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. Author manuscript; available in PMC: 2012 Oct 1.
Published in final edited form as: J Neurochem. 2011 Sep 1;119(1):153–164. doi: 10.1111/j.1471-4159.2011.07408.x

Effects of chronic nicotine on heteromeric neuronal nicotinic receptors expressed in rat primary cultured neurons

Ermelinda Lomazzo 1, Gregory P Hussmann 1, Barry B Wolfe 1, Robert P Yasuda 1, David C Perry 2, Kenneth J Kellar 1
PMCID: PMC3171599  NIHMSID: NIHMS315907  PMID: 21806615

Abstract

Nicotine increases the number of neuronal nicotinic receptors (nAChRs) in brain. This study investigated the effects of chronic nicotine treatment on nAChRs expressed in primary cultured neurons. In particular, we studied the chronic effects of nicotine exposure on the total density, surface expression and turnover rate of heteromeric nAChRs. The receptor density was measured by [125I]epibatidine ([125I]EB) binding. Untreated and nicotine-treated neurons were compared from several regions of embryonic (E19) rat brain. Twelve days of treatment with 10 µM nicotine produced a two-fold up-regulation of nAChRs. Biotinylation and whole-cell binding studies indicated that up-regulation resulted from an increase in the number of cell surface receptors as well as intracellular receptors. nAChR subunit composition in cortical and hippocampal neurons was assessed by immunoprecipitation with subunit-selective antibodies. These neurons contain predominantly α4, β2 and α5 subunits, but α2, α3, α6 and β4 subunits were also detected. Chronic nicotine exposure yielded a two-fold increase in the β2-containing receptors and a smaller up-regulation in the α4-containing nAChRs. To explore the mechanisms of up-regulation we investigated the effects of nicotine on the receptor turnover rate. We found that the turnover rate of surface receptors was > two weeks and chronic nicotine exposure had no effect on this rate.

Keywords: Nicotinic Receptors, Primary neurons, Up-regulation, Cell Surface, Receptor degradation rate

Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand-gated channels permeable to Na+, K+ and Ca2+. Heteromeric nAChRs have a pentameric structure comprised of two different types of subunits, α and β, which assemble to form different receptor subtypes. Nine α and three β neuronal nAChR subunits are known, and these associate to form either a homomeric receptor composed of α7 subunits, or simple heteromeric receptors composed of only one type of α and one type of β subunit, or mixed heteromeric receptors composed of one or more type of α subunit associated with one or more type of β subunits.

Chronic nicotine administration increases the number of neuronal nicotinic receptors. This receptor up-regulation has been shown in brains from rodents chronically treated with nicotine (Marks et al, 1983; Schwartz and Kellar, 1983; Mao et al, 2008), and in post-mortem human brains from smokers (Benwell et al, 1988; Breese et al, 1997; Perry et al, 1999), and even in heterologous systems expressing nAChRs (Xiao et al, 2004). The α4β2 subtype, which is the predominant nAChR subtype in the brain, is the most susceptible to up-regulation in vivo (Flores et al, 1992; Perry et al, 1999). The increase in nAChRs by nicotine may be directly related to its addictive mechanisms.

The mechanisms of nicotine-induced up-regulation are not yet known with certainty. Although recent evidence clearly indicates that the nicotine-induced increase in α4β2 nAChRs is related to an increase in β2 subunit protein (Gahring et al, 2010; Marks et al, 2011), transcription of mRNA and thus new protein synthesis does not seem to be directly involved (Marks et al, 1992; Peng et al, 1994; 1997; Ke et al, 1998). Therefore several reasonable hypotheses based on post-translational events have been proposed, including decreased receptor degradation (Peng et al, 1994; Kuryatov et al, 2005) and increased subunit assembly via a chaperone effect (Nashmi et al., 2003; Sallette et al, 2004; Kuryatov et al, 2005; Sallette et al, 2005; Corringer et al, 2006; Lester et al, 2009; Srinivasan et al, 2010). In addition, a change in receptor conformation that leads to increased receptor binding and function has been proposed (Govind et al, 2009).

To date, the mechanisms of up-regulation have been investigated mostly by using heterologous expression systems, such as cell lines expressing nAChR subtypes. The mechanisms of nicotine-induced up-regulation of nAChRs in neurons may differ from those in many of the heterologously expressed cell lines that are commonly used for nAChR studies. In those cell lines, probably because subunit expression is usually under control of the very active cytomegalovirus promoter, the nicotine-induced up-regulation may reach many times the basal expression (Xiao et al,, 2004) in contrast to the ~ 2-fold to 3-fold increase in nAChR density observed in brain or primary cultures (Dávila-García et al., 1999) or in heterologous cell lines under a regulated or conditional promoter (Peng et al., 1994). Consequently, we carried out studies on primary cultured neurons, which provide a model to explore some of these mechanisms in native cells. In particular, we investigated how chronic nicotine exposure affects total density, surface expression and degradation rate of heteromeric nAChRs in neurons.

Materials and methods

Materials

Timed-pregnant Sprague Dawley rats were purchased from Taconic and housed at the Georgetown University Research Resource Facility with free access to food and water. All experiments were approved by the Georgetown University Animal Care and Use Committee and were performed in accordance with the National Institutes of Health guidelines on animal care. [35S]Methionine ([35S]Met) (specific activity 1175 Ci/mmol), [3H]Epibatidine ([3H]EB) (specific activity 55.8 Ci/mmol) and [125I]EB (specific activity 2200 Ci/mmol) were obtained from Perkin Elmer Life Science (Boston, MA). In other cases [125I]EB (specific activity 100–300 Ci/mmol) was synthesized as described by Musachio et al, (1997) by [125I]-labeling of the bromopyridyl precursor of IPH, (6)-exo-2-(2-bromo-5-pyridyl)-7-azabicyclo[2.2.1]heptanes that was synthesized by Horti et al, (1996) and provided as a kind gift by Dr. Andrew Horti (Johns Hopkins University). Iodination and purification procedures of the compound were carried out as previously described (Davila-Garcia et al, 1997). Nicotine tartrate, carbamylcholine, poly-D-lysine hydrobromide and cycloheximide (CHX) were purchased from Sigma-Aldrich (St. Louis, MO). Laminin was purchased from Roche. Neurobasal media, B-27 supplement and Hank’s solution (HBSS) were purchased from Invitrogen (Gibco). EZ-link sulfo-NHS-SS biotin and neutravidine-agarose Ultralink resin were purchased from Pierce Termo-Scientific (Rockford, IL). Protein G Sepharose beads were purchased from GE Healthcare (Little Chalfont, Buckinghamshire, UK). Normal Rabbit Serum (NRS) was purchased from Calbiochem (San Diego, CA). HEK-293 cell lines stably expressing α4β2 nAChRs have been described previously (Xiao and Kellar, 2004).

Antisera and antibodies

Rabbit antisera directed at bacterially expressed fusion proteins containing partial sequences of the cytoplasmic domains of α2, α4 and α6 nAChR subunits were kind gifts from Drs. Scott Rogers and Lorise Gahring (University of Utah, Salt Lake City). These antisera have been described previously (Flores et al, 1992; Rogers et al, 1992). A monoclonal antibody (mAB 299) against the extracellular domains of α4 subunit was purchased from Sigma. An antibody directed at the C-terminal peptide sequence of the rat nAChR α3 subunit was affinity purified from rabbit serum. This antibody has been described previously (Yeh et al, 2001). A monoclonal antibody (mAb 270) to the chick β2 subunit was made from hybridoma stocks (American Type Culture Collection, Manassas, VA). This mAb has been previously developed and characterized (Whiting and Lindstrom, 1987). The specificity of all of these antibodies for immunoprecipitation of nAChRs has been reported previously (Hernandez et al, 2004; Marritt et al, 2005; Perry et al, 2007). Rabbit antisera directed at the loop and C-terminal sequences of α5 and β4 subunits were produced and characterized in our lab. For simplicity, in this paper, we use the term antibody to refer to unpurified antiserum, as well as to affinity purified antiserum and monoclonal antibody.

Primary Culture Preparation

Pregnant rats were anesthetized with isoflurane and sacrificed by decapitation at day 19 of gestation (E19). Embryos were quickly removed, decapitated and kept in sterile iced-cold HBSS buffer (pH 7.2). Embryonic brain tissues were dissected, trypsinized and dissociated in sterile conditions. Dissociated neurons were plated on poly-D-lysine and laminin pre-coated six-well dishes containing neurobasal media and cultured at 37 °C and 5% CO2.

Binding Assays

Cortical, hippocampal, striatal and thalamic neurons were cultured for 14 days and after two days in culture, fresh media with or without 10 µM nicotine tartrate was added and the cells were cultured for an additional 12 days. The media was changed and the nicotine replenished every other day. The concentration and duration of nicotine treatment were chosen to provide as robust an up-regulation of nAChRs as possible in these primary neurons in culture. Moreover, the concentration of nicotine used here is not far from the 6 µM steady state nicotine concentration found in brain from adult rats treated for 14 days with nicotine tartrate (37 µmol/kg/day) via osmotic minipump (Doura et al, 2008). The cells were then harvested and homogenized in Tris buffer (50mM Tris HCl, pH 7.0), followed by centrifugation at 35,000 × g for 10 minutes at 4°C. Pellets were washed 4–6 times, centrifuged, re-suspended in Tris HCl buffer and distributed in binding tubes containing 0.5–1 nM [125I]EB. Non-specific binding was defined in the presence of 300µM nicotine. Assay tubes were incubated for 2 hours at 24°C and bound receptors were separated from the free ligand by vacuum filtration over Brandel GF/C glass-fiber filters (Gaithersburg, MD), pre-wet with 0.5% polyethyleneimine. Filters were counted in a liquid scintillation counter.

Measurement of Surface nAChRs by Biotinylation

Cortical neurons were cultured on six-well dishes for 14 days and treated with or without 10uM nicotine for 12 days. Neurons were quickly washed twice on ice with HBSS (pH 8) and then incubated at 37 °C for 10 minutes. This procedure was repeated three times. Before biotinylation, intact cells were incubated with 1nM [125I]EB for 15 minutes on ice then washed and incubated with biotin (1mg/ml/well) for 30 minutes on ice. The biotinylation reaction was stopped with 100mM glycine. Cells were then harvested, sonicated and spun twice in fresh buffer at 35,000 × g for 10 minutes. Pellets were re-suspended in Tris HCl buffer (pH 7.0) and solubilized in 2% Triton X-100 with gentle rotation at 4°C for 4 hours, followed by centrifugation at 35,000 × g for 10 minutes. Aliquots of solubilized receptors were then distributed into tubes containing 1nM [125I]EB and a 50% slurry of neutravidine-agarose resin (40µl) for 1 hr at 4°C, followed by centrifugation at 8,000 × g for 1 minute at 4°C to precipitate the [125I]EB-labeled surface nAChRs. Supernatants were filtered over Brandel GF/B glass-fiber filters pre-wet with 0.5% polyethyleneimine and then counted in a liquid scintillation counter. The remaining pellets were washed twice with fresh Tris HCl buffer (pH 7.0) followed by centrifugation at 8,000 × g for 1 minute at 4°C. The final pellets were incubated with 200µl of 0.1 N NaOH for 30 minutes and then counted. Values obtained from un-biotinylated cells were used as control for non-specific neutravidin-agarose precipitations and were subtracted from the values obtained with biotinylated neurons.

Measurement of Surface nAChRs by Whole Cell Binding

This method is based on carbachol being a quaternary amine, which therefore does not cross cell membranes easily; thus, during short periods of incubation, it will compete only for the surface [125I]EB-labeled receptors, resulting in a direct measurement of the internal [125I]EB-labeled nAChR binding sites (Rothhut et al, 1996; Whiteaker et al, 1998; Xiao et al, 1998; Kuryatov et al, 2005). Cortical neurons were treated and washed as described above for the biotinylation procedure. Whole cell binding was performed on ice as follows: one group of cells was incubated with 1nM [125I]EB to measure total binding; a second group was incubated with 1nM [125I]EB in the presence of 300uM nicotine to measure non specific binding; a third group was incubated with 1nM [125I]EB in the presence of 10mM carbachol, a nicotinic agonist that does not readily cross cell membranes, thus allowing measurements of [125I]EB binding to nAChRs inside the cells. Whole cell binding was carried out on ice for 5 minutes, which is enough time for this relatively high concentration of [125I]EB to label nearly all of the nAChRs, even in this short incubation period. In preliminary experiments, concentration response curves with 1, 3 and 10 mM carbachol and drug incubation times up to 10 minutes yielded similar binding results (data not shown). After the incubation, the cells were quickly washed four times with Tris HCl buffer, incubated with 1ml of 0.1 N NaOH for 30 minutes and then counted. Surface nAChRs were deduced by subtraction of specific binding to internal receptors from total specific binding (internal plus surface) receptors.

Immunoprecipitation Assays

Cortical and hippocampal neurons were harvested, washed, homogenized and solubilized as described above. Aliquots of solubilized receptors were distributed into tubes containing 0.5–1nM [125I]EB and subunit-specific antibodies (Ab) or normal rabbit serum (NRS), as a control. The samples were gently rotated overnight at 4°C, and then a 50% slurry of Protein G Sepharose beads (50µl) was added. After 2 hours incubation at 4°C, the tubes were centrifuged at 8,000 × g for 1 minute at 4°C to precipitate the radiolabeled antibody-receptor complex. Supernatants were filtered over Brandel GF/B glass-fiber filters pre-wet with 0.5% polyethyleneimine and then counted in a liquid scintillation counter. The remaining pellets, containing radiolabeled receptors immunoprecipitated by the antibody and protein G, were washed twice with fresh Tris HCl buffer (pH 7.0), followed by centrifugation at 8,000 × g for 1 minute at 4°C. The final pellets were incubated with 200µl of 0.1 N NaOH for 30 minutes and then counted. The radioactivity measured in these pellets thus represented the [125I]EB-labeled nAChRs containing the specific subunit recognized by the antibody used. Values obtained from cells incubated with NRS were used as control for non-specific precipitations and were subtracted from the values obtained with subunit-specific antibodies.

Inhibition of Protein Synthesis with Cycloheximide

Striatal and hippocampal neurons were cultured for 14 days and treated with or without 10µM nicotine for 12 days, followed by incubation with 35µM cycloheximide (CHX) for 6, 24 or 48 hrs in the continued presence or absence of nicotine. Cells were then harvested, washed, homogenized and radioligand binding assays were carried out as described above. Radioligand binding in cells that did not receive CHX treatment was used as control to measure total receptor density in absence of CHX. HEK-293 cells stably expressing α4β2 nAChRs served as additional controls for the CHX treatment. These cells were treated with or without 10µM nicotine for 24 hrs, before incubation with CHX, followed by radioligand binding with [3H]EB.

[35S]Methionine Incorporation

Striatal neurons cultured for 10 days in 6-well dishes were washed twice in sterile HBSS buffer and incubated with methionine-free media for 2 hrs at 37°C. The neurons were then incubated with [35S]Met (10–20 µCi/well) in absence (control) or presence of 35µM CHX for 6 hrs (or 12 hrs in case of HEK-293 cells expressing α4β2 nAChRs) at 37°C, following which they were harvested, washed in fresh buffer and spun at 35,000 × g for 10 minutes three times. Pellets were re-suspended in 20% trichloroacetic acid (TCA) for 1 hour at 24°C and then spun at 35,000 × g for 10 minutes. Pellets containing the TCA-precipitated [35S]Met-labeled proteins were incubated with 1ml of 0.1N NaOH for 30 minutes and counted in a liquid scintillation counter along with the corresponding supernatants.

Pulse Chase Biotinylation

In these assays, the turnover rate of surface nAChRs present during nicotine exposure was measured over a period of two weeks. Striatal and cortical neurons were prepared as described above. After dissociation in sterile HBSS buffer (pH 8), cells were incubated with 1nM [125I]EB for 15 minutes followed by addition of biotin (1mg/ml/well) for 30 minutes at 37 °C. The neurons were then plated on poly-D-lysine and laminin pre-coated six-well dishes containing neurobasal media and cultured for 7 or 14 days. Nicotine-treated groups received 10µM nicotine 48 hrs after biotinylation (day 2 in culture). Both untreated and nicotine-treated cells were harvested after 7 or 14 days in culture (5 and 12 days of nicotine treatment, respectively). The cells were then washed, homogenized and solubilized as described above, followed by neutravidin-agarose precipitation in the presence of [125I]EB. Values obtained from un-biotinylated cells were used as control for non-specific neutravidin-agarose precipitations and were subtracted from the values obtained with biotinylated neurons.

Data Analysis

For radioligand binding assays measuring total receptor density, surface receptor measurements and immunoprecipitation studies, statistical analyses of the difference between group means were assessed using Student’s t-test or one-way analysis of variance followed by Bonferroni’s Multiple Comparison Test. A one-sample t-test was used to determine if values in immunoprecipitation assays and surface measurements were statistically different from zero. For the assays measuring receptor turnover rate, one phase exponential decay analysis was performed using the GRAPHPAD Prism 4.0 software package (GraphPad Software, San Diego, CA).

Results

Up-regulation of Total nAChR Density in Primary Neurons

nAChR density in primary cortical, hippocampal, striatal and thalamic neurons isolated from E19 rat brains and cultured for 14 days was measured in radioligand binding assays using [125I]EB (Fig.1). Consistent with previous studies that measured nAChRs with [3H]cytisine in primary cultures from E18 rats (Dávila-García et al, 1999), the density of nAChRs measured here in E19 primary cultures of cerebral cortex, striatum and thalamus is approximately one-third to one-half the density typically found in adult rat brain areas. In contrast, as was also found previously (Dávila-García et al., 1999), the density of nAChRs measured in these hippocampal cultures is more similar to the density typically found in the adult brain. The reason for this difference among these brain structures is not known, but it might reflect the older phylogenetic origin of the hippocampus and/or its 3 layer structure compared to the 5–6 layers in the cerebral cortex.

Fig. 1. Up-regulation of Total nAChR Density in Primary Neurons.

Fig. 1

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(A) Cortical, (B) hippocampal, (C) striatal and (D) thalamic neurons isolated from E19 rat brains were cultured for 14 days and treated with or without 10 µM nicotine for 12 days. Homogenized extracts were then labeled with 0.5–1nM [125I]EB to measure total nAChR density in both untreated and nicotine-treated groups. Non-specific binding was measured with 300 µM nicotine and subtracted. Data are expressed in fmoles of receptors per mg of total protein and are means ± SEM of 3 or 4 independent experiments. Total nAChR density in nicotine-treated groups was statistically different compared to the corresponding controls (untreated cells), except in the case of thalamic neurons (D). **p<0.01, one-way ANOVA followed by Bonferroni's multiple comparison test.

Twelve days of treatment with 10 µM nicotine yielded ~2 fold increase in the total number of receptors expressed in cultured neurons, which reached statistical significance in all areas except the thalamus (Fig. 1). These results are consistent with previous studies showing that receptor density increases up to ~2-fold in primary cultures of E18 neurons exposed to nicotine (Dávila-García et al, 1999). Similarly, nAChRs are increased 1.5- to 2-fold in adult brain areas from rats treated chronically with nicotine (Mao et al, 2008), and comparable or slightly greater increases are found in autopsied brains from human smokers (Benwell et al, 1988; Breese et al, 1997; Perry et al, 1999).

Up-regulation of Surface nAChRs in Cortical Neurons

Neutravidine-agarose precipitations were carried out on biotinylated cortical neurons in the presence of 1nM [125I]EB (Fig. 2A). Thus, radiolabeled nAChRs present in the precipitated pellets reflected the receptors that were expressed on the cell surface. [125I]EB binding in neurons biotinylated but not precipitated with neutravidine-agarose was measured in parallel to determine total receptor density. The amount of internal nAChRs was then deduced by subtraction of surface receptors from total nAChR density. These biotinylation studies indicated that ~27% of the nAChRs were on the cell surface in the untreated cortical neurons (Fig. 2A, left bars). After treatment of cortical neurons for 12 days with 10 µM nicotine, the density of nAChRs was increased by ~2.7-fold, and biotinylated cell-surface receptors represented ~30% of the total (Fig. 2A, right bars). These results indicate that chronic exposure to nicotine increases cell surface nAChRs on primary neurons ~ in proportion to the increase in total number of receptors; so that, in this case, nicotine induced 3-times more cell surface receptors on these neurons compared to control.

Fig. 2. Up-regulation of Surface nAChRs.

Fig. 2

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Cortical neurons were cultured for 14 days and treated with or without 10 µM nicotine for 12 days. (A) Neutravidine-agarose pull down assays were carried out on solubilized cells in which the surface receptors were previously biotinylated and labeled with [125I]EB. Non-specific neutravidine-agarose precipitations were measured in un-biotinylated cells and subtracted. Total nAChR density was measured with 1nM [125I]EB binding; internal nAChRs were deduced by subtraction of surface receptors from total nAChR density. (B) Whole cell radioligand binding was carried out with 1nM [125I]EB alone (total nAChR) or in presence of 10mM carbachol for 5 minutes (internal nAChRs). Surface nAChRs were deduced by subtraction of internal receptors from total nAChR density. Non-specific binding was measured with 300 µM nicotine and subtracted (A and B). Data are expressed in fmoles of receptors per mg of total protein and are means ± SEM of 5 independent experiments (A and B). Total receptor density, internal nAChRs and surface nAChRs in the nicotine-treated groups were statistically different compared to the corresponding controls (untreated cells) (A and B), *p<0.05, **p<0.01, ***p<0.001, one-way ANOVA followed by Bonferroni's multiple comparison test.

To test these results we measured cell surface receptors by a second method, which consisted of whole cell radioligand binding assays (Rothhut et al, 1996; Whiteaker et al, 1998; Xiao et al, 1998; Kuryatov et al, 2005). In this case, we used 1nM [125I]EB in the absence and presence of 10 mM carbachol, a quaternary amine that does not readily cross membranes, to measure total and internal binding sites, respectively; specific binding to the total and internal nAChR pools was calculated after subtracting non-specific binding, defined in the presence of 300 µM nicotine. Surface nAChRs were then deduced by subtraction of internal receptors from total nAChR density (Fig. 2B). A previous report found that prolonged carbachol exposure (>30 minutes) could result in some penetration of the drug into the cell (Kuryatov et al, 2005); therefore, to avoid this undesired penetration, we used a short, 5 min, incubation time (see Methods).

These whole-cell binding studies confirmed that most of the nAChRs measured in [125I]EB binding assays are in the internal pool; in these studies, ~40% of total nAChRs were found expressed on the cell surface (Fig. 2B, left bars). Chronic nicotine exposure up-regulated total nAChR density by ~ 2.5 fold, which reflected a nearly equivalent increase in the surface receptors as well as the internal receptors, without affecting their ratio (Fig. 2B, right bars). Thus, both methods used here to measure surface nAChRs lead to the same conclusion: that chronic nicotine exposure increases the number of nAChRs on the cell surface.

Immunoprecipitation Studies

The predominant heteromeric receptor in adult rat brain is the α4β2 subtype (Flores et al, 1992; Perry et al, 2002; Gotti et al, 2006; Mao et al, 2008), but developmental changes in receptor subunit composition have been found in muscle (Mishina et al, 1986; Gu and Hall, 1988) and neuronal (Zoli et al, 1995) nAChRs, as well as in GABA (Temple and Wray, 2005) and NMDA (Zhou and Baudry, 2006) receptors. To assess the predominant subunits and likely compositions of the nAChRs in these E19-derived primary neurons and determine which are up-regulated by nicotine, antibodies directed against the α2, α3, α4, α5, α6, β2 and β4 nAChR subunits were used to immunoprecipitate solubilized [125I]EB-labeled receptors (Fig. 3). In general, the density of the solubilized receptors measured with [125I]EB in these studies was lower than in primary neurons measured directly in the membrane homogenates (Fig. 1), especially in the hippocampal neurons, probably due to loss of receptors during solubilization of the very delicate embryonic tissue. Nevertheless, the results of these studies indicate that receptors containing the α4, α5 and β2 subunits predominate in cortical (Fig. 3A) and hippocampal (Fig. 3B) neurons from E19 rats. The β2 antibody immunoprecipitated most of the [125I]EB-labeled nAChRs, indicating that most of the cortical and hippocampal nAChR binding sites contain a β2 subunit.

Fig. 3. nAChR Subunit Immunoprecipitation.

Fig. 3

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(A) Cortical and (B) hippocampal neurons were cultured for 14 days and treated with or without 10 µM nicotine for 12 days. Solubilized cells were labeled with 1nM [125I]EB and immunoprecipitated with each of the subunit-selective antibodies shown. Non-specific immunoprecipitations were measured with normal rabbit serum (NRS) and subtracted. Data are expressed in fmoles of receptors per mg of total protein and are means ± SEM of 3 to 7 independent experiments. Only the β2-containing nAChRs in nicotine-treated groups were statistically different compared to the corresponding controls (untreated cells), **p<0.01, ***p<0.001, one-way ANOVA followed by Bonferroni's multiple comparison test.

The α4-containing nAChRs were measured with two antibodies directed at different sequences of the α4 subunit, the loop and C-terminal. Each of these α4 antibodies immunoprecipitated only about half the receptors immunoprecipitated by the β2 antibody. This suggests that the embryonic α4 subunit is different from its adult counterpart, resulting in a lower efficacy of the α4 antibodies or that the epitopes at which the α4 antibodies are directed may be more fragile and therefore more frequently damaged during the immunoprecipitation procedures.

The α5 antibody immunoprecipitated nearly as many nAChRs as the α4 antibody. The nAChRs containing α2, α3, α6 and β4 subunits in the cortical neurons and α2 and α3 subunits in the hippocampal neurons were detected in much lower amounts. Taken together, these results demonstrate that a β2-containing nAChR subtype predominates in primary cultures of cerebral cortex and hippocampus from E19 rats; moreover, the results suggest that this predominant receptor is an α4β2 subtype, but because of the decreased number of receptors immunoprecipitated by the α4 antibodies, that question remains open.

After chronic nicotine treatment, the β2-containing receptor density was significantly increased approximately 1.8-fold in cortical neurons (Fig. 3A) and approximately 2.3-fold in hippocampal neurons (Fig. 3B). Up-regulation of nAChRs immunoprecipitated by the α4 antibodies was much less pronounced compared to that by the β2 antibody in both cortical and hippocampal neurons and was not statistically different from the corresponding control. Nicotine treatment did not produce significant changes in the density of any other of the nAChR subunits analyzed here.

Degradation Rate of nAChRs (block of protein synthesis)

To establish the method to measure the effect of nicotine on receptor degradation rate in neurons, we first used HEK cells that express a high density of α4β2 nAChRs and respond to nicotine treatment with a large increase in nAChR density. These HEK cells were treated with 35 µM cycloheximide (CHX) for 6, 24, 48 or 72 hrs to block protein synthesis, which then allowed measurement of the decrease in nAChR density over the time course of CHX exposure compared to control cells that were incubated for the same periods without CHX. As shown in figure 4A, treatment of cells with CHX for 12 hrs decreased incorporation of [35S]methionine by 85%, indicating that this treatment was effective in inhibiting protein synthesis. In HEK cells treated with CHX but not treated with nicotine, the number of [3H]EB binding sites initially decreased rapidly, indicating a half-time for nAChR degradation of ~3 hrs; but the fall in receptors then slowed and reached a plateau at ~ 20% of control receptor density (Fig. 4B). In contrast, in HEK cells treated with CHX and 10 µM nicotine, nAChRs decreased at a much slower rate, yielding a half-time for nAChR degradation of ~ 44 hrs (Fig. 4B). These results are consistent with previous studies that examined the effects of nicotine on α4β2 nAChR degradation rates in heterologous expression systems (Peng et al, 1994; Kuryatov et al, 2005).

Fig. 4. Block of Protein Synthesis with Cycloheximide (CHX) in HEK Cells.

Fig. 4

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(A) HEK-293 cells expressing α4β2 nAChRs were loaded with 10–20 µCi [35S]Met with or without 35 µM CHX for 12 hrs. Incorporation of [35S]Met in the TCA precipitated pellets was indicative of newly synthesized proteins over a period of 12 hrs. CHX-treated cells show 85% decrease in [35S]Met incorporation compared to control (no CHX treatment). ***p<0.001 paired t-test. Data are expressed as percent of total [35S]-DPM loaded in each group and are means ± SEM of 3 independent experiments. (B) Cells were treated with or without 10µM nicotine for 24 hrs, followed by 35µM CHX treatment for 6, 24, 48 or 72 hrs in the continued presence or absence of nicotine. At the indicated times, [3H]EB binding assays were carried out on homogenized cell extracts. Cells not treated with CHX were used as controls and represent total nAChR density (100% of receptors; time=0). Data are expressed as percent of control instead of fmol/mg protein because of variability in density of the different cell batches used. Data are means ± SEM of 3 independent experiments. One phase exponential decay analysis indicated that nAChRs in nicotine-treated cells have a slower degradation rate compared to the corresponding control (cells not treated with nicotine), as shown by the half life values.

Next, we investigated the effects of nicotine on nAChR degradation rates in neurons. To do this, we first examined the effects of treatment with 35 µM CHX on protein synthesis in primary striatal neurons in culture. As shown in figure 5A, neurons not treated with CHX incorporated ~ 0.6% of the [35S]methionine loaded into the cells, indicating that new protein synthesis occurs over this time period. In contrast, in neurons treated with CHX, [35S]methionine incorporation was nearly abolished during this time period. The cells stayed attached to the cell culture flask and otherwise looked normal, indicating that the CHX treatment had not killed the cells. Striatal and hippocampal neurons were then cultured in the presence or absence of 10 µM nicotine for 10 days before treatment with CHX for 6, 24 and 48 hrs in the continued presence or absence of nicotine. Binding of [125I]EB to nAChRs was then measured in each group of cells and compared to cells not exposed to CHX.

Fig. 5. Block of Protein Synthesis with Cycloheximide (CHX) in Neurons.

Fig. 5

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(A) Striatal neurons were loaded with 10–20 µCi [35S]Met with or without 35µM CHX for 6 hrs. Incorporation of [35S]Met in the TCA precipitated pellets was indicative of newly synthesized proteins over a period of 6 hrs. CHX-treated neurons show a 98% decrease in [35S]Met incorporation compared to control (no CHX treatment). *p<0.05 paired t-test. Data are expressed as percent of total [35S]-DPM loaded in each group and are means ± SEM of 4 independent experiments. (B) Striatal and (C) hippocampal neurons were cultured for 12 days and treated with or without 10 µM nicotine for 10 days, followed by 35 µM CHX treatment for 6, 24 or 48 hrs in the continued presence or absence of nicotine. At the indicated times, [125I]EB binding assays were carried out on homogenized cell extracts. Cells not treated with CHX were used as controls and represent total nAChR density (time=0). Data are expressed in fmoles of receptors per mg of total protein and are means ± SEM of 3 independent experiments. Exponential decay analysis was not measurable since the total nAChR density did not decrease over the time course of CHX exposure in either untreated or nicotine-treated groups. Total mg of protein did not change over this period of time

As shown at the 0-time points (no CHX addition) in figures 5B and 5C, nicotine treatment before addition of CHX increased binding to nAChRs by ~1.7-fold in striatal neurons and ~2-fold in hippocampal neurons. Interestingly, inhibition of protein synthesis did not produce a significant change in the nAChR density over the 48 hrs time course of CHX exposure in either untreated or nicotine-treated neurons (Fig. 5B and 5C). This indicates that nAChRs expressed in cultured neurons are not degraded within 48 hrs and thus have a slower turnover rate compared to α4β2 nAChRs expressed in HEK cells.

Degradation Rate of Surface nAChRs (pulse-chase biotinylation)

The studies shown in figure 5 measured the degradation rate of both internal and cell surface populations of nAChRs. We next investigated the degradation rate of the surface receptors specifically which, as shown in figure 2, represent only ~27 – 40% of the total nAChR population in these neurons. Surface proteins were biotinylated and then, beginning 2 days later, cultured in the presence or absence of 10 µM nicotine for the next 5 or 12 days. The nAChRs were then labeled with [125I]EB and the surface receptors on the cultured neurons were precipitated with neutravidin-agarose. In theory, if nicotine slowed the degradation rate of the cell surface nAChRs over the 5 to 12 days of treatment, there would be more biotinylated receptors remaining to be precipitated compared to control cultures not treated with nicotine.

The effect of nicotine on the total nAChR population in neurons that were biotinylated but not precipitated with neutravidin-agarose was also measured. As shown in table 1, after being cultured with nicotine for 12 days the total population of [125I]EB binding sites was increased ~2-fold and ~2.6-fold in striatal and cortical neurons, respectively. The 5-day treatment with nicotine increased nAChRs in the cortical neurons by ~1.6-fold, but this increase did not reach statistical significance (0.1>p>0.05). Surprisingly, as shown in figure 6, there was no significant decrease in the number of biotinylated receptors in either untreated or nicotine-treated primary neurons, indicating that the cell surface population of nAChRs in the striatal and cortical neurons are not significantly degraded over 7 and 14 days in culture. Thus, the increase in the number of cell surface nAChRs measured in these primary neurons in culture with nicotine for 12 days (Fig. 2) is not likely to be due to a decrease in their degradation rate.

Table 1. Total nAChR Density from the Striatal and Cortical Neurons Shown in Figure 6.

Radioligand binding assays were carried out with 0.5–1nM [125I]EB in untreated and nicotine-treated striatal and cortical neurons used in figure 6, to measure total nAChR density. Beginning 2 days after culture, nicotine-treated neurons received nicotine for 5 or 12 days, corresponding to 7 or 14 days in culture, respectively. Data are expressed in fmoles of receptors per mg of total protein and are means ± SEM of 3 to 4 independent experiments. Total nAChR density in nicotine-treated groups was statistically different compared to the corresponding controls (untreated cells) after 14 days in culture in both types of neurons

Striatal Neurons
fmol/mg protein (mean ± S.E.M.)		 Cortical Neurons
fmol/mg protein (mean ± S.E.M.)
Days in culture Untreated Nic-treated Untreated Nic-treated
7 Days 29 ± 2.9 31 ± 2.3 19 ± 2.7 31 ± 2.7
14 Days 30 ± 1.6 61 ± 6.4*** 15 ± 2.7 39 ± 3.6**
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**

p<0.01,

***

p<0.001, one-way ANOVA followed by Bonferroni's multiple comparison test.

Fig. 6. Pulse-chase Biotinylation.

Fig. 6

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(A) Striatal and (B) cortical neurons were labeled with [125I]EB and biotinylated before being plated and before starting nicotine treatment. Nicotine-treated groups received 10 µM nicotine 48 hrs after biotinylation. Neutravidin-agarose pull down assays were carried out in the presence of [125I]EB after 7 and 14 days in culture (corresponding to 5 and 12 days of nicotine treatment, respectively). This allowed the turnover rate of surface nAChRs present before nicotine exposure to be measured over a period of two weeks. Non-specific neutravidine-agarose precipitations were measured in un-biotinylated cells and subtracted. Fractions of solubilized cell extract were used to carry out binding assays with 1nM [125I]EB to measure total nAChR density after 7 and 14 days in culture (see Table 1). Data are expressed in fmoles of surface receptors per mg of total protein and are means ± SEM of 3 or 4 independent experiments. Exponential decay analysis was not measurable since the surface nAChR density did not decrease over a period of 14 days in either untreated or nicotine-treated groups.

Taken together, these studies indicate that in primary neurons both the total population of nAChRs and those on the cell surface are remarkably stable; moreover, the data suggest that, in neurons, in contrast to heterologous expression systems (Peng et al, 1994; Kuryatov et al, 2005), the mechanisms of nicotine-induced up-regulation do not involve a decrease in degradation rate and, by extension, of turnover rate, of nAChRs.

Discussion

In these studies, we investigated the effects of chronic nicotine treatment on heteromeric nAChRs expressed in primary cultured neurons. Exposure to 10 µM nicotine for twelve days produced a statistically significant, ~ two-fold increase in receptor density in cortical, hippocampal and striatal neurons, and a somewhat smaller, non-significant increase in thalamic neurons. These effects of nicotine on nAChRs in primary neurons in culture are, in general, similar to the magnitude of the effects of chronic nicotine in adult rats in vivo and also to the magnitude of nAChR increases seen in autopsied brain from human smokers (Benwell et al, 1988; Breese et al, 1997). In contrast, the nicotine-induced increases in nAChRs typically found in HEK cell lines heterologously expressing nAChR subunits under the control of a cytomegalovirus promoter are much greater (Xiao and Kellar, 2004).

In these cortical cultures, ~ 27 to 40 percent of the nAChRs labeled with [125I]EB were on the cell surface, as measured by biotinylation and by whole cell binding. It is important to note that after nicotine-induced up-regulation, the proportion of nAChRs on the cell surface remained at ~ 30–40 percent; thus, nicotine treatment resulted in ~2-fold more cell surface nAChRs and therefore increased potential for signaling.

Our immunoprecipitation assays with subunit-selective antibodies indicate that in cortical and hippocampal neurons from E19 day rats, nAChRs containing the β2 subunit predominate. This is similar to the findings in adult rat cortex and hippocampus (Mao et al, 2008) and, in fact, in most regions of rat brain (Flores et al, 1992; Gotti et al, 2006; Millar and Gotti, 2009). Although the nAChRs containing the α4 subunit were the next most abundant receptors immunoprecipitated, they comprised only ~half of the β2-containing receptors; moreover, we obtained similar results with two different antibodies directed at different epitopes of the α4 subunit (loop and C-terminal). The α2 and α3 subunits represented a very small fraction of the total receptors in both types of neurons, and small amounts of α6 and β4 subunits were also detected in cortical neurons. The β2-containing nAChRs require an α subunit to bind agonists such as [125I]EB, and the most likely candidate is the α4 subunit. It is possible that some of the α4β2 nAChRs also contain an α5 subunit, which is associated with a significant fraction of α4β2 nAChRs in the adult cortex and hippocampus (Mao et al, 2008). Similarly, some of the α4β2 nAChRs in the hippocampus might also contain an α3 subunit, forming an α4β2α3 subtype, which has been found in the adult rat hippocampus (Lomazzo et al, 2010).

Interestingly, the α5 subunit, which is found in only ~15% of the nAChRs in the adult cortex (Mao et al, 2008), appears to be present in about 40% of the receptors in these embryonic cortical neurons. This might be directly related to the higher α5 subunit mRNA level found in developing rat brain compared to the adult (Winzen-Sehran and Leslie, 2005), and supports the suggestion that α5-containing nAChRs might influence signaling during the later developmental period of the brain, especially if they are located on the prominent GABA and glutamatergic axons (Winzer-Serhan and Leslie, 2005).

Nevertheless, the smaller fraction of receptors immunoprecipitated by the α4 antibody compared to the β2 antibody is surprising since it contrasts sharply with results in most areas of adult rat brain (Flores et al, 1992; Zoli et al, 2002; Gotti et al, 2006; Perry et al, 2007; Mao et al, 2008; Millar and Gotti, 2009; Lomazzo et al, 2010), where usually the same number of receptors are immunoprecipitated by the α4 and β2 antibodies. Furthermore, the α4β2 nAChR subtype is the most consistently up-regulated receptor by nicotine administration in vivo (Flores et al, 1992; Mao et al, 2008; Marks et al, 2011). In addition, the α4 subunit in mice has been directly implicated in the actions of nicotine on reward, tolerance and sensitization (Tapper et al, 2004). It is possible that the α4 antibodies immunoprecipitated fewer nAChRs because of the technical difficulty of carrying out quantitative immunoprecipitation on the very delicate E19 neurons, resulting in a greater loss of the critical α4 epitopes. It is also possible that the α4 subunit expressed in these embryonic cultured neurons, has structural, protein folding or post-translational characteristics different from the one in the adult brain making it resistant to measurement with our antibodies. Moreover, a splice variant in the α4 subunit has been reported (Goldman et al, 1987), and although it is unknown which variant is expressed in the embryonic brain, we cannot rule out the possibility of a developmental switch in subunits between embryonic and adult neurons. A precedent for this is the developmentally regulated switch of entire subunits that occurs in muscle nicotinic receptors, where the embryonic γ subunit is switched to ε in the adult (Mishina et al, 1986; Gu and Hall, 1988).

Chronic nicotine treatment up-regulated the β2-containing nAChRs by ~1.8-fold and ~2.3-fold in cortical and hippocampal neurons, respectively. A less pronounced up-regulation, which did not reach statistical significance, was also measured in the immunoprecipitated α4-containing receptors. Previous studies established that the α4β2 nAChR, which predominates in the adult brain, is the subtype that is most susceptible to nicotine-induced up-regulation in vivo (Flores et al, 1992; Perry et al, 1999; Mao et al, 2008), and it is likely that is the case in primary neurons also. Importantly, nicotine treatment did not increase nAChRs containing any other α or β subunits, including the α5-containing receptors, which in the cortex and hippocampus of adult brain are the α4β2α5 subtype exclusively and are resistant to nicotine-induced up-regulation (Mao et al, 2008).

Nicotine-induced up-regulation in brain occurs independently of an increase in mRNA and presumably of new protein synthesis (Marks et al, 1992). Thus, several hypotheses have been proposed to explain the increase in receptors in the absence of new protein synthesis, including decreased receptor degradation (Peng et al, 1994; Kuryatov et al, 2005), increased subunit assembly via chaperone activity of nicotine (Nashmi et al., 2003; Sallette et al, 2004; Kuryatov et al, 2005; Sallette et al, 2005; Srinivasan et al., 2010), and induced changes in the receptor conformation (Govind et al, 2009). Most of these hypotheses have been based on studies in heterologous systems that express nAChRs.

We investigated the effects of nicotine on the turnover rate of nAChRs in primary neurons to test the hypothesis that nicotine causes a decrease in the degradation rate of the receptors with a consequent increase in their number. Such an effect was first found in mouse fibroblasts expressing α4β2 nAChRs (M10 cells) by Lindstrom and colleagues (Peng et al, 1994). Moreover, nicotine was reported to slow receptor turnover rate of α7 nAChRs in brain, possibly by decreasing the activity of the ubiquitin-proteosome system (Rezvani et al, 2007).

After inhibiting protein synthesis in HEK cells that heterologously express α4β2 nAChRs, we found that the half-time for the receptors was ~ 3 h; whereas, in cells in which the nAChRs were up-regulated by treatment with nicotine, the half-time for the receptors was 44 h. These results indicate that nicotine does, in fact, decrease the degradation rate of these heterologously expressed α4β2 nAChRs in HEK cells; they are thus consistent with previous studies carried out in heterologous expression systems (Peng et al, 1994; Kuryatov et al, 2005). An additional interesting observation is that in the untreated HEK cells, after the initial rapid decrease in the number of α4β2 nAChRs, the number of receptors measured reached a plateau within 24 h. This might reflect a greater stability of some receptors due to post-translational modifications and/or anchoring by membrane proteins; alternatively, it could reflect differences in degradation rates between mature pentamers and immature forms of α4 and β2 subunit complexes, including dimers, trimers and tetramers, all of which can bind [3H]EB but have different stabilities (Kuryatov et al, 2008).

In contrast to the degradation rate of heterologously expressed nAChRs in HEK cells, in striatal and hippocampal primary neurons we found that virtually complete inhibition of protein synthesis did not produce a decrease in the density of nAChRs over a course of 48 h. This was the case in both control and nicotine-treated neurons. This indicates that nAChRs in neurons have a degradation rate longer than 48 hours. Importantly, the ~2-fold increase in nAChRs induced by the prior nicotine treatment was maintained over the 48 h time frame of CHX treatment in both striatal and hippocampal neurons. Attempts to carry out protein inhibition studies for periods longer than 48 h were unsuccessful because prolonged treatment of neurons with CHX induced visible morphological changes, including reduction in processes, decrease in diameter and cell death within 7 days (data not shown). Together these data suggest that decreased nAChR degradation does not contribute significantly to nicotine-induced up-regulation of these receptors in primary neurons.

To determine if nicotine treatment affected specifically the degradation rate of the smaller, cell surface pool of nAChRs in these neurons, which might have escaped detection, we investigated the degradation rate of these surface receptors by carrying out pulse-chase biotinylation assays. We found that cell surface nAChRs expressed in untreated striatal and cortical neurons, like the general population of receptors, are very stable and are not substantially degraded over the two week period of time studied here. Similarly, surface nAChRs existing before chronic nicotine treatment that were subsequently exposed to nicotine, are also very stable and are not significantly degraded during the two week period. Again, this is in contrast to what happens to the surface nAChRs in heterologous expression systems (Kuryatov et al, 2005; Figure 4, this paper). Therefore, our data suggest that turnover of nAChRs in neurons is very slow and likely does not occur to a significant extent within two weeks. This is perhaps not too surprising since neurons, unlike HEK cells, are not rapidly dividing. Thus, it is likely that some/many proteins produced in and utilized by neurons have a relatively long half-life, and the nAChRs measured here appear to be among those proteins.

It is interesting that even though significant turnover of surface nAChRs in neurons apparently does not occur within the two weeks studied here, a two-fold up-regulation in the total nAChR density in the cells still takes place during this period of time. This suggests, once again, that in neurons the mechanisms of nicotine-mediated up-regulation are independent of effects on the turnover rate of the receptors.

It is important to note that the pulse-chase biotinylation method used here measured only the turnover rate of surface receptors present before nicotine exposure. It would be interesting to compare these effects with those on the turnover rate of surface and internal receptors that have already been up-regulated by nicotine.

In conclusion, in primary cultured neurons it appears that nicotine-elicited up-regulation does not require a decrease in the receptor turnover rate; thus, other mechanisms must be involved in this process. Recent studies add to the accumulating evidence that up-regulation of α4β2 nAChRs involves an increase in the receptor protein (Gahring et al, 2010; Marks et al, 2011). Since up-regulation by nicotine does not involve new protein synthesis (Marks et al, 1992; Peng et al, 1997; Ke et al, 1998), it is believed that up-regulation depends on post-translational events. Much evidence supports the theory that nicotine may act as a pharmacological chaperone (Kuryatov et al, 2005) or a “maturational enhancer” (Sallette et al, 2005) to facilitate maturational processes and promote assembly of nAChR subunits or immature oligomers into final pentamers. This appealing hypotheses remains to be investigated in native cell models and/or in brain tissues.

Acknowledgements

Supported by the National Institute of Health Grant DA 012976. We thank Drs. Andrew G. Horti and Yongjun Gao (Johns Hopkins University, Baltimore, MD) for synthesizing the bromopyridyl precursor of IPH, (6)-exo-2-(2-bromo-5-pyridyl)-7-zabicyclo[2.2.1]heptanes. We thank Daniel DeGuisto and Bethany Hoegberg (George Washington University, Washington, D.C.) for their help during the iodination procedures. We thank Drs. Scott Rogers and Lorise Gahring (University of Utah, Salt Lake City, Utah) for kindly providing us with the antisera to α2, α4 and α6 nAChR subunits.

Abbreviations used

nAChR

neuronal nicotinic acetylcholine receptor

E19

embryonic day 19

HBSS

Hank’s solution

[125I]EB

[125I]Epibatidine

[3H]EB

[3H]Epibatidine

[35S]Met

[35S]Methionine

CHX

cycloheximide

TCA

trichloroacetic acid

mAb

monoclonal antibody

AB

antibody

NRS

normal rabbit serum

Footnotes

Portions of this work were previously presented as an abstract at the Society for Neuroscience annual conference in 2010 (38.18).

The authors declare that they have no conflicts of interest.

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