Cell Culture

SH-SY5Y cells (passages 14-20) were cultured as described previously (Ridley et al., 2001). In brief, cultures were maintained in Dulbecco's modified Eagle's medium/Ham's F12 medium, supplemented with 15% fetal bovine serum, 2 mM l-glutamine, 1% nonessential amino acids, 190 U/ml penicillin, and 0.2 mg/ml streptomycin. Cells were plated (1:5 dilution) into 96-well Primaria plates (Falcon, Franklin Lakes, NJ), and experiments were performed 72 h later with confluent cultures.

Lα4β2 cells were cultured as described previously (Cooper et al., 1999). Cultures were maintained in Dulbecco's modified Eagle's medium, supplemented with 10% fetal bovine serum, 2 mM l-glutamine, 100 U/ml penicillin, and 0.1 mg/ml streptomycin. Cells were plated (1:5 dilution) into 96-well Primaria plates. Expression of nAChR was induced by 1 μM dexamethasone for 7 days. Because expression of surface α4β2 nAChR is enhanced by lowering the temperature (Cooper et al., 1999), cells were transferred to a 33°C incubator 24 h before the experiment.

Binding Studies

Membrane Preparations. Rat brain P2 membranes were prepared by differential centrifugation as described previously (Davies et al., 1999). The washed pellet was resuspended in 50 mM phosphate buffer, pH 7.4, containing 1 mM EDTA, 0.1 mM PMSF, and 0.01% sodium azide (2.5 ml/g original weight) and stored in 5-ml aliquots at -20°C.

SH-SY5Y cell membranes were prepared as described previously (Sharples et al., 2000). In brief, confluent 75-cm² flasks of cells were washed with PBS, pH 7.4, and scraped into ice-cold 50 mM phosphate buffer, pH 7.4, containing 0.1 mM PMSF, and 0.01% sodium azide. Cells were washed by centrifugation (MSE, Micro Centaur, UK) for 3 min at 500g and resuspended in 10 ml of ice-cold 50 mM phosphate buffer. The suspension was sonicated (3 × 10 s) and centrifuged for 35 min at 45,000g. The pellet was resuspended in 50 mM phosphate buffer (0.5 ml per original flask of cells). Protein was determined using the Markwell method with bovine serum albumin as standard.

Saturation Binding Assays. Saturable binding of [³H]nicotine or [³H]MLA to rat brain P2 membranes was determined by incubating increasing concentrations of radioligand with 0.25 or 0.5 mg of membrane protein, respectively, in a total volume of 250 μl of 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, and 0.01% sodium azide) for [³H]nicotine binding (Sharples et al., 2000) or 50 mM phosphate buffer, pH 7.4, for [³H]MLA binding (Davies et al., 1999). Nonspecific binding was defined in the presence of 100 μM or 1 mM nicotine. Saturable binding of [³H]epibatidine to SH-SY5Y cell membranes was determined by incubating increasing concentrations of radioligand with 40 μl (95 μg of protein) of membranes in a total volume of 1 ml of 50 mM phosphate buffer, pH 7.4 (Sharples et al., 2000). Nonspecific binding was defined in the presence of 1 mM nicotine. Assays were conducted with or without the presence of 1 μM galantamine. Incubations were carried out at room temperature for 30 min followed by 60 min at 4°C. Bound radioligand was separated by rapid filtration on Whatman GFA/E filter paper (presoaked overnight in 0.3% polyethylenimine in PBS, pH 7.4), using a Brandell cell harvester. Filters were counted for radioactivity in a PerkinElmer Tri-Carb liquid scintillation counter 1500 (counting efficiency 45%).

Competition Assays. Competition binding assays were performed as described previously (Sharples et al., 2000, 2002). Rat brain P2 membranes were incubated with serial dilutions of (-)-nicotine or galantamine and [³H]nicotine (10 nM) or [³H]MLA (2 nM), and SH-SY5Y cell membranes were incubated with serial dilutions of (-)-nicotine or galantamine and [³H]epibatidine (150 pM), as described above.

Data Analysis. Dissociation constant (K_d) and maximum binding (B_max) values from saturation binding experiments were determined by nonlinear regression, fitting data points to a single-site ligand-binding model. IC₅₀ values were derived from competition binding data by fitting data points to the Hill equation using a nonlinear least-squares curve fitting facility of Sigma Plot 2.0 for Windows (SPSS Inc., Chicago, IL): % Bound = 100%/(1 + ([Ligand]/IC₅₀)^nH), where n_H is the Hill number, [Ligand] is the concentration of the competing ligand, and IC₅₀ is the concentration of competing ligand that displaces 50% of specific radioligand bound. K_i values were calculated from IC₅₀ values (Cheng and Prusoff, 1973), assuming K_d values determined in the corresponding saturation binding assays.

Calcium Fluorometry

Ca²⁺-mediated increases in fluo-3 fluorescence were monitored as described previously (Dajas-Bailador et al., 2002a). After removal of the medium from the confluent SH-SY5Y cultures, cells were washed twice with Tyrode's salt solution (137 mM NaCl, 2.7 mM KCl, 1.0 mM MgCl₂, 1.8 mM CaCl₂, 0.2 mM NaH₂PO₄, 12 mM NaHCO₃, and 5.5 mM glucose, pH 7.4) and incubated with the membrane-permeable, Ca²⁺-sensitive dye fluo-3 AM (10 μM) and 0.02% pluronic F127 for 1 h at room temperature in the dark. Cells were washed twice with Tyrode's salt solution, then 80 μl of buffer, with or without drugs, was added to each well. After 10 min of preincubation at room temperature in the dark, changes in fluorescence (excitation, 485 nm; emission, 538 nm) were measured using a Fluoroskan Ascent fluorescent plate reader (Labsystems, Helsinki, Finland). Basal fluorescence was monitored for 5 s before addition of stimulus (nicotine, KCl, or drug; 20 μl) and changes in fluorescence were monitored for a further 20 s. Responses in Lα4β2 cells were measured using the same protocol, except that responses were monitored in a high Ca²⁺ buffer (25 mM HEPES, 35 mM sucrose, 75 mM CaCl₂, pH 7.4) (Cooper and Millar, 1997) to amplify nicotine-evoked Ca²⁺ signals in cells that lack VOCC. To normalize fluo-3 signals, responses from each well were calibrated by determination of the maximum and minimum fluorescence values. At the end of each experiment, addition of 0.2% Triton (F_max) was followed by 40 mM MnCl₂ (F_min). Data were calculated as a percentage of F_max - F_min (Dajas-Bailador et al., 2002a). Values were expressed as a percentage of the response to 30 μM nicotine included in all experiments, unless otherwise stated. Maximum responses to nicotine were approximately 20% of the F_max - F_min and thus not close to saturation of the dye.

[³H]Noradrenaline Release

SH-SY5Y Cells. SH-SY5Y cells were cultured in 96-well plates until confluent. After removal of culture medium, [³H]noradrenaline (0.07 μM, 2.5 μCi/ml) was added (60 μl/well) in oxygenated Krebs-bicarbonate buffer (118 mM NaCl, 2.4 mM KCl, 2.4 mM CaCl₂, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 25 mM NaHCO₃, 10 mM d-glucose, 1 mM ascorbic acid, and 10 μM pargyline, pH 7.4), and the cells were incubated for1hat37°C. After loading the cells with [³H]noradrenaline, they were washed twice with Krebs buffer with added nomifensine (0.5 μM) to prevent reuptake of released [³H]noradrenaline. Cells were then incubated for 5 min in the same buffer, with or without mecamylamine. The buffer was replaced with Krebs buffer plus nomifensine containing nicotine and/or galantamine (80 μl/well). After 5 min, the medium containing released [³H]noradrenaline was transferred to a 96-well Optiplate (PerkinElmer Life Sciences, Zaventem, Belgium). Microscint-40 (170 μl; PerkinElmer) was added to each well and radioactivity was counted using a microbeta liquid scintillation counter (Wallac MicroBeta TriLux 1450; PerkinElmer Life Sciences, Espoo, Finland; counting efficiency, 31%). Radioactivity remaining in the cultures was determined by addition of 80 μl of 0.5 M perchloric acid and incubation for 1 h at 37°C, followed by scintillation counting. The total amount of [³H]noradrenaline present in the cells at the point of stimulation was equivalent to the tritium released plus tritium remaining. Released [³H]noradrenaline was calculated as a percentage of total radioactivity in the corresponding wells, and results were then expressed as a percentage of basal release (buffer stimulation). The average values of basal release and corresponding total radioactivity were 3189 ± 80 and 39,424 ± 809 cpm (mean ± S.E.M., n = 60) respectively. Therefore, basal release corresponds to 8.0% of the radioactivity present in culture wells at the beginning of the experiment.

Hippocampal Slices. The measurement of [³H]noradrenaline release from rat hippocampal slices was based on the method of Anderson et al. (2000). Rats (250-350 g) were killed by cervical dislocation, and hippocampi were rapidly dissected. Hippocampal slices (0.25 mm) were prepared using a McIlwain tissue chopper, washed twice with Krebs-bicarbonate buffer (as above), and loaded with [³H]noradrenaline (0.07 μM, 2.5 μCi/ml) for 30 min at 37°C. After four washes with Krebs buffer plus nomifensine (0.5 μM), slices were dispersed into 96-well Multiscreen filter plates (Millipore, Bedford, MA). Hippocampi from two animals were sufficient for one 96-well plate. Buffer was removed using a vacuum filtration unit (Millipore), leaving tissue slices on well filters. Subsequently, 70 μl of Krebs buffer containing nomifensine, with or without drugs, was added to each well and allowed to incubate for 5 min at 37°C in an atmosphere of 95% O₂ and 5% CO₂. Basal and stimulated release was collected via vacuum filtration into a 96-well Optiplate (PerkinElmer Life Sciences) and radioactivity was counted as described above. To estimate radioactivity remaining in the slices at the end of the experiment, filters were removed from the 96-well plate and counted for radioactivity in a PerkinElmer Life Sciences Tri-Carb 1500 liquid scintillation counter (counting efficiency, 44%). Total radioactivity present in the slices at the start of the stimulation was calculated as the sum of disintegrations per minute of tritium released plus disintegrations per minute of tritium radioactivity present in slices at the end of experiment, after correction for counting efficiency. Release was calculated as a percentage of total radioactivity. Values were then expressed as a percentage of the basal response (buffer stimulation). The average values of basal release and corresponding total radioactivity were 1182 ± 40 and 72,860 ± 2652 cpm (mean ± S.E.M., n = 60) respectively. Therefore, basal release corresponds to 1.62% of radioactivity present in the tissue at the beginning of the experiment.

Acetylcholinesterase (AChE) Activity

The potencies of AChEI with respect to rat brain AChE activity were assayed in P2 membranes based on the colorimetric method of Ellman et al. (1961). This measures the change in the rate of hydrolysis of a thiocholine ester, which exchanges with 5,5′-dithiobis(2-nitrobenzoate) to produce a yellow product, 5-mercapto-2-nitrobenzoate. Test compounds (final concentration, 0.1 nM-10 μM) were preincubated with P2 membranes (10 μl) in 2.5 ml of 0.1 M NaH₂PO₄, pH 8.0, for 20 min, before addition of 5,5′-dithiobis(2-nitrobenzoate) (final concentration, 0.4 mM) and the substrate acetylthiocholine iodide (0.5 mM, final concentration). The change in absorbance at 412 nm was monitored, and AChE activity was calculated according to the relationship micromoles hydrolyzed per minute per milliliter = ΔE/1.36 × 10⁴ × dilution factors, where ΔE is the change in absorbance measured and 1.36 × 10⁴ is the molar extinction coefficient. Activity in the presence of drug was calculated as a percentage of the activity determined in the absence of drug. IC₅₀ values were determined by fitting data points to the Hill equation, as described above for ligand binding.

Statistics

Data are the mean ± S.E.M. of three or more independent experiments, each with three (binding and AChE assays), four (Ca²⁺ studies), or eight (transmitter release) replicates. Statistical significance was determined using one-way ANOVA (post hoc Dunn's or Tukey's test, as indicated in figure legends) for comparison between groups and Student's t test for comparisons between treatments and respective controls. Values of p < 0.05 were taken to be statistically significant.

Binding Studies

Electrophysiological recordings of single-channel and whole-cell currents have shown that APLs, such as galantamine and physostigmine, can potentiate the effects of agonists at many subtypes of nAChR, and this effect has been attributed to an interaction with an allosteric site on the nAChR (Pereira et al., 2002). However, it has also been suggested that some AChEI interact competitively with the agonist site of fetal muscle (Cooper et al., 1996) or α4β4 nAChR expressed in Xenopus laevis oocytes (Zwart et al., 2000). To address the possibility of a competitive interaction, galantamine was examined for its ability to displace radioligand from the agonist binding site of three nAChR subtypes (Fig. 1A). Galantamine did not compete for [³H]nicotine binding to rat brain α4β2 nAChR, [³H]epibatidine binding to α3^* nAChR in human SH-SY5Y cell membranes, or [³H]MLA binding to rat brain α7 nAChR, whereas nicotine displaced radioligand binding in each case, with K_i values in the expected range (Fig. 1A). These results are consistent with the argument that galantamine acts at a site near, but distinct from, the ACh binding site on nAChR (Pereira et al., 2002; Schrattenholz et al., 1996). Through an allosteric interaction via this site, galantamine could influence agonist binding by increasing the nAChR affinity for agonist. This was examined by carrying out saturation binding assays with the same three radioligands in the presence or absence of 1 μM galantamine (Fig. 1B). Neither affinity (K_d) nor maximum binding capacity (B_max) was altered in the presence of galantamine (Table 1). These results indicate that there is no shift in nAChR affinity for agonist in response to galantamine; it is thus plausible that galantamine enhances the coupling of agonist binding to channel opening without affecting agonist binding per se. However, an alternative explanation is that saturation binding assays reflect the desensitized state of the receptor, which may not be relevant to the functional potentiation of nAChR responses.

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Fig. 1.

Ligand binding assays. A, competition binding assays were carried out with radioligand in the absence (total binding) or presence of serial dilutions of (-)-nicotine (•) or galantamine (○) as described under Materials and Methods. [³H]Nicotine (10 nM) binding to rat brain membranes defined α4β2 nAChR; [³H]epibatidine (150 pM) binding to SH-SY5Y cell membranes defined α3^* nAChR; [³H]MLA (2 nM) binding to rat brain membranes defined α7 nAChR. Nonspecific binding was determined in the presence of 100 μM, 1 mM, and 1 mM nicotine, respectively, and subtracted to give specific binding. Data points are fitted to the Hill equation and IC₅₀ values derived using the nonlinear least-squares curve fitting facility of Sigma Plot 2.0 for Windows. K_i values were determined by the method of Cheng and Prusoff (1973). Nicotine inhibited [³H]nicotine, [³H]epibatidine, and [³H]MLA binding with K_i values of 27.3 ± 1.7 nM, 122 ± 36 nM, and 12.3 ± 0.86 μM, respectively. B, saturation binding assays for [³H]nicotine binding to rat brain membranes (α4β2 nAChR), [³H]epibatidine binding to SH-SY5Y cell membranes (α3^* nAChR), and [³H]MLA binding to rat brain membranes (α7 nAChR) were carried out in the absence (•) or presence (○) of 1 μM galantamine. Nonspecific binding was defined in the presence of 100 μM, 1 mM, and 1 mM nicotine, respectively, and subtracted to give specific binding. Data points are fitted to the Hill equation for a single binding site, and binding constants determined by nonlinear regression. Data are summarized in Table 1. Values are the mean ± S.E.M. from three assays, with each point assayed in triplicate.

Nicotine-Evoked Ca²⁺ Responses

To determine whether the allosteric potentiation of nAChR was transduced into relevant cellular responses, we measured the effect of galantamine on nicotine-evoked increases in intracellular Ca²⁺ in SH-SY5Y cells loaded with the Ca²⁺-sensitive dye fluo-3 AM. As reported previously (Dajas-Bailador et al., 2002a), we observed a rapid and significant increase in fluorescence after stimulation with 30 μM nicotine (Fig. 2A), which approximates to the EC₅₀ value for this assay (Ridley et al., 2002). Preincubation with the general nAChR antagonist mecamylamine at a concentration (40 μM) to block all nAChR subtypes, completely prevented this nicotine-evoked increase in fluorescence, confirming that nAChRs mediate the response (data not shown; see Dajas-Bailador et al., 2002a). Preincubation for 5 min with a range of galantamine concentrations (0.1 - 50 μM) resulted in a bell-shaped profile of potentiation of responses to 30 μM nicotine (Fig. 2B). Galantamine at 0.5, 1, and 3 μM significantly increased the nicotine-evoked rise in fluorescence, whereas lower and higher concentrations failed to produce any significant effect compared with nicotine alone. The maximum increase of 16.9 ± 3.2% above the response to nicotine alone was produced by 1 μM galantamine. Potentiation by 0.5, 1, and 3 μM galantamine was evident throughout the 20-s time course (illustrated in Fig. 2A for 3 μM galantamine). Stimulation with galantamine alone (in the absence of nicotine) had no effect on fluorescence levels (data not shown).

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Fig. 2.

Nicotine-evoked increases in intracellular Ca²⁺: potentiation by galantamine. A, representative traces from SH-SY5Y cells loaded with fluo-3 AM and stimulated with 30 μM nicotine in the presence or absence of 3 μM galantamine. Where present, galantamine was preincubated with the cells for 5 min before addition of nicotine. Changes in fluorescence were monitored for 20 s. The nicotine-evoked increase in fluorescence in SH-SY5Y cells (B) and Lα4β2 cells (C) was determined after preincubation for 5 min with a range of galantamine concentrations. Nicotine (30 μM or 10 μM) was added and fluorescence changes after 20 s were recorded. Data are expressed as a percentage of the response to nicotine stimulation in the absence of galantamine and represent the mean ± S.E.M. of at least four independent experiments, each carried out in quadruplicate. Significantly different from nicotine stimulation; ^*, p < 0.05, Student's t test.

SH-SY5Y cells express multiple nAChR subunits (Lukas et al., 1993; Peng et al., 1994; Wang et al., 1996) and have functional α3^* and α7 nAChR that contribute to nicotine-evoked Ca²⁺ increases (Dajas-Bailador et al., 2002a; Ridley et al., 2002) but do not express α4β2 nAChR, which is the predominant subtype in the brain. To examine the ability of galantamine to modulate increases in intracellular Ca²⁺ initiated by activation of this subtype, we used a mouse fibroblast cell line expressing rat α4β2 nAChR (Lα4β2 cells; Cooper et al., 1999). Preincubation with galantamine also potentiated nicotine-evoked increases in fluo-3 fluorescence in these cells, and the effect was observed over a similar range of galantamine concentrations as in SH-SY5Y cells (Fig. 2C). The maximum potentiation (25.9 ± 9.4% increase) was produced by 1 μM galantamine.

Galantamine was compared with other AChEI for their abilities to modulate nicotine-evoked Ca²⁺ responses in SHSY5Y cells. Preincubation with physostigmine (the first compound recognized as an APL at nAChRs; Shaw et al., 1985; Pereira et al., 1993, 2002) also potentiated nicotine-evoked increases in fluorescence but only at a single concentration (1 μM; Fig. 3A). The magnitude of the increase in nicotine-evoked fluorescence in the presence of physostigmine (18.9 ± 4.9% above the response to nicotine alone) was comparable with that observed with 1 μM galantamine (Fig. 2B). However, unlike galantamine, high concentrations of physostigmine (>30 μM) produced a significant decrease in nicotine-evoked fluorescence (Fig. 3A). To investigate whether the potentiation of nAChR function was related to the ability to inhibit AChE, we examined nicotine-evoked responses after incubation with donepezil and rivastigmine, two structurally unrelated AChEI presently used in the treatment of AD. Neither rivastigmine nor donepezil potentiated responses to nicotine. At the highest concentration tested, rivastigmine (50 μM) produced a partial inhibition of the nicotine-evoked increase in fluo-3 fluorescence (Fig. 3B), whereas preincubation with donepezil generated a significant reduction of nicotine-evoked responses at concentrations of 3 μM and above, with an almost complete block at the highest concentrations tested (Fig. 3C). The IC₅₀ value for this inhibition by donepezil was 3.9 μM.

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Fig. 3.

The effect of AChEI on nicotine-evoked increases in intracellular Ca²⁺. SH-SY5Y cells were loaded with fluo-3 AM and preincubated for 5 min with a range of concentrations (0.3-50 μM) of physostigmine (A), rivastigmine (B), or donepezil (C). Nicotine (30 μM) was added and fluorescence changes after 20 s were recorded. Data are expressed as a percentage of the response to nicotine stimulation in the absence of AChEI, and represent the mean ± S.E.M. of at least four independent experiments, each carried out in quadruplicate. Significantly different from nicotine stimulation; ^*, p < 0.05, Student's t test.

The nicotine-evoked Ca²⁺ signals in SH-SY5Y cells are generated through the sequential activation of various sources of extracellular and intracellular Ca²⁺, including VOCC and intracellular stores (Dajas-Bailador et al., 2002a). To assess if the potentiation or inhibition of nicotine-evoked responses was occurring through the modulation of downstream processes not necessarily dependent on nAChR activation, we compared the effect of AChEIs (0.5-30 μM) on the increases in fluorescence evoked by a general depolarising stimulus (KCl, 40 mM) in SH-SY5Y cells. The magnitude of the response to this concentration of KCl was comparable with that elicited by 30 μM nicotine (Ridley et al., 2002). Neither galantamine nor physostigmine nor rivastigmine produced any significant effect on the KCl-evoked increase in fluorescence, whereas in the presence of 10 and 30 μM donepezil, KCl-evoked responses were significantly decreased, to 75 ± 3.9% and 53.2 ± 6.1% of KCl control, respectively (data not shown). Thus, of the four AChEI examined, only donepezil exhibited any effect on nAChR-independent increases in Ca²⁺.

[³H]Noradrenaline Release

SH-SY5Y Cells. One of the many functional consequences of increasing intracellular Ca²⁺ concentrations is the exocytosis of neurotransmitter. Therefore, we examined whether the allosteric modulation of nAChR could be observed downstream of the evoked Ca²⁺ signals by analyzing the effects of galantamine and other AChEI on nicotine-evoked [³H]noradrenaline release from SH-SY5Y cells. Nicotine produced a significant and concentration-dependent increase of [³H]noradrenaline release (see Fig. 4) that was Ca²⁺-dependent (data not shown) and blocked by the nAChR antagonist mecamylamine (see below). Coincubation with galantamine (0.3-30 μM) resulted in a bell-shaped potentiation of [³H]noradrenaline release evoked by 5 μM nicotine (Fig. 4A), reminiscent of the effects of galantamine on nicotine-evoked Ca²⁺ responses in these cells (Fig. 2B). Significant potentiation was observed with 0.5, 1 and 3 μM galantamine in both assays. However, the extent of potentiation was relatively greater than that observed for the Ca²⁺ responses, with 0.5, 1, and 3 μM galantamine producing an increase in [³H]noradrenaline release that was approximately 75% above that evoked by 5 μM nicotine alone. At a lower concentration of nicotine (3 μM), galantamine produced a similar potentiation (Fig. 4B), whereas responses to a higher nicotine concentration (10 μM) were unaffected by the presence of galantamine (Fig. 4C). The responses to 5 μM nicotine alone and 5 μM nicotine plus galantamine (0.5, 1, and 3 μM) were prevented by mecamylamine (40 μM; Fig. 4A), indicating that the potentiation occurred through nAChR. Consistent with this view, galantamine (1 μM) had no significant effect on [³H]noradrenaline release in the absence of nicotine (Fig. 4A).

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Fig. 4.

The effect of galantamine on nicotine-evoked [³H]noradrenaline release in SH-SY5Y cells. SH-SY5Y cells were loaded with [³H]noradrenaline and the release of tritium evoked by incubation for 5 min with increasing concentrations of nicotine [5 μM (A), 3 μM (B), and 10 μM (C)] as described under Materials and Methods. Nicotine-evoked [³H]noradrenaline release was determined in the absence or presence of a range of galantamine concentrations. Galantamine (1 μM) was tested in the absence of nicotine (A). The effect of 5 μM nicotine on [³H]noradrenaline release in the presence and absence of galantamine was also examined in the presence of mecamylamine (40 μM; A). Data are expressed as a percentage of basal release in the absence of nicotine or galantamine and represent the mean ± S.E.M. of at least five independent experiments, each with eight replicates. Significantly different from nicotine stimulation; ^*, p < 0.05, Student's t test. In the presence of mecamylamine (A), there was no significant difference in release between nicotine, nicotine plus galantamine, and basal condition (Student's t test). There was no significant increase in [³H]noradrenaline release after incubation with galantamine alone (A), compared with basal release or with release in the presence of nicotine plus mecamylamine (Student's t test).

In contrast to galantamine, neither physostigmine, rivastigmine, nor donepezil (0.3-30 μM) produced any significant modulation of [³H]noradrenaline release from SH-SY5Y cells stimulated with 5 μM nicotine (data not shown). This was unexpected in the case of donepezil, in view of its inhibition of nicotine- (Fig. 3C) and KCl-evoked increases in intracellular Ca²⁺. However, if some of donepezil's inhibitory effect is caused by the blockade of a secondary source of Ca²⁺, and VOCC would be a prime candidate, it is possible that VOCC do not contribute to exocytosis in response to 5 μM nicotine in SH-SY5Y cells; this would explain the lack of any significant block by donepezil. To explore this possibility, we compared the effects of donepezil (30 μM) and CdCl₂ (100 μM; sufficient to block VOCC without inhibiting nAChR; Rathouz and Berg, 1994) on [³H]noradrenaline release evoked by increasing concentrations of nicotine (Fig. 5). At 5 μM nicotine, neither CdCl₂ nor donepezil affected the amount of nicotine-evoked [³H]noradrenaline release, consistent with a lack of involvement of VOCC. At 10 μM nicotine, CdCl₂ had no effect, whereas donepezil inhibited the response to nicotine by 34%. The response to 100 μM nicotine was significantly reduced by both CdCl₂ and donepezil, by 46% and 73%, respectively. These results suggest that VOCC contributes only to nicotine-evoked [³H]noradrenaline release at the highest agonist concentration tested, when CdCl₂ and donepezil are both effective blockers. The greater inhibition observed with donepezil, compared with that of CdCl₂, suggests that it has alternative modes of action: possibly a direct effect on nAChR and/or an indirect effect at a downstream target governing a secondary source of Ca²⁺.

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Fig. 5.

The effect of donepezil and CdCl₂ on nicotine-evoked [³H]noradrenaline release in SH-SY5Y cells. SH-SY5Y cells were loaded with [³H]noradrenaline and the release of tritium was evoked by 5-min incubation with 5, 10, or 100 μM nicotine in the presence or absence of donepezil (30 μM) and CdCl₂ (100 μM). Significantly different from nicotine-evoked release in the absence of drugs; ^*, p < 0.05, Student's t test. Significantly different from response in the presence of CdCl₂; #, p < 0.05, one-way ANOVA (post hoc Dunn's test). In all cases, data represent the mean ± S.E.M. of at least five independent experiments, each with eight replicates.

Hippocampal Slices. To determine whether the allosteric potentiation of nAChR-mediated transmitter release observed in SH-SY5Y cells is also pertinent to the central nervous system, we examined the effect of galantamine on nAChR-evoked [³H]noradrenaline release from rat hippocampal slices. In this experimental model, stimulation with nicotine (1, 5, and 10 μM) produced a significant and concentration-dependent increase in [³H]noradrenaline release (Fig. 6) that was much greater (relative to basal release) than in the cell line (Fig. 4). Stimulation with 1 and 5 μM nicotine in the presence of galantamine (0.5, 1, and 3 μM) resulted in a significant potentiation of [³H]noradrenaline release compared with the response induced by nicotine alone (Fig. 6). In agreement with the data from SH-SY5Y cells, galantamine did not potentiate release evoked by a higher concentration of nicotine (10 μM; Fig. 6). However, in contrast to studies with the cell line, galantamine (1 μM) alone produced a significant increase in [³H]noradrenaline release in the absence of nicotine (Fig. 7; 31 ± 9.4% increase above basal). This release could be a consequence of AChE inhibition, such that any ACh released from the hippocampal slices would be preserved and hence could activate presynaptic nAChR (or muscarinic receptors). Evidence for the participation of nAChR in the release of [³H]noradrenaline evoked by galantamine alone was provided by the partial blockade by mecamylamine (Fig. 7A). This mechanism would augment the effects of submaximal nicotine concentrations and confound interpretation of the potentiation observed with galantamine. Simply subtracting the galantamine-alone response from the release of [³H]noradrenaline provoked by nicotine plus galantamine would be inappropriate, because nicotine will release further ACh from the hippocampal preparation (Wilkie et al., 1996); hence, the AChEI-dependent component will be increased.

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Fig. 6.

Effect of galantamine on nicotine-evoked [³H]noradrenaline release from hippocampal slices. Rat hippocampal slices were loaded with [³H]noradrenaline, and the release of tritium was evoked by 5-min incubation with 1, 5, or 10 μM nicotine in the presence or absence of increasing concentrations of galantamine. Data are expressed as a percentage of basal release and represent the mean ± S.E.M. of at least six independent experiments, each with eight replicates. Significantly different from nicotine stimulation; ^*, p < 0.05, Student's t test.

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Fig. 7.

Effect of AChEI on basal [³H]noradrenaline release (A) and nicotine-evoked [³H]noradrenaline release (B) from rat hippocampal slices. Rat hippocampal slices were loaded with [³H]noradrenaline and incubated for 5 min with (A) rivastigmine, donepezil, physostigmine or galantamine (1 μM). Galantamine was examined in the presence and absence of mecamylamine (40 μM). The tritium released was expressed as a percentage of basal release in the absence of drugs. All of the AChEI tested significantly increased the release of [³H]noradrenaline above basal levels (^*, p < 0.05, Student's t test); the effect of galantamine was significantly greater than that of rivastigmine (#, p < 0.05, one-way ANOVA, post hoc Tukey's test). In the presence of mecamylamine, the release evoked by galantamine was significantly less than that evoked by galantamine alone (#, p < 0.05, Student's t test). B, rat hippocampal slices were incubated with rivastigmine (1 μM) to inhibit AChE before stimulation for 5 min with nicotine (1 μM) in the presence or absence of galantamine (0.5, 1, and 3 μM) and measurement of [³H]noradrenaline release. In parallel, slices were stimulated with nicotine in the absence of any AChE. Significantly different from the response to nicotine + rivastigmine; ^*, p < 0.05, Student's t test. Data represent the mean ± S.E.M. of at least five independent experiments, each with eight replicates.

Consistent with the hypothesis that AChE inhibition results in the release of [³H]noradrenaline, rivastigmine, physostigmine, and donepezil (1 μM) all significantly increased the amount of tritium released (Fig. 7). The potencies of these compounds as AChEI were determined in parallel for rat brain cholinesterase (Table 2); rivastigmine and donepezil were 8- and 20-fold more potent, respectively, than galantamine, which is in general agreement with literature values (Perry et al., 1988; Santos et al., 2002). At the concentration employed in the [³H]noradrenaline release assay (1 μM, Fig. 7), all of the compounds would have completely blocked AChE. To dissect the contribution of AChE inhibition to the observed galantamine potentiation of nicotine-evoked [³H]noradrenaline release in hippocampal slices, we examined galantamine's effects in the presence of 1 μM rivastigmine. The rationale for this experiment is that rivastigmine is essentially without effect on nAChR-mediated responses (Fig. 2B) but is a potent inhibitor of AChE (Table 2). Thus, incubation with rivastigmine would inhibit AChE without directly compromising nAChR activation, allowing any nicotinic action of galantamine to be revealed. As shown in Fig. 7B, galantamine still potentiated nicotine-evoked [³H]noradrenaline release in the presence of rivastigmine. However, the effect was considerably less than in the absence of the independent inhibition of AChE; 1 μM galantamine enhanced nicotine-evoked [³H]noradrenaline release by 43 ± 9.5% and 14 ± 3.0% in the absence or presence, respectively, of rivastigmine.