for [³H]-DA release from olfactory tubercles (EC₅₀ values of 39.6 ± 9.5 µM for SIB-1765F and 0.67 µM for nicotine, respectively. The sensitivity of SIB-1765F-induced [³H]-DA release to Mec, a noncompetitive NAChR antagonist (Gurney and Rang, 1984; Arcava et al., 1987; Rapier et al., 1988), and DHE, a competitive NAChR antagonist (Williams and Robinson, 1984), clearly demonstrates the involvement of NAChR in these responses. As shown previously for ()-nicotine (Sacaan et al., 1995), [³H]-DA release induced by SIB-1765F was insensitive to blockade by d-TC.

SIB-1765F did not inhibit the uptake of [³H]-DA into rat striatal synaptosomes; hence any contribution of DA uptake blockade to SIB-1765F-induced [³H]-DA release is likely to be insignificant. Similarly the possible role of muscarinic receptor antagonism by SIB-1765F is also unlikely due to the inability of atropine (a muscarinic acetylcholine receptor antagonist) to release [³H]-DA (data not shown).

Analysis of [³H]-NE release in various brain regions shows a different profile for SIB-1765F compared to ()-nicotine. In the hippocampus, at concentrations as high as 1 mM, SIB-1765F was only 27% as efficacious as ()-nicotine. The inability of SIB-1765F to attenuate ()-nicotine-induced [³H]-NE release indicates that SIB-1765F was not a partial agonist in this response. In PFC and thalamus, SIB-1765F appeared to be equiefficacious with ()-nicotine. The release of [³H]-NE in response to SIB-1765F in the PFC was dependent on extracellular calcium and sensitive to blockade by Mec indicating a NAChR-mediated exocytotic release of [³H]-NE.

()-Nicotine is also known to increase DA turnover as measured by changes in DOPAC levels (Andersson et al., 1979, Balfour, 1982), a sensitive index of nerve terminal DA metabolism. In our investigation, the NAChR agonists, ()-nicotine, (±)-epibatidine and SIB-1765F were evaluated for their effects on DA metabolism in rat striatum and olfactory tubercles. The ex vivo biochemical studies indicate that ()-nicotine, SIB-1765F and (±)-epibatidine increase DA metabolism in two distinct DA-ergic projection areas, e.g., striatum and olfactory tubercles. These results are in agreement with earlier in vitro and in vivo studies (Amano et al., 1989; Andersson et al., 1981; Kubo et al., 1989; Imperato et al., 1986; Sacaan et al., 1995). Microdialysis studies suggest a greater release of DA induced by ()-nicotine (Imperato et al., 1986) and SIB-1765F (fig. 8) in mesolimbic DA terminal fields compared to nigrostriatal terminal fields. The sensitivity of ()-nicotine, SIB-1765F and (±)-epibatidine to Mec implicates the activation of NAChR. Interestingly, the racemate and the isomers of epibatidine were equiefficacious at a single dose at increasing DA metabolism. These results are consistent with a lack of stereoselectivity for epibatidine isomers in ligand binding and functional assays (Rupniak et al., 1994). In addition, these ex vivo studies also indicate rapid brain penetration of ()-nicotine, SIB-1765F and (±)-epibatidine.

A comparison of the releasing effects of SIB-1765F and ()-nicotine on [³H]-DA and [³H]-NE indicate differences suggesting mediation via distinct NAChR subtypes. The striatal [³H]-DA release contrasts with hippocampal [³H]-NE release in the following ways: 1) striatal [³H]-DA release induced by nicotine is Mec and DHE sensitive and insensitive to blockade by d-TC whereas 2) the hippocampal [³H]-NE release is Mec and d-TC sensitive, but insensitive to blockade by DHE (Sacaan et al., 1995). In addition, recently Clarke and Reuben (1996) reported that chlorisondamine preferentially blocks nicotine-evoked NE release and proposed that hippocampal NE release may be modulated by NAChR composed of 34 subunits. These observations are consistent with differential anatomical distribution of mRNA for NAChR subunits (Wada et al., 1989; Seguela et al., 1993; Duvoisin et al., 1989; Sargent, 1993). The differential effects of SIB-1765F and ()-nicotine at displacing the binding of (±)-[³H]-epibatidine to NAChR supplement the evidence that SIB-1765F is a subtype selective ligand with a profile distinct from ()-nicotine. These differences in selectivity provide a logical explanation for the different in vivo pharmacological profiles of SIB-1765F and ()-nicotine (Menzaghi et al., 1995, and Menzaghi et al., Accompanying papers).

In summary, our investigation demonstrates that SIB-1765F is a subtype selective NAChR agonist and displays a distinct pharmacological profile from ()-nicotine and (±)-epibatidine as shown in radioligand binding studies, calcium flux studies and in vitro and in vivo neurotransmitter release studies.