Introduction

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Multiple nAChR subtypes are present in the brain, each having a unique distribution (reviewed in Lukas, 1998). nAChRs in the brain are suggested to play roles in attention and memory, in mood and emotion, and in sensation and perception (reviewed in Paterson and Nordberg, 2000). These nAChRs also have been implicated in nicotine dependence (Balfour, 1994) and in neurodegenerative and mood disorders (reviewed in Mihailescu and Drucker-Colin, 2000b).

Either through therapeutic use of nicotinic ligands to treat neuropsychiatric disorders or via habitual use of tobacco products, there will be or is sustained exposure of the brain and body to nicotinic ligands. Any effects of such exposure are likely mediated by effects on numbers and function of nAChRs. Historically, much attention has focused on the phenomenon of quantitative increases in nAChR-like radioligand binding sites ("up-regulation") following chronic nicotine exposure (reviewed in Gentry and Lukas, 2002). However, up-regulation is predominantly attributable to changes in internal rather than cell surface pools of nAChR-like radioligand binding sites (Ke et al., 1998; Whiteaker et al., 1998), and its physiological relevance is unclear (Gentry and Lukas, 2002). By contrast, effects on nAChR function are of obvious, primary importance in determining physiological and behavioral effects of sustained exposure to either nicotine or nicotinic therapeutic agents (Gentry and Lukas, 2002).

The literature predominantly indicates that sustained exposure to nicotinic agonists induces losses in nAChR function. nAChR "desensitization", which descriptively refers to a rapid-in-onset, quickly and fully reversible loss of function during or following brief (seconds) exposure to nicotinic agonists, was first discovered at the nerve-muscle junction by Katz and Thesleff (1957). Subsequently, other processes involving longer-lasting loss of nAChR function following longer-term (minutes to days) nicotine exposure became recognized for nAChR subtypes found in the periphery (Boyd, 1987; Lukas, 1991; Ke et al., 1998; Reitstetter et al., 1999). Most studies also found that sustained nicotinic agonist exposure induces more than one phase of functional loss for the predominant, high-affinity nicotine-binding nAChR in the brain, composed of 4 and 2 subunits (Flores et al., 1992), whether expressed naturally or heterologously in the oocyte system (Collins et al., 1990a; Marks et al., 1993; Hsu et al., 1996; Fenster et al., 1997; Gopalakrishnan et al., 1997; Olale et al., 1997; Gentry and Lukas, 2002). The longer-lasting losses of nAChR function have been called "specific chronic desensitization" (Ochoa et al., 1990; Collins et al., 1994), "stable desensitization" (Mihailescu and Drucker-Colin, 2000a), "functional down-regulation" (Marks et al., 1993), "long-lasting inactivation" (Kawai and Berg, 2001), or "persistent inactivation" (Lukas, 1991; Lukas et al., 1996; Ke et al., 1998). Nevertheless, further evolution in the understanding of nAChR functional state transitions is required to identify mechanisms and to specify processes involved in "desensitization" or any of the other, apparently distinct processes of longer-lasting nAChR functional loss. For example, there still is ambiguity about whether and to what extent "desensitization" due to conversion to a closed-channel form is "contaminated" with agonist-mediated open-channel block; it is not yet clear whether phosphorylation or other processes account for loss of nAChR function with agonist exposure (Gentry and Lukas, 2002). That is, before tangible definitions of desensitization or of other, longer-lasting phases of nAChR functional loss can be formulated beyond postulated conversions of agonist-receptor complexes to inactive forms, explanations for these phenomena require refinement.

Although most studies have shown functional loss for 42-nAChR on sustained exposure to nicotine, a recent electrophysiological study of human 42-nAChR heterologously expressed in a mammalian human embryonic kidney-derived cell line reported an enhancement of function under some experimental conditions following chronic exposure to nicotine (Buisson and Bertrand, 2001). This finding poses new questions about whether and how different expression systems and details of experimental design influence functional responsiveness of individual nAChR subtypes to chronic nicotinic ligand exposure.

This study examines functional responses of human 42- and 44-nAChR heterologously expressed in the SH-EP1 human epithelial line following pretreatment with nicotinic ligands. Whereas the 42-nAChR subtype is of obvious importance, nAChR 4 subunit messages as detected by in situ hybridization in nonhuman primate brain also are abundant and colocalize with 4 subunit messages (Quik et al., 2000). Moreover, 4 and 4 subunits combine to form a functional nAChR ion channel with high affinity for nicotine (Eaton et al., 2000). The current studies examined and contrasted effects of nicotine as a membrane-permeant agonist, carbamylcholine (carb) as an ionic nAChR agonist, and mecamylamine (mec) as a prototypical nAChR antagonist with potential as an aid to smoking cessation (Rose et al., 1998) and a therapeutic (Sanberg et al., 1997). A preliminary report of these findings has appeared (Gentry and Lukas, 2001).

	Materials and Methods

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Drug Dilutions. All drugs were prepared fresh on the day of the assay as 10 mM stock solutions in cell culture medium (see cell culture) and diluted appropriately for each experiment. Efflux assay buffer consisted of 130 mM NaCl, 5.4 mM KCl, 2 mM CaCl₂, 5 mM glucose, and 50 mM HEPES, pH 7.4. Ringer's buffer consisted of 115 mM NaCl, 5 mM KCl, 1.8 mM CaCl₂, 1.3 mM MgSO₄, and 33 mM Tris supplemented with 1.5 mM NaN₃. Buffer components as well as ()-nicotine bitartrate, carb Cl, and mec HCl were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Model Cell Lines and Cell Culture. The present study used low-passage (less than 50) SH-EP1 human epithelial cell clones stably and heterologously expressing human 42- or 44-nAChRs to examine functional responsiveness of receptors following pretreatment with nicotine or other nicotinic ligands. These clonal cell lines were generated using techniques that have been previously reported (Peng et al., 1999; Eaton et al., 2000; Lukas et al., 2002). Briefly, transfection with pcDNA3.1-zeo-human 4 and pcDNA3.1-hygro-human 2 constructs created in our laboratory [subcloned using human 4(S452) and 2 subunit cDNAs kindly provided by Dr. Ortrud Steinlein, Rheinishce-Friedrich-Wilhelms-Universitaet, Bonn, Germany] followed by isolation of a stable, high-expressing clonal line was accomplished to create SH-EP1-h42 cells. Transfection with pCEP4-hygro-human 4 and pcDNA3.1-zeo-human 4 constructs created in our laboratory (the latter was subcloned using a human 4 subunit cDNA kindly provided by Dr. Jon Lindstrom, University of Pennsylvania, Philadelphia, PA) also, followed by isolation of a high-expressing clone, was used to generate SH-EP1-h44 cells. Cells were maintained at 37°C, under 95% O₂/5% CO₂ in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum (Hyclone Laboratories, Logan, UT), 10% horse serum (Invitrogen, Carlsbad, CA), 1% sodium pyruvate (Cellgro AK; Mediatech Inc., Herndon, VA), 2% glutamine penicillin-streptomycin (Irvine Scientific, Santa Ana, CA), and 0.02% amphotericin B (Sigma). Cell culture medium was also supplemented with 0.01% hygromycin (Calbiochem, San Diego, CA) and 0.03% zeocin (Invitrogen) to maintain stable expression of 4 and 2 or 4 subunits.

Assays of nAChR Function. ⁸⁶Rb⁺ efflux assays (Lukas and Cullen, 1988; Lukas et al., 2002) were performed using SH-EP1-h42 or SH-EP1-h44 cells with some assay modifications to account for the nicotinic ligand pretreatment protocol. Cells were cultured (~2 × 10⁵ cells per 15.5-mm-diameter well; ~150 µg of total cell protein per well) on Falcon 24-well culture plates (BD Biosciences, San Jose, CA) precoated with poly(D-lysine), mol. wt. 70,000 to 150,000 (Sigma). Cells were grown overnight to confluence, verified using light microscopy. ⁸⁶RbCl (3.8 mCi/mg or 0.2 cpm/fmol at 40% counting efficiency) was obtained from PerkinElmer Life Sciences (Boston, MA).

Conditions for ⁸⁶Rb⁺ Loading. Prior to initiating experiments, a control study compared effects of three different methods for 15-min ligand pretreatment: 1) simply adding a concentrated aliquot of ligand in media to cells maintained in ⁸⁶Rb⁺ loading media, 2) using the flip plate method to replace ⁸⁶Rb⁺ loading media with ⁸⁶Rb⁺-free media containing ligand, and 3) using the flip plate method to apply ligand in ⁸⁶Rb⁺ loading medium. Regardless of the method used, double-normalized results obtained (see Data Analysis below) were nearly identical (data not shown). This indicated that final results were not influenced by differences in ⁸⁶Rb⁺ loading during ligand pretreatment.

Data Analysis. To control for possible influences of ligand pretreatment and recovery conditions on ⁸⁶Rb⁺ loading, data were subjected to a double normalization process. Background radioactivity was subtracted from all samples. Amounts of ⁸⁶Rb⁺ loaded and present intracellularly at the start of the acute agonist challenge to initiate the functional efflux assay were determined as the sum for each sample of released (extracellular) and remaining (intracellular) ⁸⁶Rb⁺ present at the conclusion of the functional assay. Specific ⁸⁶Rb⁺ efflux was defined as total ⁸⁶Rb⁺ efflux minus nonspecific ⁸⁶Rb⁺ efflux (described above). Normalized specific ⁸⁶Rb⁺ efflux was then expressed as a percentage of specific ⁸⁶Rb⁺ efflux divided by ⁸⁶Rb⁺ loaded. Double normalized specific ⁸⁶Rb⁺ efflux was then expressed as a percentage of normalized specific ⁸⁶Rb⁺ efflux for experimental samples subjected to ligand pretreatment divided by normalized specific ⁸⁶Rb⁺ efflux for control samples not subjected to ligand pretreatment. Data were plotted as means ± S.E.M. using Prism 3.0 software (GraphPad Software Inc., San Diego, CA). Results from three or more independent experiments are shown. Appropriate to sample size and degrees of freedom, unpaired t tests (two comparisons) or one-way ANOVA followed by Bonferroni post-tests (three comparisons) or Tukey's post hoc tests (greater than three comparisons) were used to evaluate statistical significance of important data sets. Most of the statistically significant differences between and within panels of data are obvious, but statistically significant differences that are not as evident are specified in the narrative, whereas remarks about similarity between such sets of data refer to differences that are not statistically significant at the 95% confidence interval.

Radiolabeled Epibatidine Binding Studies. Competition for [³H]epibatidine (EBDN) binding sites on cell membranes prepared from SH-EP1-h42 cells (Lukas et al., 2002) was utilized as a sensitive method for quantifying residual nicotine in efflux buffer samples collected during successive rinses of cells pretreated for 48 h with nicotine. Assays were performed in a total volume of 200 µl of Ringer's buffer in a 96-well plate format. Each well used to define the concentration of nicotine in one rinse sample of efflux buffer by competition for EBDN binding sites contained 50 µl of a 400 pM concentration of EBDN (final concentration = 100 pM), 50 µl of efflux buffer from one rinse sample, and 100 µl of SH-EP1-42 cell membrane suspension (diluted to ensure that binding site concentrations were no more than 10 pM). Total control binding was defined as the binding of EBDN in the absence of any rinse sample, which was substituted for by a 50-µl aliquot of Ringer's-NaN₃ buffer. Following a 1-h incubation at room temperature (~22°C), cell membrane-bound EBDN was separated from unbound EBDN using a cell harvester system (Inotech Biosystems International, Inc., Rockville, MD) by filtration through GF/C glass fiber filters (Inotech Biosystems International, Inc.) that had been presoaked in a 0.1% polyethyleneimine solution at 4°C for at least 30 min. Cell membranes embedded in the filters were subsequently rinsed twice with ice-cold Ringer's buffer before being transferred to Ready Safe Liquid Scintillation Cocktail (Beckman Coulter, Inc., Fullerton, CA) for determination of [³H] content by coincidence counting on a Wallac MicroBeta TriLux microplate liquid scintillation counter (PerkinElmer Life Sciences). Counts obtained were transformed for presentation to a percentage of control specific EBDN binding defined in each assay.

	Results

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Determination of Residual Drug Levels. Because a recurring technical critique of some studies of effects of chronic drug treatment regards possible effects of residual pretreatment drug on the parameter being measured, experiments were conducted to ensure that the rinse method used in our nicotine pretreatment-acute challenge protocol was sufficient to remove pretreatment ligands, thereby allowing nAChR to recover from effects of drug pretreatment in a drug-free environment. Other studies showed that rinses of 30- to 60-s duration were adequate to allow equilibration in nicotine distribution between intra- and extracellular spaces (J. Fryer and R. D. Lukas, unpublished observations). Independent experiments were completed to measure residual nicotine levels present in buffers used to rinse SH-EP1-h42 cells after nicotine pretreatment based on the ability of residual drug to activate nAChR-mediated ⁸⁶Rb⁺ efflux from fresh SH-EP1-h42 cells (Fig. 1). These functional assays demonstrated that nicotine levels in first-rinse samples were >100-fold lower than the initial nicotine concentration (i.e., there was a more than 2 log unit, rightward shift in the dose-response profile for the first-rinse sample from that for acute nicotine effects). Residual nicotine present in second-rinse samples elicited ion flux only if initial nicotine concentrations were 1 mM, but with efficacy equivalent to 0.3 to 1 µM nicotine, indicating a 1000- to 3000-fold removal of drug. Levels of residual nicotine present in third-rinse samples had been reduced to below those capable of eliciting any ⁸⁶Rb⁺ efflux; i.e., below 100 nM, a greater than 10,000-fold reduction. The efficiency of removal of nicotine in the second rinse appeared to be slightly lower following longer pretreatment times of 1 or 48 h relative to 20 s of nicotine pretreatment, but no pretreatment time-dependent difference in nicotine removal was observed after the third rinse. Thus, three rinses were routinely used to process samples.

View larger version (34K): [in this window] [in a new window]   Fig. 1.   Functional bioassay to assess effectiveness of removal of pretreatment nicotine. SH-EP1 cells heterologously expressing human 42-nAChR were pre-exposed to nicotine at the indicated concentration (abscissa, log M scale) for 20 s (top), 1 h (center), or 48 h (bottom). Pre-exposure medium was aspirated, and the pre-exposed cells were subjected to four successive rinses of 2 ml of drug-free assay buffer. The first rinse was for 1 min, and each subsequent rinse was for 2 min. Biologically active nicotine in the rinse buffers was assayed based on its ability to elicit function of human 42-nAChRs heterologously expressed in fresh SH-EP1 cells not previously exposed to nicotine (specific 86Rb+ efflux; ordinate, percentage of control; see Materials and Methods). nAChR function was measured in the presence of fresh, acute nicotine () to generate a standard curve or in buffers collected from successive cell rinses (first rinse, ; 2nd rinse, ; third rinse, ; fourth rinse, ). Smooth curves were drawn through data points, which are means ± S.E.M. from at least three separate experiments. Concentration-response profiles were used to determine nicotine levels in rinse buffers based on comparison with the standard curve and indicate progressively lower levels of residual nicotine with each rinse.

View larger version (39K): [in this window] [in a new window]   Fig. 2.   Evaluation of residual nicotine levels after cell pretreatment based on radioligand binding competition assays. SH-EP1 cells heterologously expressing human 42-nAChRs were pre-exposed to nicotine at the indicated concentration (abscissa, log M scale) for 48 h. Samples were processed and rinse medium was collected as described in the legend to Fig. 1. Biologically active nicotine collected in rinse buffers was tested based on its ability to inhibit specific [3H]epibatidine binding (ordinate, percentage of control; see Materials and Methods) to cellular membrane fractions from fresh SH-EP1-h42 cells. Assays done in the acute presence of fresh nicotine () were used to generate a standard nicotine dose-inhibition curve against which pretreatment nicotine dose-inhibition curves were compared for samples from successive cell rinses (first rinse, ; second rinse, ; third rinse, ; fourth rinse, ). Smooth curves were drawn through data points, which were means ± S.E.M. from at least two separate experiments. Concentration-response profiles were used to determine nicotine levels in rinse buffers based on comparison with the standard curve and indicate progressively lower levels of residual nicotine with each rinse.

Effects of Nicotine Pretreatment on SH-EP1 Cell Human 42-nAChR Function. Effects of prolonged nicotine exposure on heterologously expressed 42-nAChRs were examined. 42-nAChRs are the predominant form of high-affinity nicotine binding site in the brain, making them not only physiologically and pharmacologically important, but also the most likely effectors in nicotine dependence. Pre-exposure of SH-EP1-h42 cells for 1 min to nicotine at concentrations as low as 1 µM produced an ~20% loss of human 42-nAChR function when tested with an acute challenge dose of carb after 1 min of recovery from nicotine pre-exposure (Fig. 3, top-left). Losses of function for 1-min pretreatment with 1 or 10 µM nicotine reversed (i.e., function elicited by an acute challenge dose of carb returned to control levels) after 1 h of recovery. However, after 1 h (or less) of recovery, losses of function were still evident in cells pre-exposed to 100 µM (~20% loss for 1-h recovery) or 1 mM (~30% loss for 1-h recovery) nicotine. In the absence of tangible definitions of desensitization and phases of longer-lasting losses of nAChR function induced by sustained nicotinic agonist exposure, explicit, operational definitions provided by Ke et al. (1998) distinguishing desensitization as a loss of nAChR function that is reversed during a 5-min period of recovery from agonist exposure from persistent inactivation, which was defined as a loss of nAChR function that is not reversed during a 5-min period of recovery from agonist exposure, were applied for their empirical utility. Thus, levels of persistent inactivation surviving the 5-min recovery period were ~10% for pretreatment with 1 µM nicotine and ~55% for pretreatment with 1 mM nicotine.

View larger version (55K): [in this window] [in a new window]   Fig. 3.   Time- and concentration-dependent effects of nicotine exposure on function of 42-nAChR measured using 86Rb+ efflux assays. SH-EP1 cells heterologously expressing human 42-nAChRs were pre-exposed to nicotine at the indicated concentrations (abscissa, log M scale) for 1 min (top-left), 15 min (top-right), 1 h (bottom-left) or 24 h (bottom-right). Pre-exposure medium was aspirated and the pre-exposed cells were subjected to three successive rinses with 2 ml of drug-free assay buffer. The first and second rinses were for 1 min, and the third rinse consumed the remainder of the experimentally defined recovery time. Recovery times were 1 min (), 3 min (), 5 min (), 15 min (), 30 min (), or 60 min (). Specific 86Rb+ efflux (ordinate, percentage of control), measured as described under Materials and Methods, was determined by eliciting nAChR function using acute (2 min) exposure to 1 mM carb. A 100% response was defined by untreated control cells' response to 1 mM carb. Linear curves are drawn through data points (means ± S.E.M. from at least three separate experiments). Over much of the dose profile, 1-min pretreatment results were significantly different from other results for either 15-min, or 1- or 24-h pretreatments. One- and 24-h pretreatment results were not statistically different from each other (ANOVA followed by Tukey's post hoc test, p < 0.05).

Effects of Nicotine Pretreatment on SH-EP1 Cell Human 44-nAChR Function. Studies of effects of chronic nicotine exposure on function of human 44-nAChRs were also conducted because nAChR 4 subunit messages in nonhuman primate brain have been reported to be abundant and to colocalize with 4 subunit messages and because human 44-nAChRs also have high affinity for nicotine. Upon prolonged nicotine exposure, heterologously expressed, human 44-nAChRs in transfected SH-EP1 cells became less responsive to subsequent activation by 1 mM carb (Fig. 4), and patterns of functional changes were similar to those of 42-nAChRs. Following 1 min of exposure to 1 mM nicotine, an ~75% reduction in efflux from 44-nAChRs was observed (Fig. 4, top-left). Functional response remained at ~50% of normal when cells were allowed to recover for 1 h in drug-free buffer. A dose of 100 µM nicotine led to ~50% reduction in 44-nAChR function that did recover substantially (to ~20% loss) following 1 h in drug-free buffer. One-minute pretreatment with nicotine at 1 µM or less did not induce persistent inactivation. Five-minute recovery allowed 44-nAChR function to return to ~80%, 70%, or 40% of control, respectively, after 10 µM, 100 µM, or 1 mM nicotine pre-exposure.

View larger version (51K): [in this window] [in a new window]   Fig. 4.   Time- and concentration-dependent effects of nicotine exposure on function of 44-nAChR measured using 86Rb+ efflux assays. SH-EP1 cells heterologously expressing human 44-nAChR were pre-exposed to nicotine at the indicated concentrations (abscissa, log M scale) for 1 min (top-left), 15 min (top-right), 1 h (bottom-left), or 24 h (bottom-right). Samples were processed as described in the legend to Fig. 3. Recovery times were 1 min (), 3 min (), 5 min (), 15 min (), 30 min (), or 60 min (). Specific 86Rb+ efflux (ordinate, percentage of control) was measured as described in the legend to Fig. 3 (means ± S.E.M. from at least three separate experiments). Results were not significantly different for 1- or 24-h pretreatments at equivalent nicotine doses and recovery times, but these results and results from 1- and 15-min pretreatments were significantly different from each other over the entire dose profile (ANOVA followed by Tukey's post hoc test, p < 0.01).

Effects of Carbamylcholine Pretreatment on SH-EP1 Cell Human 42-nAChR Function. carb was chosen for study as a representative ionic agonist to allow comparisons of its ability and that of nicotine as a membrane-permeant agonist to induce changes in nAChR function. One-minute pretreatments with 0.1 or 1 mM carb caused a reduction in heterologously expressed, human 42-nAChR function of 15% and 25%, respectively, when measured with a subsequent, acute carb challenge (Fig. 5, top-left). In both cases, receptor function fully recovered within 30 min. One-min pretreatment with carb concentrations of 10 µM or less had no effect. Only 100 µM or 1 mM pretreatments produced persistent inactivation.

View larger version (53K): [in this window] [in a new window]   Fig. 5.   Time- and concentration-dependent effects of carb exposure on function of 42-nAChR measured using 86Rb+ efflux assays. SH-EP1 cells heterologously expressing human 42-nAChRs were pre-exposed to carb at the indicated concentrations (abscissa, log M scale) for 1 min (top-left), 15 min (top-right), 1 h (bottom-left) or 24 h (bottom-right). Otherwise, samples were processed as described in the legend to Fig. 3. Recovery times were 1 min (), 3 min (), 5 min (), 15 min (), 30 min (), or 60 min (). Specific 86Rb+ efflux (ordinate, percentage of control) was measured as described in the legend to Fig. 3 (means ± S.E.M. from at least three separate experiments). For 1-min pretreatments at equivalent carb doses and recovery times, results were significantly different (less functional loss for 1-min pretreatment) for 1-min recovery at high carb doses, but 15-min, 60-min, and 24-h pretreatments were not significantly different from each other across the dose profile (ANOVA followed by Tukey's post hoc test, p < 0.01). Comparing results for studies of 42-nAChR, carb pretreatment produced significantly less functional inhibition across all dose profiles compared with effects of nicotine under equivalent dose and pretreatment and recovery times (ANOVA followed by Bonferroni post hoc test, p < 0.05) (see Fig. 3).

Effects of Carbamylcholine Pretreatment on SH-EP1 Cell Human 44-nAChR Function. Following 1 min of carb exposure, heterologously expressed, human 44-nAChRs functioned normally except when pretreated with concentrations of carb greater than 100 µM (Fig. 6, top-left). One-minute exposure to 1 mM carb and 1-min recovery led to a reduction of efflux response (~20% decline). Receptor function returned to normal after 30 min in drug-free buffer. Greater 44-nAChR functional effects were observed following 15 min of carb exposure (Fig. 6, top-right). After 1 h of incubation, pretreatment concentrations of carb as low as 10 µM began to have an effect on 44-nAChR function (Fig. 6, bottom-left), although function recovered fully within 1 h of drug-free recovery. Functional losses of ~60 to 70% occurred for 1-h pretreatment with 0.1 to 1 mM carb, and function recovered to only ~80% of control after 1 h of recovery. Increasing carb exposure time from 1 to 24 h did not change 44-nAChR functional inactivation (Fig. 6, bottom-right), although the range for functional loss remaining at 1 h of recovery extended to 100 nM carb (~10% loss; ~25% at 0.01 to 1 mM carb). Persistent inactivation first became evident for 1-min pretreatment with 1 mM carb, for 15 min to 1 h of pretreatment with 10 µM carb, or for 24-h pretreatment with 100 nM carb.

View larger version (46K): [in this window] [in a new window]   Fig. 6.   Time- and concentration-dependent effects of carb exposure on function of 44-nAChR measured using 86Rb+ efflux assays. SH-EP1 cells heterologously expressing human 44-nAChRs were pre-exposed to carb at the indicated concentrations (abscissa, log M scale) for 1 min (top-left), 15 min (top-right), 1 h (bottom-left), or 24 h (bottom-right). Otherwise, samples were processed as described in the legend to Fig. 3. Recovery times were 1 min (), 3 min (), 5 min (), () 15 min, () 30 min, or () 60 min. Specific 86Rb+ efflux (ordinate, percentage of control) was measured as described in the legend to Fig. 3 (means ± S.E.M. from at least three separate experiments). Linear curves are drawn through data points (means ± S.E.M. from at least three separate experiments). Results were not significantly different for 1- or 24-h pretreatments at equivalent carb doses and recovery times, but these profiles and those for 1- and 15-min pretreatments were significantly different from each other across the dose profile (ANOVA followed by Tukey's post hoc test, p < 0.05). carb pretreatment produced significantly less functional loss of 44-nAChR than did nicotine under all otherwise equivalent conditions tested (ANOVA followed by Bonferroni post hoc test, p < 0.05) (see Fig. 4). Moreover, 1-min or 15-min carb pretreatment produced significantly greater functional loss for 42-nAChR than for 44-nAChR (unpaired t test, p < 0.01) (see Fig. 5).

Effects of Mecamylamine Pretreatment on SH-EP1 Cell Human 42-nAChR Function. Studies were done to determine whether chronic exposure to mec could induce long-lasting nAChR functional changes, in part because of its potential utility as a smoking cessation aid and therapeutic, but also because its noncompetitive antagonism might occur through open-channel block, possibly mimicking effects of nicotine acting at high concentrations and/or chronically. Pretreatment with mec induced functional losses in heterologously expressed, human 42-nAChRs that persisted after removal of the drug from media (Fig. 7). It should be noted, however, that experiments evaluating effectiveness of rinses for removing mec after pretreatment (not shown) indicated that our protocol yielded an ~1000-fold reduction in mec concentration, meaning that for samples pretreated with 1 mM mec, enough residual drug could remain to antagonize as much as 50% of receptor function. Nevertheless, these studies showed that residual mec levels would be less than those needed to acutely produce nAChR functional block if pretreatment with mec was done at concentrations of 100 µM or less. Therefore, although efficiency of mec removal was lower than for removal of nicotine, any functional loss observed following pretreatment with lower concentrations of mec can be attributed to desensitization and/or persistent inactivation rather than channel block. 42-nAChRs were functioning at 25% of normal following 100 µM mec pre-exposure for 1 min and 1 min of recovery, but 5 to 60 min of recovery allowed function to return to ~85% of control levels (Fig. 7, top-left). Pretreatment for 1 min with mec at concentrations less than 10 µM had negligible effects on 42-nAChR function. Persistent inactivation evident after 5 min of recovery was about 15% for 1-min pretreatment with 10 µM mec.

View larger version (52K): [in this window] [in a new window]   Fig. 7.   Time- and concentration-dependent effects of mec exposure on function of 42-nAChR measured using 86Rb+ efflux assays. SH-EP1 cells heterologously expressing human 42-nAChRs were pre-exposed to mec at the indicated concentrations (abscissa, log M scale) for 1 min (top-left), 15 min (top-right), 1 h (bottom-left) or 24 h (bottom-right). Otherwise, samples were processed as described in the legend to Fig. 3. Recovery times were 1 min (), 3 min (), 5 min (), 15 min (), 30 min (), or 60 min (). Specific 86Rb+ efflux (ordinate, percentage of control) was measured as described in the legend to Fig. 3 (means ± S.E.M. from at least three separate experiments). Results for 1- or 24-h pretreatments at equivalent mec doses and recovery times were not significantly different, but these profiles and those for 1- and 15-min pretreatments were significantly different from each other across the dose profile (ANOVA followed by Tukey's post hoc test, p < 0.01). mec produced less functional loss of 42-nAChR than did nicotine, but more than carb, across virtually all conditions (ANOVA followed by Bonferroni post hoc test, p < 0.01; see Figs. 3 and 5).

Effects of Mecamylamine Pretreatment on SH-EP1 Cell Human 44-nAChR Function. One minute of pretreatment with as much as 10 µM mec had no effect on subsequently assessed function of heterologously expressed, human 44-nAChRs (Fig. 8, top-left). Following 1 min of 1 mM mec exposure and 1-min recovery, 44-nAChR functional response was 50% of untreated control. Functional assessment after 5 min of recovery found 44-nAChRs to function at 70% of normal, and recovery was to 95% of control after 0.5 to 1 h in drug-free medium. No significant differences were observed if mec pretreatment times were extended to 15 min (Fig. 8, top-right). One or 24 h of pretreatment with 1 mM mec yielded only modest increases in the extent of functional loss (Fig. 8, bottom, left and right). However, pre-exposure to 100 µM mec for 1 h induced an ~30% loss in function, although recovery occurred within 5 to 60 min of drug-free incubation.

View larger version (44K): [in this window] [in a new window]   Fig. 8.   Time- and concentration-dependent effects of nicotine exposure on function of 44-nAChR measured using 86Rb+ efflux assays. SH-EP1 cells heterologously expressing human 44-nAChRs were pre-exposed to mec at the indicated concentrations (abscissa, log M scale) for 1 min (top-left), 15 min (top-right), 1 h (bottom-left), or 24 h (bottom-right). Otherwise, samples were processed as described in the legend to Fig. 3. Recovery times were 1 min (), 3 min (), 5 min (), 15 min (), 30 min (), or 60 min (). Specific 86Rb+ efflux (ordinate, percentage of control) was measured as described in the legend to Fig. 3 (means ± S.E.M. from at least three separate experiments). Linear curves are drawn through data points (means ± S.E.M. from at least three separate experiments). Results were not significantly different for 1- or 15-min pretreatments at equivalent mec doses and recovery times, but results for pretreament for 1 h, compared with 1 or 15 min, at 100 µM mec were significantly different for 1- and 3-min recovery times (ANOVA followed by Tukey's post hoc test, p < 0.05). mec pretreatment produced significantly less functional loss of 44-nAChRs than did nicotine under all otherwise equivalent conditions tested (see Fig. 4) and than did carb for 1- or 24-h pretreatments (ANOVA followed by Bonferroni post hoc test, p < 0.01; see Fig. 6). For nearly all conditions tested, mec pretreatment produced significantly greater functional loss for 42-nAChRs than for 44-nAChRs (unpaired t tests, p < 0.05; see Fig. 7).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Major Findings. One of the major findings of this study is that pre-exposure to nicotine induces losses in function of human 42- or 44-nAChRs heterologously expressed in the SH-EP1 cell line. Losses in function, measured at maximally efficacious challenge doses of acute agonist, are substantial after pretreatment with concentrations of nicotine (~100 nM-1 µM) found in the plasma of human smokers (Russell et al., 1980). These losses in function are even more pronounced after pretreatment at higher doses of nicotine, i.e., at concentrations that have acute functional efficacy equivalent to acetylcholine when acting at active synapses. Using the operational definition of persistent inactivation as the loss of function after 5 min of drug-free incubation (Ke et al., 1998), the present study found that 1 h of nicotine pretreatment at "smoker doses" of 100 nM to 1 µM induces 60 to 80% loss of human 4*-nAChR function, and recovery to 90% of control levels requires ~1 h after drug removal.

Ligand and nAChR Subtype Selectivity of Functional Inactivation. As an ionic species, carb does not freely traverse the membrane lipid bilayer and gain access to the intracellular space as readily as nicotine does. The finding that carb induces 4*-nAChR receptor desensitization and inactivation suggests that ligand access to cytoplasmic or other buried domains is not required for functional inactivation. Nevertheless, whether expressed in absolute terms or normalized to agonist acute functional efficacy and potency, nicotine has more persistent inactivation potency (1-h pretreatment, 5-min recovery) than carb at either 42- or 44-nAChRs (Table 1). This suggests that the ability of nicotine to access the cell interior may indeed play a role in its strong induction of persistent inactivation. Whether expressed absolutely or after normalization to agonist acute potency, persistent inactivation potency of carb is higher (>6- to >20-fold) at 42- than at 44-nAChRs (Table 1), suggesting an influence of nAChR  subunit sequence on this effect.

View this table: [in this window] [in a new window]   TABLE 1 Comparisons between nicotinic ligand functional inactivation and acute activation/inhibition potencies Concentrations of the indicated ligands acting at the specified nAChR subtypes (column 1) needed to induce persistent inactivation (50% loss of function after 1 h of drug pretreatment and 5 min of drug-free recovery; PI-IC50 in µM; column 2) were compared with concentrations needed to acutely induce 50% of maximal functional activation (for agonists nicotine and carbamylcholine; A-EC50 in µM; column 3) or to acutely inhibit agonist-induced function by 50% (for the antagonist mecamylamine; A-IC50 in µM; column 3). Persistent inhibition potency normalized to acute ligand potency was then calculated as the dimensionless ratio between persistent inactivation IC50 and either acute EC50 or IC50 values [PI-IC50/A-EC(IC)50; column 4]. nAChR function was determined using 86Rb+ efflux assays (see Materials and Methods). Acute functional EC50 or IC50 values are from Peng et al. (1999) or Eaton et al. (2000), respectively, for 42- or 44-nAChR, and persistent inactivation IC50 values are from the present study.

Comparisons to Other in Vitro Findings. Findings in the present study using the SH-EP1 human epithelial cell expression system are consistent with numerous reports of functional inactivation of 42-nAChRs from different species in diverse experimental systems following chronic nicotine exposure (Peng et al., 1994; Hsu et al., 1996; Fenster et al., 1997; Olale et al., 1997). Other nAChR subtypes that undergo functional inactivation following prolonged nicotine exposure include human muscle-type 1*-nAChR naturally expressed in TE671/RD cells, 7-nAChR and 3*-nAChR expressed in Xenopus oocytes, and ganglionic 3*-nAChR naturally expressed in rat PC12 or human SH-SY5Y cells (reviewed in Gentry and Lukas, 2002). Additionally, evidence from in vivo studies that allow evaluation of nicotine effects and recovery times on the order of days are consistent with the hypothesis that chronic nicotine inactivates brain nAChR (reviewed in Gentry and Lukas, 2002).

Relationships Between Chronic Nicotine-Induced Functional Inactivation and nAChR Numerical Up-Regulation. Several factors can account for changes in the level of measurable nAChR function. Among these are enhanced/reduced conductance of each channel, enhanced/reduced frequency of channel opening, or longer/shorter channel open time. Among these possibilities, changes brought about by nicotinic ligands may be influenced by membrane lipid composition effecting conformational equilibria of nAChR (Baenziger et al., 2000), electrostatic interactions (Song and Pedersen, 2000), enzymatic carboxymethylation (Kloog et al., 1980), conformation changes (Baenziger and Chew, 1997), phosphorylation (Hsu et al., 1997; Fenster et al., 1999a), endocytosis/exocytosis (Peng et al., 1994), or number of nAChRs on the cell surface (Ke et al., 1998).

Perspectives. The current evaluation of effects of prolonged nicotinic ligand exposure on function of human 42- and 44-nAChR provides a comprehensive characterization of nAChR desensitization and persistent inactivation and adds to findings indicating that all nAChR subtypes experience functional loss in response to chronic ligand exposure. Knowledge of nAChR subtype selectivity with regard to rates, extents, and nicotinic ligand concentration dependence of nAChR activation, desensitization, and persistent inactivation is critical toward development of rational approaches for nicotinic drug therapies in treatment of neuropsychiatric disorders and successful approaches to smoking cessation recognizing that tobacco use is a form of nicotine self-medication (reviewed in Gentry and Lukas, 2002). This is because, as the current studies show, acute and more chronic effects of nicotinic drugs are integrated across nAChR subtypes, drug exposure time, and drug dose at sites of nAChR expression, inducing some balance between activation and inactivation not only of nAChR subtypes, but also of the excitatory or inhibitory effects they have on specific neuronal processes and functions.