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The Journal of Neuroscience, April 15, 2000, 20(8):2783-2791

UB-165: A Novel Nicotinic Agonist with Subtype Selectivity Implicates the 42* Subtype in the Modulation of Dopamine Release from Rat Striatal Synaptosomes

Christopher G. V. Sharples¹, Sergio Kaiser¹, Lev Soliakov¹, Michael J. Marks², Allan C. Collins², Mark Washburn³, Emma Wright⁴, James A. Spencer⁴, Timothy Gallagher⁴, Paul Whiteaker², and Susan Wonnacott¹

¹ Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, United Kingdom, ² Institute for Behavioral Genetics, University of Colorado, Boulder, Colorado 80309, ³ SIBIA Neurosciences Inc., La Jolla, California 92037-4641, and ⁴ School of Chemistry, University of Bristol, Bristol BS8 1TS United Kingdom

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Presynaptic nicotinic acetylcholine receptors (nAChRs) on striatal synaptosomes stimulate dopamine release. Partial inhibition by the 32-selective -conotoxin-MII indicates heterogeneity of presynaptic nAChRs on dopamine terminals. We have used this -conotoxin and UB-165, a novel hybrid of epibatidine and anatoxin-a, to address the hypothesis that the -conotoxin-MII-insensitive subtype is composed of 4 and 2 subunits. UB-165 shows intermediate potency, compared with the parent molecules, at 42* and 3-containing binding sites, and resembles epibatidine in its high discrimination of these sites over 7-type and muscle binding sites. (±)-Epibatidine, (±)-anatoxin-a, and (±)-UB-165 stimulated [³H]-dopamine release from striatal synaptosomes with EC₅₀ values of 2.4, 134, and 88 nM, and relative efficacies of 1:0.4:0.2, respectively. -Conotoxin-MII inhibited release evoked by these agonists by 48, 56, and 88%, respectively, suggesting that (±)-UB-165 is a very poor agonist at the -conotoxin-MII-insensitive nAChR subtype. In assays of ⁸⁶Rb⁺ efflux from thalamic synaptosomes, a model of an 42* nAChR response, (±)-UB-165 was a very weak partial agonist; the low efficacy of (±)-UB-165 at 42 nAChR was confirmed in Xenopus oocytes expressing various combinations of human nAChR subunits. In contrast, (±)-UB-165 and (±)-anatoxin-a were similarly efficacious and similarly sensitive to -conotoxin-MII in increasing intracellular Ca²⁺ in SH-SY5Y cells, a functional assay for native 3-containing nAChR. These data support the involvement of 42* nAChR in the presynaptic modulation of striatal dopamine release and illustrate the utility of exploiting a novel partial agonist, together with a selective antagonist, to dissect the functional roles of nAChR subtypes in the brain.

Key words: neuronal nicotinic acetylcholine receptor; presynaptic nicotinic modulation; dopamine release; rat striatal synaptosomes; Xenopus oocytes; SH-SY5Y cells; -conotoxin

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Nicotinic acetylcholine receptors (nAChRs) are widely distributed in the vertebrate CNS. With a few recently reported exceptions (Alkondon et al., 1998; Frazier et al., 1998), most nAChRs in the brain do not appear to mediate synaptic transmission. Instead, their primary function may be modulatory (Role and Berg, 1996). One locus for modulation is the nerve terminal, where presynaptic nAChRs can promote transmitter release and hence influence resting tone or synaptic efficacy.

Presynaptic nAChRs facilitate the release of many neurotransmitters, in numerous brain regions, via various nAChR subtypes (Wonnacott, 1997). nAChR heterogeneity arises from the pentameric assembly of receptors from numerous  and  subunits (2-7, 2-4) expressed in the mammalian brain (Role and Berg, 1996; Lukas et al., 1999). Defining the subunit composition of native nAChR is a major challenge; this quest is hampered by a lack of subtype-specific tools. A recent advance has been the identification of Conus toxins that target particular neuronal nAChR subtypes (McIntosh et al., 1999). -Conotoxin-MII, with specificity for 32-containing nAChRs (Cartier et al., 1996), partially inhibits the nicotinic stimulation of [³H]-dopamine release from striatal preparations (Kulak et al., 1997; Kaiser et al., 1998), indicating heterogeneity of the nAChR mediating this response.

The nicotinic modulation of [³H]-dopamine release from striatal preparations has been exploited as a model system for examining native nAChR responses (Soliakov and Wonnacott, 1996; Grady et al., 1997) and evaluating novel ligands (Holladay et al., 1997) and is pertinent to physiological and pathological processes (Dani and Heinemann, 1996; Decker and Arneric, 1998). Nicotinic agonists elicit dopamine release from rodent striatal synaptosomes and slices in a concentration-dependant manner, and this response is blocked by nicotinic antagonists, including mecamylamine and dihydroerythroidine (Grady et al., 1992; El-Bizri and Clarke, 1994; Sacaan et al., 1995; Soliakov et al., 1995). Insensitivity to the 7-selective antagonists -bungarotoxin (Bgt) (Rapier et al., 1990; Grady et al., 1992) and -conotoxin-ImI (Kulak et al., 1997) argues against the direct involvement of 7 nAChR. The loss of [³H]-nicotine binding sites from the striatum after 6-hydroxydopamine lesion of the nigrostriatal pathway is consistent with 42 nAChRs on striatal terminals (Clarke and Pert, 1985). Alternatively, sensitivity to neuronal bungarotoxin was interpreted in favor of 3-containing nAChRs (Grady et al., 1992). These disparate views are reconciled by the nAChR heterogeneity implicit in the partial inhibition by -conotoxin-MII (Kulak et al., 1997; Kaiser et al., 1998). The selectivity of this toxin is consistent with an nAChR containing 3 and 2 subunits (Cartier et al., 1996; Kaiser et al., 1998); the 42* nAChR is a candidate for the -conotoxin-MII-insensitive component of dopamine release evoked by nicotinic agonists.

Here we report studies with the novel nicotinic ligand UB-165 (Wright et al., 1997) (see Fig. 1) that support this interpretation. UB-165 is a hybrid of anatoxin-a and epibatidine, with potency at rat brain [³H]-nicotine binding sites that is intermediate between the values of the parent molecules. We have extended the characterization of UB-165 and have exploited its limited agonism at 42 nAChRs to examine the putative contribution of this subtype to the nicotinic stimulation of striatal dopamine release.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Materials

Cell culture. SH-SY5Y cells were from European Collection of Animal Cell Cultures (Porton Down, Salisbury, Wiltshire, UK) and were cultured as described by Murphy et al. (1991). M10 cells were provided by Dr. P. Whiting (Merck, Sharp and Dohme Research Center, Harlow, Essex, UK) and were cultured as described previously (Whiteaker et al., 1998). Cell culture media were provided by Life Technologies (Paisley, Renfrewshire, Scotland), and tissue culture plastic ware was obtained from Becton Dickinson UK Ltd. (Oxford, UK) and Sterilin (Stone, Staffordshire, UK).

Drugs and reagents. ()-[³H]-Nicotine (3.0 TBq/mmol in ethanol) and (±)-[³H]-epibatidine (2.1 TBq/mmol in ethanol) were provided by Dupont NEN (Stevenage, Herts, UK) and stored at 20°C. ⁸⁶RbCl was obtained from Dupont NEN (Herts, UK or Boston, MA); it was stored at 20°C and used within 1 month. [7,8-³H]-dopamine (specific activity, 1.78 TBq/mmol) was purchased from Amersham International (Buckinghamshire, UK) and stored at 20°C. Na¹²⁵I from Amersham International was used to iodinate Bgt to a specific activity of 26 TBq/mmol. (±)-Epibatidine was purchased from RBI (Natick, MA), and (±)-anatoxin-a was obtained from Tocris Cookson (Bristol, UK). Racemic UB-165 (Wright et al., 1997) and -conotoxin-MII (Cartier et al., 1996; Kaiser et al., 1998) were synthesized as previously described. All other drugs and reagents were provided by Sigma (Poole, Dorset, UK).

Tissue preparations

Rat brain membranes. P2 membranes from whole rat brain (minus cerebellum) were prepared as previously described (Davies et al., 1999). Briefly, brains were homogenized (10% w/v) in ice-cold 0.32 M sucrose, pH 7.4, containing 1 mM EDTA, 0.1 mM PMSF, and 0.01% NaN₃, and centrifuged at 1000 × g for 10 min. The supernatant fraction (S1) was decanted and retained on ice. The pellet (P1) was resuspended in ice-cold 0.32 M sucrose (5 ml/g original weight) and recentrifuged at 1000 × g for 10 min. The supernatant was combined with S1 and centrifuged at 12,000 × g for 30 min. The pellet (P2) was resuspended (2.5 ml/g original weight) in phosphate buffer (50 mM potassium phosphate, pH 7.4, containing 1 mM EDTA, 0.1 mM PMSF, and 0.01% NaN₃), and washed twice by centrifugation at 12,000 × g for 30 min. The washed pellet was resuspended in phosphate buffer (2.5 ml/g original weight) and stored in 5 ml aliquots at 20°C.

Rat muscle extract preparation. A Triton X-100 extract of muscle from the hindlimbs of Wistar rats was prepared as previously described (Garcha et al., 1993).

SH-SY5Y cell membrane preparation. SH-SY5Y cells, grown to confluency in 175 cm² flasks, were washed briefly with warm PBS containing (in mM): (150 NaCl, 8 K₂HPO₄, 2 KH₂PO₄, pH 7.4, 37°C) and scraped into cold phosphate buffer. Cells were washed by centrifugation for 3 min at 500 × g and resuspended in 10 ml of ice-cold phosphate buffer. The suspension was homogenized for 10 sec using an Ultraturax and centrifuged for 30 min at 45,000 × g. The pellet was resuspended in phosphate buffer (0.5 ml per original flask).

Radioligand binding assays

()-[³H]-nicotine competition binding assays: rat brain membranes. P2 membranes (250 µg protein) were incubated in a total volume of 250 µl in HEPES buffer (20 mM HEPES, pH 7.4, containing 118 mM NaCl, 4.8 mM KCl, 2.5 mM CaCl₂, 200 mM Tris, 0.1 mM PMSF, 0.01% (w/v) NaN₃) (Romm et al., 1990) with 10 nM ()-[³H]-nicotine and serial dilutions of test drugs. Nonspecific binding was determined in the presence of 100 µM ()-nicotine. Samples were incubated for 30 min at room temperature followed by 1 hr at 4°C. The reaction was terminated by filtration through Whatman GFA/E filter paper (presoaked overnight in 0.3% polyethyleneimine in PBS), using a Brandel Cell Harvester. Filters were counted for radioactivity in 5 ml Optiphase "Safe" scintillant in a Packard Tricarb 1600 scintillation counter (counting efficiency 45%).

(±)-[³H]-epibatidine competition binding assays. SH-SY5Y cells. SH-SY5Y membranes (30 µg protein) were incubated in a total volume of 2 ml in 50 mM phosphate buffer with 150 pM (±)-[³H]-epibatidine and serial dilutions of test drugs. Nonspecific binding was determined in the presence of 100 µM ()-nicotine. Samples were incubated for 2 hr at 37°C. They were filtered and counted for radioactivity as described above.

[¹²⁵I]-Bgt competition binding assays: rat brain membranes. P2 membranes (250 µg protein) were incubated in a total volume of 250 µl in 50 mM phosphate buffer with 1 nM [¹²⁵I]-Bgt and serial dilutions of test drugs (Davies et al., 1999). Nonspecific binding was determined in the presence of 10 µM Bgt. Samples were incubated for 3 hr at 37°C. Ice-cold PBS (0.5 ml) was then added, and the samples were centrifuged for 3 min at 10,000 × g. Pellets were washed by resuspension in 1.25 ml PBS and centrifugation as before. The resultant pellets were counted for radioactivity in a Packard Cobra II auto-gamma counter.

[¹²⁵I]-Bgt competition binding assays: rat muscle extract. Rat muscle extract (1.5 mg protein) was incubated in a total volume of 500 µl in 2.5 mM sodium phosphate buffer, pH 7.4, with 1 nM [¹²⁵I]-Bgt and serial dilutions of test drugs (Garcha et al., 1993). Nonspecific binding was determined in the presence of 10 µM Bgt. Samples were incubated for 2 hr at 37°C. Bound radioligand was separated by filtration through Whatman GF/C filters (presoaked in 0.3% polyethyleneimine in PBS overnight) using a Millipore vacuum manifold. Filters were washed three times with 3 ml cold PBS supplemented with 0.1% BSA and counted for radioactivity in a Packard Cobra II auto-gamma counter.

[³H]-epibatidine binding to M10 cells. Total numbers of nicotinic binding sites in M10 cells were measured in situ, in cultures in 24-well plates incubated with 500 pM (±)-[³H]-epibatidine for 2 hr, as previously described (Whiteaker et al., 1998). Competition binding assays were performed similarly, using 200 pM [³H]-epibatidine and serial dilutions of test drugs.

Upregulation of [³H]-epibatidine binding sites in M10 cells

M10 cells grown were grown in 24-well plates to ~70% confluency and were then incubated for 48 hr at 37°C with dexamethasone (to induce nAChR expression) in medium containing serial dilutions of test drugs (Whiteaker et al., 1998). Control cells, treated with dexamethasone in medium without the addition of nicotinic agents, were incubated in parallel. A rigorous washing procedure, in which medium was replaced three times at hourly intervals, was used to ensure removal of nicotinic drugs (Whiteaker et al., 1998) before assaying for [³H]-epibatidine binding sites as outlined above.

Superfusion of rat striatal synaptosomes for [³H]-dopamine release

P2 synaptosomes were prepared from rat striata, loaded with [³H]-dopamine (0.1 µM, 0.132 MBq/ml) for 15 min at 37°C, and superfused in open chambers as previously described (Soliakov et al., 1995; Kaiser et al., 1998). Synaptosomes were superfused with Krebs bicarbonate buffer containing (in mM): 118 NaCl, 2.4 KCl, 2.4 CaCl₂, 1.2 MgSO₄, 1.2 K₂HPO₄, 25 NaHCO₃ and 10 glucose, titrated to pH 7.4 with 95% O₂/5% CO₂, supplemented with 1 mM ascorbic acid, 8 µM pargyline, and 0.5 µM nomifensine to prevent dopamine degradation and reuptake. Agonists were applied for 40 sec. In antagonist studies, -conotoxin-MII (112 nM) or mecamylamine (10 µM) was added to the superfusion buffer 10 min before the application of agonist and maintained throughout the remainder of the experiment. Experiments always included chambers stimulated in parallel with 1 µM (±)-anatoxin-a, as a standard for normalization of data between experiments.

⁸⁶Rb⁺ efflux from thalamic synaptosomes

⁸⁶Rb⁺ efflux experiments were performed essentially as described by Marks et al. (1996). P2 synaptosomes were prepared from mouse or rat thalamus by homogenization in 0.32 M sucrose in 5 mM HEPES, pH 7.5, and differential centrifugation (Soliakov et al., 1995). Synaptosomes were loaded with ⁸⁶Rb⁺ (sufficient to give ~70 MBq per chamber) by incubation for 30 min at 22°C in uptake buffer containing (in mM): 140 NaCl, 1.5 KCl, 2.0 CaCl₂, 1.0 MgS0₄, 25 HEPES, 20 glucose, pH 7.5. Uptake was terminated, and unincorporated ⁸⁶Rb⁺ was removed by transferring aliquots (mouse, 25 µl; rat, 35 µl) to glass fiber filters for gentle filtration and washing. One thalamus provided sufficient material for up to 8 (mouse) or 12 (rat) filters. Each filter was placed in an open chamber of a superfusion apparatus (Marks et al., 1993; Soliakov et al., 1995). Samples were perfused at a rate of 2.5 ml/min with physiological buffer [135 mM NaCl,, 1.5 mM KCl, 2.0 mM CaCl₂, 1.0 mM MgS0₄, 20 mM glucose, 25 mM HEPES, pH 7.5, containing 0.1% (w/v) BSA; 5 mM CsCl and 50 nM tetrodotoxin]. After perfusion for 6 min, 12 samples were collected at 30 sec intervals. Agonist stimulation (60 sec) was given 90 sec after the start of sample collection. Where used, antagonists were present in the perfusion buffer throughout the experiment.

Xenopus oocyte preparation and recording

Stage V-VI oocytes were isolated from anesthetized Xenopus laevis frogs and enzymatically defolliculated by gentle shaking with collagenase [Worthington (Freehold, NJ), Type II, 1.7 mg/ml for 90 min; then Sigma (St. Louis, MO), Type II, 1.7 mg/ml for 30 min] in a Ca²⁺-free Barth's solution. After defolliculation, oocytes were incubated at 16-19°C in a solution containing (in mM): 77.5 NaCl, 2 KCl, 1.8 CaCl₂, 1 MgCl₂, 5 HEPES, pH 7.5, adjusted with NaOH, and supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 50 mg/ml gentimycin, and 5% heat-inactivated horse serum. Oocytes were injected the next day with 10-50 ng of mRNA encoding x and x nAChR subunits in an injection volume of 50 nl. The human nAChR subunits 2, 3, 4-2, 2, 4, and 7 were cloned from cDNA libraries prepared from human brain and the human IMR32 neuroblastoma cell line (Elliott et al., 1996). For two-way combinations, RNA was injected at a ratio of 1:1 (25 ng of each subunit per egg). RNA for 7 was injected at a concentration of 25 ng per egg.

Oocytes were examined for functional expression 2-5 d after mRNA injection using two-electrode voltage-clamp techniques described previously (Chavez-Noriega et al., 1997). Agonist-induced currents were elicited at a holding potential of 60 mV. The recording solution contained (in mM): 115 NaCl, 2.5 KCl, 1.8 CaCl₂, 10 HEPES, 0.001 atropine, pH 7.3. Recording electrodes (0.5-2.5 M resistance) were filled with 3 M KCl. Perfusion solutions were gravity fed into the recording chamber (capacity, 100 µl) at a rate of ~6-10 ml/min. All recordings were performed at room temperature (19-23° C). Signals were amplified, digitized (100-500 Hz), and filtered (at 40-200 Hz).

Calcium fluorimetry

SH-SY5Y cells, grown to confluency in 175 cm² flasks, were removed by incubation in Ca²⁺-free PBS for 3 min at 37°C. The cell suspension was centrifuged for 3 min at 500 × g, and the pellet was resuspended in 3-4 ml Ca²⁺-free HEPES buffer containing (in mM): 145 NaCl, 5 KCl, 1 MgCl₂, 0.5 Na₂HPO₄, 5.5 glucose, and 10 HEPES, pH 7.4, containing 5 µM fura-2 AM. The suspension was incubated in darkness at room temperature for 45 min, and excess fura-2 AM was removed by centrifugation for 3 min at 500 × g followed by three wash/centrifugation steps. Cell density was adjusted to 1-2 × 10⁶ cells/ml, and a 2 ml aliquot of cell suspension was placed in a cuvette in a PTI dual-excitation spectrophotofluorimeter (Photon Technology International, South Brunswick, NJ). To the cell suspension, 2 mM CaCl₂ was added before applying a nicotinic agonist ((±)-UB-165 or (±)-anatoxin-a). Where used, the antagonist -conotoxin-MII (112 nM) or mecamylamine (10 µM) was added to the cuvette 5 min before the addition of agonist. Excitation at 340 and 380 nm and emission at 510 nm were monitored. Calibration was performed in each experiment by adding sequentially 0.5% (v/v) Triton X-100 and 10 mM EDTA to derive maximum and minimum fluorescence ratios, respectively. Results were normalized within each experiment with respect to a maximally stimulating concentration of agonist.

Data analysis

Competition binding. IC₅₀ values were calculated by fitting data points to the Hill equation, using the nonlinear least squares curve fitting facility of Sigma Plot V2.0 for windows. K_i values were derived from IC₅₀ values according to the method of Cheng and Prusoff (1973), assuming K_d values of 10 and 1 nM for [³H]-nicotine and [¹²⁵I]-Bgt binding to rat brain membranes and 0.12 nM for [¹²⁵I]Bgt binding to rat muscle, respectively.

⁸⁶Rb⁺ efflux. Agonist-induced ⁸⁶Rb⁺ efflux was calculated as the fractional release above baseline on agonist stimulation (Marks et al., 1996). Basal efflux was defined as cpm collected in the fractions immediately before and after stimulation, and the basal rate of ⁸⁶Rb⁺ efflux was determined as an exponentially decaying curve fitted to these data points. Agonist-stimulated efflux was calculated as the cpm above the calculated baseline during the period of agonist application. To correct for interexperimental variation in synaptosomal ⁸⁶Rb⁺ uptake, agonist-stimulated efflux was divided by the calculated baseline efflux. Values for ⁸⁶Rb⁺ efflux from mouse thalamic synaptosomes are more than double those from corresponding rat preparations (see Fig. 5), reflecting the higher density of [³H]-nicotine binding sites in mouse thalamus. Curve fitting of ⁸⁶Rb⁺ efflux data from mouse thalamic synaptosomes was performed using the nonlinear least squares curve fitting facility of Sigma Plot V5.0 for DOS.

Upregulation. Upregulation profiles were fit to a logistic equation (Whiteaker et al., 1998) using the nonlinear least squares curve fitting facility of Sigma Plot V2.0 for Windows, to give the EC₅₀ for the upward phase of the dose-response curve and U_max, the maximum upregulation produced by the drug tested.

Dopamine release. Evoked [³H]-dopamine release was calculated as the area under the peak, after subtraction of the baseline, as described previously (Kaiser et al., 1998). Data points for agonist dose-response relationships (after subtraction of nonspecific release determined in the presence of mecamylamine) were fitted to the Hill equation y = (a  d) /[1 + (k/x)ⁿ] + d, where a is the asymptotic maximum, d is the asymptotic minimum, k is the agonist concentration at the inflection point (EC₅₀), x is the ligand concentration, and n is the slope parameter (Hill number).

Data for inhibition by antagonist are represented as percentages of the corresponding controls, assayed in parallel in the absence of antagonist. One-way ANOVA-post hoc Bonferroni test was used to determine the significance of differences from control.

Two-electrode voltage-clamp recordings. Full dose-response curves were obtained from individual oocytes and normalized relative to the response to an EC₅₀ concentration of ACh recorded in the same oocyte. Sigmoidal concentration-response curves were fit to the Hill equation using Origin 4.0 (Microcal Software).

Calcium fluorimetry. The intracellular Ca²⁺ concentration ([Ca²⁺]_i) was calculated from the fluorescence ratio of fura-2 (A₃₄₀/A₃₈₀, given as R below) according to the Grynkiewicz equation (Grynkiewicz et al., 1985) [Ca²⁺]_i = K_d * [(R  R_min)/(R_max  R)]*(S_f2/S_b2), where K_d is the dissociation constant for Ca²⁺ binding to fura-2, R_min is the fluorescence ratio under nominally "zero" free Ca²⁺ conditions, R_max is the fluorescence ratio under saturating Ca²⁺ conditions, and S_f2/S_b2 is the ratio of fluorescence values of Ca²⁺-free and Ca²⁺-saturated fura-2, measured at the wavelength used to monitor Ca²⁺-free fura-2.

For dose-response curves, agonist responses were calculated as the percentage of the change in intracellular Ca²⁺ produced by a maximally stimulating concentration of the same agonist, assayed in parallel. EC₅₀ values were calculated by fitting data points to the Hill equation, using the nonlinear least squares curve fitting facility of Sigma Plot V2.0 for Windows.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Competition binding assays

(±)-UB-165 was compared with (±)-anatoxin-a and (±)-epibatidine (see Fig. 1 for structures) for its ability to displace the binding of a number of nicotinic radioligands (Fig. 2, Table 1). Radioligand binding assays, at least those using radiolabeled agonists, reflect the desensitized state of the nAChRs. Most high-affinity [³H]-nicotine binding sites in rat brain are considered to represent the 42 subtype (Zoli et al., 1998). (±)-Anatoxin-a and (±)-epibatidine inhibited [³H]-nicotine binding with K_i values of 1.25 ± 0.20 and 0.02 ± 0.005 nM, respectively. (±)-UB-165 displayed an intermediate potency, with a K_i value of 0.27 ± 0.05 nM (Fig. 2A, Table 1). Comparable K_i values were derived for inhibition of [³H]-epibatidine binding to chicken 42 nAChRs in M10 cells (Table 1). (±)-Anatoxin-a and (±)-epibatidine competed with [¹²⁵I]-Bgt for binding to putative 7-type nAChRs in rat brain membranes, with K_i values of 1840 ± 260 and 233 ± 69 nM, respectively. (±)-UB-165 exhibited slightly lower potency than (±)-anatoxin-a, having a K_i value of 2790 ± 370 nM (Fig. 2B).

View larger version (10K): [in this window] [in a new window]   Figure 1.   Molecular structures of ()-epibatidine and (+)-anatoxin-a and the enantiomer of UB-165 that corresponds to (+)-anatoxin-a.

View larger version (38K): [in this window] [in a new window]   Figure 2.   Competition binding assays for [3H]-nicotine binding sites (A) and [125I]-Bgt binding sites (B) in rat brain membranes, [3H]-epibatidine binding sites (C) in human SH-SY5Y cell membranes, and [125I]-Bgt binding sites (D) in rat muscle extract. Preparations were incubated with radioligand and increasing concentrations of (±)-UB-165 (), (±)-anatoxin-a (), or (±)-epibatidine () as described in Materials and Methods. Each point is the mean of at least three separate determinations ± SEM. Data points were fitted to the Hill equation.

View this table: [in this window] [in a new window]   Table 1.   Affinities for radioligand binding sites corresponding to various nAChR subtypes

[³H]-Epibatidine labels 3-containing nAChRs in human neuroblastoma SH-SY5Y cells (Wang et al., 1996). The concentration of [³H]-epibatidine used (150 pM) was chosen to preferentially label 32-containing nAChRs [including 32 and 325 combinations (Wang et al., 1996)], but K_i values have not been derived because of the inherent nAChR heterogeneity. (±)-UB-165 displaced [³H]-epibatidine from SH-SY5Y cell membranes with an IC₅₀ value of 20 ± 0.7 nM, intermediate between that of (±)-anatoxin-a (IC₅₀ = 155 ± 25 nM) and (±)-epibatidine (IC₅₀ = 0.34 ± 0.06 nM) (Fig. 2C). (±)-UB-165 was also examined for its ability to displace [¹²⁵I]-Bgt from rat muscle extract (Fig. 2D). Its K_i value of 990 ± 240 nM was similar to that of (±)-epibatidine (K_i = 610 ± 160 nM), whereas (±)-anatoxin-a was the most potent competing ligand, with a K_i value of 85 ± 41 nM.

Thus the rank order of potencies at the rat and chicken 42 and human 3-containing nAChR subtypes is (±)-epibatidine > (±)-UB-165 > (±)-anatoxin-a. In contrast, (±)-UB-165 was the least potent at 7-type nAChRs and muscle nAChRs: rank orders of potencies are (±)-epibatidine > (±)-anatoxin-a  (±)-UB-165 and (±)-anatoxin-a > (±)-epibatidine  (±)-UB-165, respectively. The ability of each of these ligands to discriminate between nAChR subtypes was expressed as an affinity ratio, relative to the value at the rat brain [³H]-nicotine binding site, which was defined as 1 (Table 1). From comparison of these affinity ratios, (±)-UB-165 resembles (±)-epibatidine in its marked preference for 42* nAChRs compared with 7-type and muscle nAChRs.

Upregulation of 42 nAChRs

(±)-Epibatidine and (±)-anatoxin-a differ in their abilities to upregulate 42 nAChRs in the M10 cell line: (±)-epibatidine is a partial upregulator whereas (±)-anatoxin-a is fully efficacious (Whiteaker et al., 1998). (±)-UB-165 was compared with (±)-anatoxin-a and (±)-epibatidine in this assay, to determine which of the parent compounds it most resembles with respect to upregulation (Fig. 3). Dose-response profiles for upregulation produced by (±)-UB-165 and (±)-anatoxin show that they are similarly efficacious, with maximum upregulation of 237 ± 47 and 221 ± 27% above control levels, respectively. (±)-UB-165 was a more potent upregulator than (±)-anatoxin-a, with EC₅₀ values of 25.3 ± 17.7 and 985 ± 515 nM, respectively. In contrast, (±)-epibatidine achieved a maximum upregulation of only 76 ± 6% above control, although it was the most potent of the three ligands, with an EC₅₀ value of 1.2 ± 0.2 nM. The ability of (±)-UB-165 to upregulate nicotinic binding sites is consistent with it behaving as an agonist at 42 nAChRs, because all agonists that we have examined have produced some degree of upregulation in this system, whereas antagonists produce little if any upregulation (Whiteaker et al., 1998).

View larger version (26K): [in this window] [in a new window]   Figure 3.   Upregulation of [3H]-epibatidine binding sites expressed in M10 cells. M10 cells were cultured in the presence of dexamethasone and (±)-UB-165 (), (±)-anatoxin-a (), or (±)-epibatidine () for 48 hr. After extensive washing, [3H]-epibatidine binding was performed on M10 cells in situ. Upregulation was expressed as specific [3H]-epibatidine binding above that of control cells treated in parallel with dexamethasone alone. (±)-Epibatidine was a partial agonist for upregulation compared with (±)-UB-165 and (±)-anatoxin-a. Each point is the result of at least three separate determinations ± SEM. Data points were fitted to a logistic equation (Whiteaker et al., 1998), giving EC50 values of 1.2 ± 0.2, 25.3 ± 17.7, and 985 ± 515 nM, and percentage maximum upregulation above control of 76.4 ± 5.7, 237 ± 47, and 221 ± 28%, for (±)-epibatidine, UB-165, and (±)-anatoxin-a, respectively.

Presynaptic nicotinic stimulation of [³H]-dopamine release from rat striatal synaptosomes

(±)-UB-165 was compared with (±)-anatoxin-a, (±)-epibatidine, and ()-nicotine for their abilities to promote [³H]-dopamine release from rat striatal synaptosomes (Fig. 4A). Of these four compounds, (±)-epibatidine was the most efficacious and most potent (EC₅₀ value = 2.42 ± 0.44 nM), whereas ()-nicotine (EC₅₀ value = 1.59 ± 0.38 µM) was the least potent but had higher efficacy than (±)-anatoxin-a. Although (±)-UB-165 was slightly more potent than (±)-anatoxin-a (EC₅₀ values = 88 ± 18 and 134 ± 26 nM, respectively), it was much less efficacious than the other agonists, achieving a maximum of specific, nAChR-mediated [³H]-dopamine release that was only 42% of that of (±)-anatoxin-a and 21% of that of (±)-epibatidine.

View larger version (21K): [in this window] [in a new window]   Figure 4.   Nicotinic stimulation of [3H]-dopamine release from rat striatal synaptosomes. A, Dose-response curves for the release of [3H]-dopamine evoked by increasing concentrations of (±)-epibatidine (), ()-nicotine (), (±)-anatoxin-a (), and (±)-UB-165 (). Each agonist concentration was tested in the presence and absence of mecamylamine to determine specific nAChR-mediated release, as described in Materials and Methods. Responses were normalized to the response to 1 µM (±)-anatoxin-a, determined in parallel. Values are the mean ± SEM of at least three independent assays. The rank order of potencies (EC50 values) is (±)-epibatidine (2.4 ± 0.4 nM) > (±)-UB-165 (88 ± 18 nM)  (±)-AnTx-a (134 ± 26 nM) > ()-nicotine (1595 ± 377 nM). B, The inhibition by -conotoxin-MII (112 nM; hatched bars) of [3H]-dopamine release evoked by maximally effective concentrations of agonists is compared with inhibition by mecamylamine (10 µM; black bars). Synaptosomes loaded with [3H]-dopamine were perfused with antagonist for 10 min before stimulation with (±)-epibatidine (0.1 µM), ()-nicotine (10 µM), (±)-anatoxin-a (1 µM), or (±)-UB-165 (1 µM). Responses were normalized to the response to 1 µM (±)-anatoxin-a, determined in parallel. Values are the mean ± SEM of at least three independent assays.

nAChRs mediating the presynaptic nicotinic stimulation of [³H]-dopamine release from striatal terminals appear to be heterogeneous, based on the partial inhibition by the 32-selective toxin -conotoxin-MII (Kulak et al., 1997; Kaiser et al., 1998). A maximally effective concentration of this toxin (112 nM) was compared with mecamylamine (10 µM) for their abilities to inhibit [³H]-dopamine release evoked by maximally effective concentrations of the four agonists mentioned above (Fig. 4B). The results confirm the differences in efficacy noted above. Although mecamylamine inhibited agonist-evoked [³H]-dopamine release to the same extent in each case (the residual release being the nonspecific component that is elicited by a buffer pulse without agonist), -conotoxin-MII produced varying degrees of inhibition. The mecamylamine-sensitive response to (±)-anatoxin-a was reduced by 56% by the toxin, in agreement with our previous findings (Kaiser et al., 1998), whereas the mecamylamine-sensitive [³H]-dopamine release evoked by (±)-epibatidine and ()-nicotine was inhibited by 47.9 and 32.4%, respectively. However, the mecamylamine-sensitive response to (±)-UB-165 was almost completely blocked (87.8%) by -conotoxin-MII. This suggests that (±)-UB-165 has very little efficacy at the other subtype(s) of nAChRs responsible for [³H]-dopamine release. Because the 42 subtype is a candidate for this role, we examined the behavior of (±)-UB-165 in a neurochemical assay for this putative subtype.

⁸⁶Rb⁺ efflux from thalamic synaptosomes

The efflux of ⁸⁶Rb⁺ from preloaded mouse thalamic synaptosomes in response to nicotinic agonists is proposed to reflect the activation of 42 nAChRs (Marks et al., 1993, 1996, 1999). (±)-UB-165 was compared with (±)-epibatidine for its ability to elicit ⁸⁶Rb⁺ efflux from this preparation (Fig. 5A,B). Although (±)-epibatidine was a potent and efficacious agonist in this assay (Fig. 5A), as reported previously (Marks et al., 1996), (±)-UB-165 produced very little response when tested over the concentration range examined in the [³H]-dopamine release assay (Fig. 5B). Indeed, 1 µM (±)-UB-165 elicited <15% of the maximum ⁸⁶Rb⁺ efflux provoked by (±)-epibatidine. The interpretation that (±)-UB-165 is a partial agonist with respect to ⁸⁶Rb⁺ efflux is supported by the ability of (±)-UB-165 (1 µM) to shift the dose-response curve for (±)-epibatidine to the right (Fig. 5A). Moreover, increasing concentrations of (±)-UB-165 progressively inhibited the response to a maximally effective concentration of (±)-epibatidine (100 nM) (Fig. 5B). The low efficacy of (±)-UB-165 in eliciting ⁸⁶Rb⁺ efflux was verified in rat thalamic synaptosomes (Fig. 5C). Maximally effective concentrations of (±)-UB-165, (±)-anatoxin-a, and (±)-epibatidine with respect to [³H]-dopamine release were compared. Their relative efficacies in stimulating ⁸⁶Rb⁺ efflux resemble their efficacies in evoking -conotoxin-MII-insensitive [³H]-dopamine release (Fig. 4B). Indeed, the slight increase in ⁸⁶Rb⁺ efflux in response to (±)-UB-165 was blocked by mecamylamine but was insensitive to -conotoxin-MII (Fig. 5C).

View larger version (27K): [in this window] [in a new window]   Figure 5.   Nicotinic stimulation of 86Rb+ efflux from thalamic synaptosomes. P2 synaptosomes from mouse (A, B) or rat (C) thalamus were loaded with 86Rb+ and superfused; 86Rb+ efflux is a measure of nAChR activation, primarily of the 42 subtype (Marks et al., 1993). A, Dose-response curve of 86Rb+ efflux stimulated by (±)-epibatidine, in the absence () or presence () of 1 µM (±)-UB-165. B, Dose-response curve of 86Rb+ efflux stimulated by (±)-UB-165, in the absence () or presence () of 100 nM (±)-epibatidine. C, Comparison of 86Rb+ efflux from rat thalamic synaptosomes stimulated by maximally effective concentrations of (±)-UB-165 (1 µM), (±)-anatoxin-a (10 µM), and (±)-epibatidine (100 nM) (and buffer control). (±)-UB-165 and (±)-anatoxin-a were also tested in the presence of -conotoxin-MII (112 nM; hatched bars) and mecamylamine (10 µM; black bars). Values are the mean ± SEM of at least three independent assays.

Activation of inward currents in Xenopus oocytes expressing nAChRs of defined subunit combination

To confirm that UB-165 has low efficacy at 42 nAChRs, and to assess its efficacy at other nAChR subtypes, it was examined for its ability to elicit inward currents in Xenopus oocytes expressing pairwise combinations of human  and  subunits, or homo-oligomeric nAChRs of 7 subunits (Fig. 6). (±)-UB-165 was most potent in activating 44 and 24 nAChRs (EC₅₀ = 0.05 µM), followed by 34 (0.27 µM), 32 (3.9 µM), and 7 (6.9 µM), and was similarly efficacious at these subtypes. In contrast, (±)-UB-165 failed to elicit significant currents from oocytes expressing 42 or 22 nAChRs.

View larger version (26K): [in this window] [in a new window]   Figure 6.   Concentration-response curves for (±)-UB-165 at recombinant human nAChRs. Pairwise combinations of human  and  subunits, or 7 alone, were expressed in Xenopus oocytes. Current responses in each oocyte were normalized to an EC50 concentration of ACh. EC50 values yielded by logistical curve fits were 3.9 µM for 32 (), 0.05 µM for 24 (), 0.27 µM for 34 (), 0.05 µM for 44 (), and 6.9 µM for 7 (). There was little activation of 22 () and 42 () subtypes. Data points represent mean ± SD of three to six experiments.

Activation of Ca²⁺ fluxes in the SH-SY5Y cell line

The functional potency of (±)-UB-165 at native 3-containing nAChRs was investigated in the human neuroblastoma cell line SH-SY5Y. Because this cell line does not express 4 subunits, it was used to examine the efficacy of UB-165 at native nAChRs other than the 42* subtype. Receptor activation was assayed as an increase in intracellular Ca²⁺, measured quantitatively in suspensions of SH-SY5Y cells using fura-2 (Fig. 7). (±)-UB-165 and (±)-anatoxin-a were compared: both agonists increased intracellular Ca²⁺, and this effect was totally blocked by 10 µM mecamylamine (Fig. 7A,B). Determination of dose-response relationships showed that (±)-UB-165 was more potent than (±)-anatoxin-a, with EC₅₀ values of 154 ± 20 and 530 ± 92 nM, respectively (Fig. 7C). Comparison of maximally effective concentrations of each drug (1 µM (±)-UB-165 and 10 µM (±)-anatoxin-a) showed them to be comparably efficacious in this assay (Fig. 7D). Increases in intracellular Ca²⁺ evoked by these maximally stimulating drug concentrations were partially blocked to the same extent by 112 nM -conotoxin-MII (43.9 ± 8.3 and 49.8 ± 10.0% inhibition of (±)-UB-165- and (±)-anatoxin-a-evoked Ca²⁺ responses, respectively) (Fig. 7D).

View larger version (53K): [in this window] [in a new window]   Figure 7.   Nicotinic stimulation of increases in intracellular Ca2+ in SH-SY5Y cells. SH-SY5Y cells were loaded with 5 µM fura-2 AM in Ca2+-free buffer for 45 min before addition of 2 mM CaCl2 followed by (±)-anatoxin-a or (±)-UB-165. Antagonists were added 5 min before the addition of Ca2+ and agonist. Representative fluorimetry traces in response to 10 µM (±)-anatoxin-a (A) and 1 µM (±)-UB-165 (B). The bottom trace in A and B shows the response in the presence of 10 µM mecamylamine. C, Dose-response relationships of increases in intracellular Ca2+ induced by (±)-anatoxin-a () and (±)-UB-165 (). For each curve, results were normalized to the responses produced by a maximally effective concentration of agonist, determined in parallel in the same experiment. Data points are the mean ± SEM of three independent assays and were fitted to the Hill equation, giving EC50 values of 154 and 530 nM for (±)-UB-165 and (±)-anatoxin-a, respectively. D, Comparison of efficacies of maximally stimulating concentrations of (±)-UB-165 (1 µM) and (±)-anatoxin-a (10 µM), and inhibition by -conotoxin-MII (112 nM, black bars). Values are the mean of three independent experiments ± SEM and were normalized to the response to (±)-UB-165 within each separate experiment.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The potent nicotinic agonists epibatidine and anatoxin-a are structurally related in that they both incorporate an azobicyclic core (Fig. 1); UB-165 is a hybrid molecule comprising the bulkier azobicyclo[4.2.1]nonane moiety of anatoxin-a attached to the chloropyridyl substituent of epibatidine (Wright et al., 1997). At 42* and 3-containing nAChR binding sites (defined by [³H]-nicotine binding to brain membranes and [³H]-epibatidine binding to SH-SY5Y cell membranes, respectively), UB-165 has intermediate potency, compared with the parent compounds (Fig. 2). It resembles epibatidine in its high degree of discrimination between 42* binding sites on the one hand versus 7 and muscle sites on the other (Table 1). In contrast, UB-165 is more like anatoxin-a with regard to its enantiospecificity (Wright et al., 1997) and its efficacy in upregulating 42 nAChRs in M10 cells (Fig. 3). From these properties and its structural features it was not possible to predict the very low efficacy of UB-165 with regard to striatal [³H]-dopamine release, measured in perfused synaptosome preparations in vitro. Use of the 32-selective -conotoxin-MII (Cartier et al., 1996) has enabled us to show that this low efficacy of UB-165 arises from its almost complete inability to activate the -conotoxin-MII-insensitive component of nAChR-stimulated [³H]-dopamine release. The failure of UB-165 to activate 42*-mediated ⁸⁶Rb⁺ efflux from thalamic synaptosomes and to elicit responses in oocytes expressing 42 nAChRs provides support for the proposition that presynaptic 42* nAChRs on striatal dopaminergic terminals contribute to the nicotinic stimulation of [³H]-dopamine release, in addition to 32-containing nAChRs (Kulak et al., 1997; Kaiser et al., 1998).

Neurons in the substantia nigra pars compacta of the rat express mRNA for 3, 4, 5, 6, 2, and 3 (Wonnacott, 1997) and possibly 4 and 7 subunits (Charpantier et al., 1998), and some or all of these may contribute to nAChRs on dopaminergic terminals in the striatum. The challenge of defining the subunit composition of native nAChRs responsible for particular physiological responses is important for understanding the significance of subunit heterogeneity, rules of assembly, and functional implications arising from receptor subtypes with differing properties. Nicotine-evoked [³H]-dopamine release from striatal synaptosomes is absent from 2 null mutant mice (Grady et al., 1998), implicating the 2 subunit in all nAChRs in dopaminergic terminals governing this response in the mouse. Sensitivity to -conotoxin-MII (Kulak et al., 1997; Kaiser et al., 1998), a toxin with specificity for the 32 subunit combination expressed in Xenopus oocytes (Cartier et al., 1996; Kaiser et al., 1998) is compatible with the requirement for 2 to be present. This does not exclude the presence of additional types of subunit in an nAChR containing 3 and 2 subunits. The restricted distributions of 6 and 3 mRNA in the CNS, with high expression in the substantia nigra pars compacta (Deneris et al., 1989; Le Novère et al., 1996; Charpantier et al., 1998), together with the erstwhile "orphan" status of the 6 and 3 subunits, makes them prime candidates for complex combinations with other subunits such as 3 and 2 (Fucile et al., 1998; Groot-Kormelink et al., 1998).

A striking observation from the studies with -conotoxin-MII is its incomplete inhibition of nicotinic agonist-evoked [³H]-dopamine release. Kulak et al. (1997) found a block of 34-49% of ()-nicotine-stimulated [³H]-dopamine release, whereas Kaiser et al. (1998) reported 56% inhibition of mecamylamine-sensitive release evoked by (±)-anatoxin-a. The present study reproduces these findings and extends the analysis to other agonists. A partial inhibition (48%) of [³H]-dopamine release evoked by (±)-epibatidine was observed, whereas the response evoked by (±)-UB-165 was almost totally inhibited by -conotoxin-MII (Fig. 4). This suggests that (±)-UB-165 primarily activates only the -conotoxin-MII-sensitive nAChRs associated with dopaminergic terminals, which accords with an 32-containing nAChR. The magnitude of the -conotoxin-MII-sensitive portion of evoked [³H]-dopamine release is rather similar between agonists (with the exception of (±)-epibatidine, for which considerable variability was encountered), whereas the -conotoxin-MII-insensitive portion varies in the ratio 1:0.88:0.40:0.04 for (±)-epibatidine, ()-nicotine, (±)-anatoxin-a, and (±)-UB-165, respectively. This variation in the -conotoxin-MII-insensitive responses to different agonists is largely responsible for their different efficacies with respect to total release (Fig. 4B).

The hypothesis, elaborated in the introductory remarks, states that the -conotoxin-MII-insensitive component of nAChR-evoked striatal dopamine release is mediated by 42* nAChRs. The low efficacy, and complete block by -conotoxin-MII, of UB-165-evoked [³H]dopamine release (Fig. 4) leads to the prediction that (±)-UB-165 should be a very poor agonist at this receptor subtype. nAChR-evoked ⁸⁶Rb⁺ efflux from mouse thalamic synaptosomes is attributed to an 42* nAChR (Marks et al., 1993, 1996), and, in agreement with this prediction, UB-165 is a very weak, partial agonist in this assay (Fig. 5). In rat thalamic synaptosomes, (±)-epibatidine, (±)-anatoxin-a, and (±)-UB-165 stimulated mecamylamine-sensitive ⁸⁶Rb⁺ efflux with relative efficacies of 1:0.39:0.08, very comparable to the ratio of efficacies for -conotoxin-MII-insensitive [³H]-dopamine release (see above). Moreover, Type II responses in rat hippocampal neurons, attributed to an 42 nAChR, show different efficacies with different agonists, with the ratio 1:0.6:0.3 for (+)-epibatidine, ()-nicotine, and (+)-anatoxin-a, respectively (Alkondon and Albuquerque, 1995), similar to those found here for these agonists, with respect to -conotoxin-MII-insensitive [³H]-dopamine release and ⁸⁶Rb⁺ efflux.

Results from Xenopus oocytes expressing various recombinant nAChR subtypes verify that (±)-UB-165 has low efficacy at 42 nAChRs. The only other subunit combination examined at which (±)-UB-165 was inefficacious was 22 (Fig. 6), but the lack of expression of 2 in substantia nigra (Wada et al., 1989) excludes this as a contributor to the presynaptic modulation of striatal dopamine release. The electrophysiological analysis confirms that UB-165 is a full agonist at 32 nAChRs. This is consistent with the data from a functional assay for native 3-containing nAChRs [increases in intracellular Ca²⁺ in SH-SY5Y cells (Fig. 7)], in which (±)-anatoxin-a and (±)-UB-165 were equally efficacious and equally sensitive to -conotoxin-MII. SH-SY5Y cells expresses mRNA for 3, 5, 7, 2, and 4 nAChR subunits (Lukas et al., 1993; Peng et al., 1994). Therefore the 42 nAChR is not present in this cell line. Nevertheless, inhibition by -conotoxin-MII was incomplete (~50%) (Fig. 7), and this presumably reflects the presence of additional nAChR subtypes that lack an 32 interface, in particular 4-containing nAChRs. Heterogeneous combinations of 3, 5, 2, and 4 subunits in SH-SY5Y cells have been proposed, on the basis of immunoisolation of nAChRs using subunit-specific antibodies (Wang et al., 1996). Notably, 56% of 3-containing nAChRs were found to contain the 2 subunit. The -conotoxin-MII-insensitive response is likely to arise from activation of 34-containing nAChRs that lack the 2 subunit (although a contribution from 7-type nAChRs cannot be ruled out). 34 nAChRs expressed in Xenopus oocytes are insensitive to the concentration of -conotoxin-MII used in this study (Cartier et al., 1996; Kaiser et al., 1998) but are fully activated by UB-165 (Fig. 6). Moreover, the comparable efficacies of (±)-UB-165- and (±)-anatoxin-a demonstrate that the -conotoxin-MII-insensitive component of nAChR-mediated Ca²⁺ increases in SH-SY5Y cells is clearly different from the -conotoxin-MII-insensitive component of nAChR-mediated [³H]-dopamine release from striatal synaptosomes.

The weak partial agonism of UB-165 at 42* nAChRs contrasts with its full efficacy in upregulating 42 nAChRs in M10 cells (Fig. 3). Lower concentrations of agonist are needed to induce upregulation, compared with those required to elicit functional responses. Together with the general inability of antagonists to prevent agonist-induced upregulation, a higher affinity state of the nAChRs than the active conformation is implicated as the trigger for the upregulation of binding sites (Whiteaker et al., 1998; Fenster et al., 1999). Whether this state is the high-affinity desensitized state measured in ligand binding assays remains controversial. This result demonstrates that although UB-165 lacks functional efficacy at 42* nAChRs, long-term exposure to this drug would upregulate this receptor subtype with potential functional consequences.

UB-165 is not unique as a partial agonist in stimulating [³H]-dopamine release (Holladay et al., 1997) and ⁸⁶Rb⁺ efflux (Marks et al., 1996). For example RJR 2429 has a structure comparable to UB-165, comprising an azobicyclo[2.2.2]octane core coupled to