Introduction

Top Summary Introduction Procedures Results Discussion References

Molecular cloning has disclosed a diversity of nAChR subunits that are expressed in the vertebrate central nervous system: to date, 11 subunits (2-9 and 2-4) have been found (Sargent, 1993; Elgoyhen et al., 1994). Immunoprecipitation studies indicate that >90% of high affinity nicotinic agonist binding in the rat brain corresponds to a receptor composed of 4 and 2 subunits (Flores et al., 1992). Chronic nicotine treatment in vivo up-regulates the numbers of brain nAChRs identified by high affinity tritiated agonist binding in mouse (Marks et al., 1985) and rat (Flores et al., 1992; Schwartz and Kellar, 1985). Chronic nicotine treatment over 5-21 days produced concentration-dependent increases of 50-100% above control. The extent of the response varied in a brain region specific manner (Marks et al., 1985, 1992; Sanderson et al., 1993). After cessation of nicotine treatment for 4-8 days, nAChR levels returned to control values (Marks et al., 1985; Schwartz and Kellar, 1985), demonstrating the reversibility of this phenomenon. Up-regulation of [³H]nicotine binding sites also has been observed in human brain tissue from tobacco smokers compared with controls from nonsmokers (Breese et al., 1997). This phenomenon may be relevant to tolerance, sensitization, and withdrawal, features considered to contribute to the development and/or maintenance of nicotine dependence. The up-regulation of nicotinic binding sites by nicotine has been termed paradoxical (Wonnacott, 1990) because chronic agonist exposure has traditionally been predicted to down-regulate receptor numbers (Creese and Sibley, 1981). This model was based on the down-regulation of G protein-coupled receptors but has been found to apply to certain ligand-gated ion channels, notably -aminobutyric acid_A receptors: for example, in cortical neurons, both subunit polypeptide and mRNA are down-regulated by chronic treatment with -aminobutyric acid (Mhatre and Ticku, 1994).

Up-regulation of neuronal nAChR has also been demonstrated in in vitro systems, including cells transfected with 4 and 2 subunits (Peng et al., 1994; Zhang et al., 1994; Bencherif et al., 1995; Gopalakrishnan et al., 1997). The M10 cell line consists of mouse fibroblasts stably transfected with chick 4 and 2 subunits under the control of a dexamethasone-sensitive promoter (Whiting et al., 1991): treatment of M10 cells with nicotine for 2-3 days increases 42 nAChR by 2-3-fold, as judged by [³H]nicotine binding to immunoisolated material (Peng et al., 1994) or membrane preparations (Zhang et al., 1994; Bencherif et al., 1995). Up-regulation of human 42 nAChR expressed in human embryonic kidney 293 cells (Gopalakrishnan et al., 1997) (measured by [³H]cytisine binding to membrane preparations) is similar to that seen in the M10 cell line. This phenomenon is not unique to the 42 subtype of nAChR: -bungarotoxin binding sites, considered to correspond to 7-type nAChRs, are up-regulated after chronic nicotine treatment in vivo (Pauly et al., 1991) and in vitro (Barrantes et al., 1995), and 3-type nAChRs in SHSY-5Y human neuroblastoma cells have recently been shown to exhibit the same response to chronic agonist (Peng et al., 1997). Up-regulation of 7- and 3-type nAChRs requires higher nicotine concentrations and therefore may be less relevant to changes produced by smoking doses of nicotine.

Studies in vivo (Marks et al., 1985; Schwartz and Kellar, 1985; Sanderson et al., 1993) and in vitro (Peng et al., 1994) have established that up-regulation of [³H]nicotine binding sites reflects an increase in receptor numbers rather than a change in receptor affinity for the ligand. This increase is probably not the result of increased transcription because mRNA levels of the constituent subunits are unchanged (Marks et al., 1992; Peng et al., 1994; Zhang et al., 1994; Bencherif et al., 1995). This result implies a post-transcriptional mechanism: altered translation rates (Gopalakrishnan et al., 1997), a decrease in receptor turnover (Peng et al., 1994), recruitment from a finite pool of preexisting receptors (Bencherif et al., 1995), and improved efficiency of receptor assembly from constituent subunits (Rothhut et al., 1996) have been proposed. As well as these conflicting views of the mechanism underlying up-regulation, how chronic nicotine initiates the response remains unclear. Originally it was proposed that up-regulation might be linked to desensitization of the nAChR (Marks et al., 1985; Schwartz and Kellar, 1985). Under the functional model of Katz and Thesleff (1957), chronic nicotine treatment would convert the receptor from an active to a desensitized state. Up-regulation would then be an adaptive mechanism to compensate for this loss of function, but the fact that up-regulation can be provoked in cell lines with no opportunity for natural cholinergic stimulation, and hence no function to "lose," tends to refute this argument. Up-regulation must be an intrinsic molecular response, as opposed to a specifically neuronal cellular response. Indeed, Peng et al. (1994) showed that nicotine concentrations that promote up-regulation are considerably greater than those needed for desensitization of the 42 nAChR.

Here, we conducted a detailed pharmacological evaluation of the up-regulation of 42 nAChR in M10 cells, with respect to a wide spectrum of agonists and antagonists, which were analyzed over a broad concentration range. This homogeneous in vitro system facilitates a quantitative dose-response analysis that is untenable in vivo. Agonists showed a diversity of up-regulatory effects that were mediated at the cell surface. The conditions producing up-regulation are consistent with a state of the receptor that is different from either the high affinity desensitized or activated states.

	Experimental Procedures

Top Summary Introduction Procedures Results Discussion References

Materials

Tissue culture. M10 cells were provided by Dr. Paul Whiting (MSD Research Center, Harlow, Essex, UK). Tissue culture reagents were obtained from GIBCO BRL (Paisley, Renfrewshire, Scotland). Tissue cultureware was purchased from Becton Dickinson UK (Oxford, UK) or Sterilin (Stone, Staffs, UK). Maintenance medium consisted of DMEM supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine, and 0.5 mg/ml geneticin as a selection agent. Induction medium was composed of DMEM supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine, and 1 µM dexamethasone to induce receptor expression.

Drugs and reagents. (±)-[³H]Epibatidine (57 Ci/mmol in ethanol) was obtained from DuPont NEN (Stevenage, Herts, UK) and stored at 20°. (±)-Anatoxin-a was provided by Prof. T. Gallagher (School of Chemistry, University of Bristol, UK). ABT-418 [(S)-3-methyl-5-(1-methyl-2-pyrrolidinyl)isoaxole] was provided by Dr. S. Arneric (Abbott Laboratories, Chicago, IL). All other drugs were purchased from Sigma Chemical (Poole, Dorset, UK) or RBI (Natick, MA). Drugs were dissolved in distilled water or ethanol as appropriate and stored at 20°. All chemical reagents used were supplied by BDH/Merck (Poole, Dorset, UK), Sigma Chemical, or FSA Supplies (Loughborough, UK).

Methods

Cell culture. Routine culture of M10 cells was carried out as described by Whiting et al. (1991). Briefly, cells were grown at 37° in a humidified incubator (95% O₂/5% CO₂ atmosphere). M10 cell stocks were maintained in 75-cm² flasks containing 25 ml of maintenance medium. Cells were seeded onto 75-cm² flasks or 24 × 16-mm well plates at ~20% confluency (15,000 cells/cm²). Receptor production was induced when cells had reached ~70% confluency (typically 2-3 days) by replacement of maintenance medium with induction medium (in which geneticin was replaced with 1 µM dexamethasone).

Quantification of [³H]epibatidine binding sites in M10 cells in situ. [³H]Epibatidine binding was performed on cells in 24-well plates. All steps were carried out at 37° unless otherwise stated. Medium was removed by aspiration, and each well was washed by the addition and aspiration of 1 ml of sterile PBS (150 mM NaCl, 8 mM K₂HPO₄, 2 mM KH₂PO₄, pH 7.4). [³H]Epibatidine (500 pM in maintenance medium; 1 ml/well) was added. Nonspecific binding was assayed in the presence of 100 µM ()-nicotine. Samples were incubated for 2 hr with gentle agitation before aspiration of the assay solution and washing with four changes of PBS to remove unbound ligand. To measure bound ligand, the cells were dissolved overnight in 1 ml/well of Markwell reagent A [2% (w/v) Na₂CO₃, 1% (w/v) sodium dodecyl sulfate, 0.1 M NaOH, 0.16% (w/v) Na/K tartrate]. Samples (700 µl) were mixed with 5 ml of Optiphase Safe liquid scintillant and counted for tritium (Tricarb 1600 liquid scintillation counter; Packard, Meriden, CT; counting efficiency, 45%). Protein was determined by the method of Markwell et al. (1978) using 200 µl of the remaining cell solution. Specific cpm [³H]epibatidine bound/well were converted to fmol bound/mg protein. [³H]Epibatidine binding to M10 cells induced for 48 hr gave 326 ± 46 fmol/mg (protein) (mean ± standard error, 10 independent assays).

Kinetic analysis of [³H]epibatidine binding to M10 cells in situ. On and off rates were determined at 4°, 20°, and 37°. At equilibrium, bound [³H]epibatidine (200 pM) accounted for <5% of total ligand added, so ligand depletion effects were not significant.

(1)

View larger version (27K): [in this window] [in a new window]   Fig. 1.   Time dependence of [3H]epibatidine association to and dissociation from M10 cells in situ. Experiments were performed at 4°(), 20° () and 37° (). a, Association data were derived from cultures incubated with 200 pM [3H]epibatidine in the presence and absence of 100 µM ()-nicotine for the times indicated. Specific binding was calculated as a percentage of equilibrium (maximum) binding determined by incubation at 37o for 2 hr. Values were fit to a double exponential increase model (see Experimental Procedures). b, Dissociation data were derived from cultures pre-equilibrated with 200pM [3H]epibatidine in the presence and absence of 100 µM ()-nicotine, followed by incubation with excess unlabelled ()-nicotine for the times indicated. Specific binding was calculated as a percentage of the initial (equilibrium) binding and values were fit to a single exponential decrease model (see Experimental Procedures). Logarithmic transformation of the data (insets) shows good agreement between the data and the model, confirming the validity of this approach. Points, mean ± standard error of at least three independent determinations.

(2)

(3)

Chronic treatment of M10 cells with nicotinic drugs. To examine the effects of chronic exposure to nicotinic drugs, cells were incubated for 48 hr in induction medium supplemented with the desired concentrations of nicotinic agonist or antagonist. To assess the effects of antagonists or partial agonists on agonist-induced up-regulation, both compounds were added simultaneously. A control, consisting of cells treated with induction medium alone, was included in each experiment.

(4)

Competition assays of [³H]epibatidine binding to M10 cells in situ. [³H]Epibatidine displacement binding assays were performed using a modified version of the standard binding protocol. [³H]Epibatidine (200 pM) was prepared in DMEM, and this was used to make serial dilutions of nicotinic drugs. All steps were carried out at room temperature to reduce metabolically dependent transport of ligands, and incubations were extended to 3 hr to ensure that equilibrium binding of [³H]epibatidine was attained. The IC₅₀ value for each drug was calculated by fitting to Hill equation, using the nonlinear least-squares curve fitting facility of SigmaPlot for Windows

(5)

where n_H is the Hill number, [Ligand] is the concentration of the displacing ligand, and the IC₅₀ is the concentration at which the ligand tested displaces half of the specific [³H]epibatidine binding. IC₅₀ values obtained from individual experiments were used to calculate the affinity constant (K_i) for each ligand according to the method of Cheng and Prusoff (1973):

(6)

where [Ligand] is the concentration of the labeled ligand, and K_d is the equilibrium displacement binding constant.

Preparation of M10 cell membranes. M10 cell membranes were prepared by a modification of the procedure described by Whiting et al. (1991). After induction of M10 cells in 75-cm² flasks (48-72 hr), medium was removed, followed by washing with two changes (5 ml, 37°) of PBS. The cells were harvested in 5 ml of homogenization buffer (ice-cold PBS containing 10 mM EDTA, 10 mM EGTA, 1 mM phenylmethylsulfonyl fluoride) and collected by gentle centrifugation (5 min, 500 × g). After resuspension into 5 ml of homogenization buffer, the cells were disrupted by sonication (three times for 10 sec). The resulting membrane preparation was collected by centrifugation (100,000 × g, 15 min), and the supernatant fraction was discarded. The membrane pellet was resuspended in an additional 5 ml of homogenization buffer (hand-held glass homogenizer, six strokes) before recentrifugation (100,000 × g, 15 min), resuspension in homogenization buffer containing glycerol (1:1 v/v; 1 ml/75-cm² flask of cells), and storage at 20° in 1-ml aliquots.

[³H]Epibatidine binding to M10 membranes. Competition assays of [³H]epibatidine binding to membrane preparations were terminated by filtration. Membranes (duplicate 25-µl samples) were incubated with [³H]epibatidine (200 pM in DMEM, 2 ml) for 2 hr at 37° in the presence and absence of nicotinic ligands. Nonspecific binding was assayed in the presence of 100 µM ()-nicotine. Samples were filtered and washed on Whatman GFA/E filters soaked in polyethyleneimine (0.3% w/v, 24 hr), using a Brandel (Semat, St. Albans, Herts, UK) cell harvester. Protein was determined according to the method of Markwell et al. (1978). The mean density of [³H]epibatidine binding sites in M10 membranes was 712 ± 49 fmol/mg of protein (mean ± standard error, three independent assays). IC₅₀ and K_i values were determined as for M10 cells in situ.

Estimation of surface [³H]epibatidine binding sites. The contribution of surface nAChRs to the total population of [³H]epibatidine binding sites was determined in M10 cells in situ using a modified version of the protocol for cells chronically treated with nicotinic ligands. Cells in 24-well plates were induced in the presence or absence of up-regulating concentrations of agonists and then extensively washed as described above. [³H]Epibatidine binding (200 pM) was performed either in plain medium (to measure the total population) in the presence of ACh (3 µM, a sufficient concentration to completely block [³H]epibatidine binding to M10 membrane preparations and thus cell surface sites) or in the presence of ()-nicotine (3 µM, to measure nonspecific binding).

Time course of nicotinic ligand entry by M10 cells in situ. To determine the extent to which ligands that were impermeant in the acute binding protocols might permeate M10 cells during the standard up-regulation protocol (48 hr at 37°), competition binding experiments were performed at time points during the up-regulation process on M10 cells in situ. Cells were induced in 24-well plates for 72 hr to ensure equilibrium levels of nAChR expression. Spent induction medium was aspirated from the cells and replaced with fresh induction medium (0.5 ml). At intervals during the next 48 hr, the induction medium was replaced by drug solutions prepared by dilution in induction medium (0.5 ml). A control of cells grown in induction medium alone for an additional 48 hr was included in each experiment.

(7)

Statistical analysis. Up-regulation and surface population data were examined by one- and two-factor analyses of variance, using SigmaStat for Windows V2.0. The Tukey-B test was used for multiple pairwise comparisons, whereas comparisons versus control were performed using Dunnett's test.

	Results

Top Summary Introduction Procedures Results Discussion References

Kinetic studies. 42 nAChRs in membrane preparations and M10 cells in situ were quantified by binding of [³H]epibatidine. Its binding to M10 cells gave an excellent signal-to-noise ratio and reproducible data (Whiteaker et al., 1996), but its low picomolar affinity for 42 nAChR (Houghtling et al., 1995) poses a problem of ligand depletion at low concentrations, which can distort K_d determinations from saturation binding experiments. This prompted derivation of the K_d value for [³H]epibatidine binding to M10 cells in situ by an independent method, based on binding rate constants. Rates of association and dissociation were measured at 4°, 20°, and 37° (Fig. 1, Tables 1 and 2).

Agonist-induced up-regulation. The abilities were compared of a wide spectrum of nicotinic agonists to up-regulate numbers of [³H]epibatidine binding sites in M10 cells. ()-Nicotine, (±)-epibatidine, MCC, (±)-anatoxin-, ()-cytisine, ABT-418, and TMA were coadministered with 1 µM dexamethasone for 48 hr. All of the agonists provoked dose-dependent up-regulation (Fig. 2), which exhibited an inverted U-shaped profile (except in the case of ABT-418, which did not reach a distinct maximum up-regulation value within the concentration range tested). The dose-response profiles vary among compounds; differences in profile show no correlation with agonist potency. For instance, the slopes of the up and down phases of (±)-epibatidine-induced up-regulation are extremely shallow compared with those produced by (±)-anatoxin- or TMA. Averaged up-regulation data were fit to a logistic model (Fig. 2): the calculated values of maximum up-regulation, EC₅₀ (concentration giving half-maximum up-regulation) and IC₅₀ (concentration at which up-regulation declines to half the maximum) are recorded in Table 3.

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Agonist induced up-regulation of [3H]epibatidine binding sites in M10 cells. Nicotinic agonists were administered during nAChR induction (48 hr). After extensive washing to remove nicotinic ligands, numbers of [3H]epibatidine binding sites were determined by binding to cells in situ. a, Epibatidine () and MCC () are partial agonists for up-regulation compared with ()-nicotine (). b, All other compounds tested [()-cytisine (), ABT-418 (), (±)-anatoxin-a () and TMA ()] up-regulate with full efficacy compared with nicotine. Points, mean ± standard error of at least three independent determinations, using separate cultures. For each culture, percentage up-regulation was determined relative to a control of cells induced for 2 days with no agonist present. Data were fitted to a logistic model (see Experimental Procedures).

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Up-regulation by fixed ()-nicotine concentrations in the presence of (±)-epibatidine, MCC and dTC. Drugs were co-administered with dexamethasone during the nAChR induction period (48 hr). Specific [3H]epibatidine binding to M10 cells in situ is expressed as a percentage above control values determined in parallel for cells induced in the absence of nicotinic ligands. a, Up-regulation caused MCC alone () and co-administered with ()-nicotine (1 µM [() or 100 µM ()]. ()-Nicotine alone increased [3H]epibatidine binding to induced M10 cells by 83.5 ± 14.2% (mean ± standard error, 1 µM) and 193 ± 29.3% (100 µM) above control. b, Up-regulation caused by (±)-epibatidine alone () and co-administered with ()-nicotine [1 µM () or 100 µM ()]. Up-regulation by 1 µM ()-nicotine alone was 85.0 ± 26.1%, by 100 µM ()-nicotine alone was 171.7 ± 21.0% above control. c, Up-regulation produced by ()-nicotine [1 µM () or 100µM ()] in the presence of dTC. To allow direct comparison of the effects of dTC on up-regulation by different concentrations of ()-nicotine, up-regulation has been converted to a percentage of that produced by the appropriate concentration of ()-nicotine in the absence of dTC. Points, mean ± standard error of at least three separate experiments, using separate cultures.

Antagonist-induced up-regulation. The ability of the antagonists DHE, MLA, dTC, HEX, DEC, and mecamylamine to induce up-regulation was also examined by coadministration with 1 µM dexamethasone for 48 hr. Generally, these compounds had very little effect on the density of [³H]epibatidine binding sites except at high concentrations (Fig. 4, a and b). In contrast to the up-regulation produced by agonists, there was no common pattern of dose response, so no attempt was made to fit these data to a model.

View larger version (31K): [in this window] [in a new window]   Fig. 4.   Effects of nicotinic antagonists on the up-regulation of [3H]epibatidine binding sites in M10 cells. Drugs were administered with dexamethasone during the 48-hr nAChR induction period. Specific [3H]epibatidine binding to M10 cells in situ is expressed as a percentage above control values determined in parallel in cells not exposed to any nicotinic ligand. a and b, Effects of antagonists alone. a, `Competitive' antagonists: MLA () and DHE () produced mild up-regulation at high concentrations, whereas dTC () was ineffective. b, Noncompetitive antagonists: HEX () and DEC () have no effect below 1 mM, but mecamylamine () is down-regulatory at high concentrations. c and d, Effects of antagonists on up-regulation produced by increasing concentrations of (±)-anatoxin-a (control, ). c, MLA (30 µM; ), DHE (100 µM; ) and dTC (100 µM; ). Only dTC blocked anatoxin-a induced up-regulation (p < 0.001, two-way analysis of variance). d, HEX (1 mM; ), DEC (1 mM; ) and mecamylamine (100 µM; ). Points, mean ± standard error of at least three independent determinations, using separate cultures. Data in c and d were fitted to a logistic model (see Experimental Procedures).

Surface versus intracellular [³H]epibatidine binding sites. As a prerequisite to determining whether up-regulation is initiated through cell surface or intracellular nAChR, we addressed the distribution of receptors in M10 cells. Because [³H]epibatidine is a readily cell-permeant radioligand that will access both populations, a quantitative evaluation of nAChR distribution was sought by comparing the abilities of permeant and impermeant ligands to displace [³H]epibatidine binding in intact cells and membrane preparations. Competition binding assays were carried out with all of the compounds examined in the up-regulation studies, plus ACh (Fig. 5, Table 4). In the case of M10 membrane preparations, each of the compounds (with the sole exception of mecamylamine) was capable of fully displacing [³H]epibatidine binding, with Hill slopes of 1.0-1.5. K_i values are typical of these compounds at 42-type nAChR (Houghtling et al., 1995; Gopalakrishnan et al., 1996), with agonists generally giving K_i values in the nanomolar range, whereas antagonists yielded K_i values in the micromolar range. (±)-Epibatidine gave a K_i value of 11 pM, in excellent agreement with the K_d value of 10.8 pM derived from the kinetic analysis of binding to intact cells.

View larger version (20K): [in this window] [in a new window]   Fig. 5.   [3H]Epibatidine displacement binding. The ability of compounds to displace [3H]epibatidine (200 pM) binding from either intact M10 cells in situ (filled symbols) or M10 membrane preparations (open symbols) was assessed in equilibrium binding assays. Where displacement was observed, data were fitted to the Hill equation (see Experimental Procedures). a, Cell permeable ligands: (±)-epibatidine (, ), ()-nicotine (, ) and dTC (, ). b, Cell impermeable ligands, MCC (, ), ACh (, ) and tetramethylammonium (, ). Points, mean ± standard error of at least three independent determinations.

View this table: [in this window] [in a new window]   TABLE 4 Binding affinities of nicotinic ligands at the 42 nAChR expressed in M10 cells

Is up-regulation mediated by cell surface or intracellular nAChRs? Given the large proportion of intracellular binding sites and the freely permeant nature of drugs such as nicotine that have commonly been used to promote up-regulation, it is pertinent to inquire whether interaction with surface nAChRs provides the trigger for up-regulation or whether binding to intracellular sites can also effect this response. From the ability of ligands to access the intracellular pool (Fig. 5, Table 4), MCC and TMA can be defined as impermeant, yet both provoke up-regulation (Fig. 2, Table 3). Although MCC was found to be a partial up-regulator compared with ()nicotine, TMA was as effective as the permeant agonists. However, it remained possible that over the extended period of chronic drug exposure in the up-regulation protocol, these "impermeant" ligands might gain access to the interior of the cell. Therefore, the rate of entry of nicotinic ligands into M10 cells was assessed by determining their ability to compete for [³H]epibatidine binding sites in M10 cells in situ at intervals throughout the 48-hr exposure to the compounds: displacement of a large proportion of [³H]epibatidine binding would indicate entry of the ligand into the cell interior. Equilibrium [³H]epibatidine binding was determined in the presence of 10 µM TMA and 100 µM MCC, concentrations sufficient to displace [³H]epibatidine binding to M10 cell membranes (Fig. 5) while evoking minimal up-regulation themselves (Fig. 2). The antagonist dTC also was examined, at a concentration (1 mM) sufficient to displace all [³H]epibatidine binding to M10 cell membranes (Fig. 5).

View larger version (14K): [in this window] [in a new window]   Fig. 6.   Time course of nicotinic ligand uptake by M10 cells. a, The rate of entry of nicotinic ligands into M10 cells was assessed by determining their ability to compete for [3H]epibatidine binding in induced (1 µM dexamethasone, 72 hr) M10 cells in situ after increasing periods of time. Equilibrium binding in the presence of TMA (10 µM, ), MCC (100 µM, ) and dTC (1 mM, ) was measured over a period of 48 hr. Specific [3H]epibatidine binding was assessed as percentage of control (cells untreated with drugs). b, From the degree of inhibition observed, the intracellular concentration of drug could be estimated by reference to the affinity of the ligand for [3H]epibatidine binding sites in M10 membranes (see Experimental Procedures). Intracellular levels of TMA were undetectable. Points, mean ± standard error of at least three independent determinations, using separate cultures.

	Discussion

Top Summary Introduction Procedures Results Discussion References

In this study, we investigated whether up-regulation of 42 nAChRs is triggered at the cell surface and examined the state of the receptor coupled to up-regulation. To this end, we used M10 cells exposed to a variety of drug conditions and monitored receptor numbers, properties, and distribution using [³H]epibatidine binding to intact M10 cells in situ and to M10 membrane preparations. Previous studies of 42 nAChR up-regulation in vitro have used [³H]agonist and [¹²⁵I]mAb binding to immunoisolates (Peng et al., 1994) or membrane preparations (Zhang et al., 1994; Bencherif et al., 1995) or subunit-specific [¹²⁵I]mAb binding to intact cells (Rothhut et al., 1996) (or both) to quantify receptor expression. [³H]Agonist binding has the advantage of recognizing only fully assembled receptors, whereas study of cells in situ allows cellular receptor populations to be quantified reliably because there is no possibility of changes in subpopulation proportions arising from processing of the sample. In this study, [³H]epibatidine binding to M10 cells in situ combines the advantages of both approaches; nAChRs expressed on the cell surface can be discriminated using an impermeant competing ligand.

Agonist induced up-regulation. We examined up-regulation by a wide spectrum of drugs over a broader range of concentrations than has previously been documented; where there is overlap, the data presented here are in good agreement with those of previous reports (Peng et al., 1994; Zhang et al., 1994). The bell-shaped dose-response curves for up-regulation are reminiscent of 42 nAChR activation profiles in M10 cells (Thomas et al., 1993) and Xenopus laevis oocytes (Vibat et al., 1995). Correlation plots (Fig. 7) show that there is no correlation between the EC₅₀ value for up-regulation and either maximum up-regulation observed (Fig. 7c) or the IC₅₀ value for the downturn in the dose-response profile (Fig. 7d). There is, however, good correlation between the EC₅₀ value for up-regulation and both the EC₅₀ value for activation (Fig. 7a) and the K_I value for binding (Fig. 7b). However, up-regulation of 42 nAChRs in M10 cells requires chronic exposure to agonist concentrations intermediate between those that produce receptor activation on acute application (typically in or near the micromolar range) and those producing desensitization without prior activation (typically nanomolar). Other considerations also make the hypothesis that activation may provoke up-regulation untenable. First, in contrast to their efficacy in activating nAChRs, (±)-epibatidine and MCC are partial agonists for up-regulation: the maximum up-regulation induced by ()-nicotine is approximately double that elicited by (±)-epibatidine and over six times greater than that produced by MCC (Table 3). Both enantiomers of epibatidine behaved as potent full agonists at a number of nAChR subtypes composed of human or chicken nAChR subunits and expressed in X. laevis oocytes (Gerzanich et al., 1995), (±)-epibatidine was more efficacious than ()-nicotine at chicken (Vibat et al., 1995) and human 42 nAChR (Gopalakrishnan et al., 1996) and at least as efficacious as ()-nicotine in activating "type III" currents, attributed to 42-type nAChRs, in rat hippocampal cultures (Alkondon and Albuquerque, 1995). Marks et al. (1996) found both enantiomers of epibatidine to be approximately twice as effective as ()-nicotine in stimulating ⁸⁶Rb⁺ efflux from mouse thalamic synaptosomes (a response considered to be mediated by 42-type nAChRs). These authors found MCC to be even more efficacious than epibatidine. Second, dose-response profiles for activation of nAChR in M10 cells do not exactly match the dose dependence of up-regulation. For instance, ⁸⁶Rb⁺ flux induced by ()-nicotine is maximal at 3-5 µM (Thomas et al., 1993; Court et al., 1994), whereas maximum up-regulation is produced by 30-50 µM ()-nicotine (Fig. 2).

View larger version (42K): [in this window] [in a new window]   Fig. 7.   Correlations between agonist potency for up-regulation and other parameters. EC50 values for up-regulation are plotted against (a) EC50 values for activation (Table 3); (b) Ki values for binding to membranes (Table 4); (c) maximum up-regulation (Table 3) and (d) IC50 values for the downturn of the up-regulation dose-response profiles (Table 3). Linear regression gives correlation coefficients of 0.78 for EC50 (activation); 0.97 for Ki (binding); 0.20 (for maximum up-regulation); 0.52 for IC50 (up-regulation).

Antagonist effects on up-regulation. If agonist activation of nAChR is the trigger for up-regulation, antagonists (particularly competitive antagonists) should block agonist-induced up-regulation while themselves having no effect. However, with the exception of dTC, none of the antagonists tested prevented agonist-induced up-regulation (Fig. 4). dTC was an effective antagonist of up-regulation, as previously reported (Peng et al., 1994; Zhang et al., 1994), but inhibition by dTC was independent of nicotine concentration (Fig. 3c) and the lack of a sideways shift in the (±)-anatoxin- dose-response curve in the presence of dTC (Fig. 4c) suggests a noncompetitive mode of action. This is in contrast to the competitive antagonism by dTC of activation of chicken 42 nAChR in X. laevis oocytes (Bertrand et al., 1990). The lack of inhibition by DHE is consistent with the findings of Zhang et al. (1994). Mecamylamine has been reported to up-regulate numbers of ()-[³H]nicotine binding sites in M10 cells (Peng et al. 1994) and in vivo (Abdulla et al., 1996; Pauly et al., 1996). We found no effect of mecamylamine: similarly, chronic mecamylamine (alone or in combination with agonist) has no effect on the up-regulation of 3- and 7-type nAChRs expressed in the SHSY-5Y cell line (Peng et al., 1997) or 7-type nAChR in hippocampal neurons (Wonnacott and Rogers, 1996).

[³H]Epibatidine displacement binding studies. It is no longer tenable that up-regulation of nAChR is mediated by the high affinity desensitized state of the receptor as originally proposed (Marks et al., 1985; Schwartz and Kellar, 1985). Although there is a positive correlation between the rank order of K_I values from competition binding assays and EC₅₀ values for up-regulation for nicotinic agonists (Fig. 7b), the concentrations required to promote up-regulation are 1 or 2 orders of magnitude greater than the binding affinities (which represent the desensitized state of the nAChR), in agreement with Peng et al. (1994) and Gopalakrishnan et al. (1997). At agonist concentrations at which the high affinity desensitized state predominates, very little or no up-regulation is observed. Moreover, this view is consistent with in vivo evidence: Rowell and Li (1997) measured brain nicotine levels and numbers of [³H]nicotine binding sites in rats chronically treated with nicotine over 10 days. In treatment regimens that produced brain nicotine concentrations sufficient to achieve a complete desensitization of receptors in other studies (Marks et al., 1994), there was no statistically significant up-regulation of [³H]nicotine binding sites: up-regulation required higher concentrations of nicotine. This refutes the candidature of the classic desensitized state as an up-regulatory trigger.