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Division of Pharmacology, College of Pharmacy (R.B.F., S.B.M., D.B.M.)
Department of Neuroscience, College of Medicine and Public Health (R.T.B.), The Ohio State University, Columbus, Ohio
Department of Molecular Genetics and Microbiology, The University of Texas at Austin, Austin, Texas (P.D.G.)
Abstract
Several pathological conditions involve alterations in expression of neuronal nicotinic acetylcholine receptors (nAChRs). Although some studies have addressed processes involved with muscle nAChR expression, knowledge of the regulation of neuronal nAChRs is particularly sparse. The following studies were designed to investigate cellular mechanisms involved with expression of neuronal 34 nAChRs. Catecholamine secretion assays and receptor binding studies coupled with receptor alkylation were used to study the nAChR regulation and turnover. Alkylation of adrenal nAChRs results in a rapid and complete loss of receptor-mediated neurosecretion and surface [3H]epibatidine binding sites. After alkylation, both neurosecretory function and nAChR binding slowly (24eC48 h) return to prealkylation levels. When cells are treated with the protein synthesis inhibitor puromycin, after alkylation, receptor-mediated neurosecretion does not recover. Long-term treatment (24eC48-h) with puromycin, in the absence of alkylation, results in a slow, time-dependent shift to the right, followed by a downward shift, in the nicotine concentration-response curve, documenting a disappearance of surface nAChRs. Puromycin treatment alone also results in a loss to both surface and intracellular [3H]epibatidine binding sites. nAChR 4 subunit levels are significantly decreased after treatment with puromycin. These data support a constitutive turnover of adrenal 34 nAChRs, requiring continual de novo synthesis of new receptor protein.
Nicotinic acetylcholine receptors (nAChRs) are ligandgated ion channels that have essential physiological roles in the central and peripheral nervous systems and are thought to be the primary mediators of nicotine addiction. Neuronal nAChRs have also been associated with a variety of other neurological disease states, all of which involve changes in expression or distribution of the nAChRs (Lindstrom, 1997). Several processes have been described that regulate neuronal nAChR expression and directly influence their functional activity. These processes have a temporal component. Loss of receptor function via desensitization occurs rapidly, usually within seconds or minutes. Receptor down-regulation develops over several hours. Receptor up-regulation occurs with some neuronal nAChR subtypes and also develops over several hours. Finally, tolerance and dependence generally take days to weeks to develop. Several mechanisms are probably involved with these processes, including alterations in receptor internalization, receptor recycling, receptor degradation, and/or receptor synthesis.
At least three separate steps in the receptor expression/turnover process may regulate the number of nAChRs expressed on the cell surface. These include alterations in nAChR formation and assembly (Mitra et al., 2001), nAChR transport to the cell surface (Rothhut et al., 1996; Keller et al., 2001), or nAChR stabilization in the cellular membrane (Peng et al., 1994). The rate-limiting processes involved in nAChR turnover remain to be determined and may vary with different disease conditions. For example, stabilization within cellular membranes is thought to occur during prolonged agonist exposure, resulting in nAChR up-regulation (Peng et al., 1994, 1997), a condition seen in tobacco addiction. However, a decrease in nAChR expression in specific brain regions is associated with Alzheimer's disease (Warpman and Nordberg, 1995; Hellstrom-Lindahl et al., 1999). Understanding how neurons regulate the expression of nAChRs may lead to methods for manipulating nAChR levels that could be used in the treatment of several conditions, including Alzheimer's disease.
Few studies have addressed the question of surface trafficking or turnover of neuronal nAChRs. Several studies have investigated the surface trafficking of muscle nAChRs (e.g., Marchand et al., 2002). Neuronal nAChRs may traffic in a manner similar to their muscle counterpart. However, because multiple heteromeric (34, 354, 3524, 42, and 452) and homomeric (7, 8, and 9) subtypes (Lukas et al., 1999) of neuronal nAChRs are expressed, the mechanisms for regulation and trafficking could vary between subtypes. The "rules" that govern the trafficking and surface expression of any nAChR subtype have not yet been defined. Most of what is known about nAChR trafficking and turnover comes from studies using transfected cells or oocytes. The interpretation of results using cell lines is complicated by the finding that receptor expression and assembly are both host cell- and receptor subtype-dependent (Sweileh et al., 2000). Furthermore, marked differences in surface trafficking have been seen when comparing native with recombinant systems expressing 7 nAChRs (Kassner and Berg, 1997). These findings highlight GAPs in the understanding of neuronal nAChR regulation and indicate the importance of using native systems to examine questions relating to receptor regulation.
The ability to study neuronal nAChRs in their native environment and directly correlate cellular and molecular changes in nAChRs with changes in neurosecretion is a distinct advantage of using primary cultured bovine adrenal chromaffin cells. Evidence exists that these cells contain multiple populations of neuronal nAChRs. The 7-containing nAChRs are thought to be expressed (Wilson and Kirshner, 1977; Garcia-Guzman et al., 1995) and functional (Lopez et al., 1998) on adrenal chromaffin cells, but the primary nAChR subtype responsible for catecholamine release in these cells is the mAb35 nAChR (Gu et al., 1996). Binding studies support the characterization of these nAChRs as 34 nAChRs (Free et al., 2002). The demonstration that bovine chromaffin cells contain mRNA for 3, 5, and 4 nAChRs further supports this subunit composition (Criado et al., 1992; Garcia-Guzman et al., 1995; Campos-Caro et al., 1997; Wenger et al., 1997; Free et al., 2002). Therefore, cultured bovine chromaffin cells present an ideal model to investigate regulation of native neuronal nAChRs.
Our laboratory has recently demonstrated that cultured adrenal chromaffin cells contain a substantial population of fully assembled intracellular nAChRs (Free and McKay, 2003). The studies presented here seek to determine the contribution of preassembled intracellular pools of nAChRs to the complement of functional surface receptors and to deduce the mechanisms by which chromaffin cells regulate 34 nAChR surface expression. Our data support the necessity of constitutive de novo protein synthesis for the regulation of neuronal nAChR expression and suggest that the preassembled intracellular pool may not play a primary role in the normal maintenance of 34 nAChR surface expression.
Materials and Methods
Materials. Nicotine hydrogen tartrate, puromycin, 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB), and components of N2+ media were obtained from Sigma-Aldrich (St. Louis, MO). Dithiothrietol was purchased from Amersham Biosciences Inc. (Piscataway, NJ). Dulbecco's modified Eagle's medium (DMEM) and DMEM/Ham's F-12 were obtained from Invitrogen (Carlsbad, CA). Bromoacetylcholine bromide was purchased from Sigma/RBI (Natick, MA). [3H]Norepinephrine ([3H]NE, specific activity 12.0eC15.0 Ci/mmol) and (±)-[5,6-bicycloheptyl-3H]epibatidine (specific activity 66.6 Ci/mmol) were purchased from PerkinElmer Life and Analytical Sciences (Boston, MA). mAb35 (anti-acetylcholine receptor monoclonal antibody) was obtained from a hybridoma cell line purchased from American Type Culture Collection (Manassas, VA). The cells were cultured and the antibody was concentrated and purified using techniques described previously (Gu et al., 1996).
Isolation and Primary Culture of Bovine Adrenal Chromaffin Cells. Adrenal chromaffin cells were dissociated from intact glands and plated in supplemented DMEM, as described previously (Maurer and McKay, 1994). Cells were plated on 24-well plates at a density of 1 to 2 x 105 cells per well for functional studies and 5 x 105 cells per well for intact cell binding studies. Two days after plating, media were replaced with a modified, serum-free N2+ medium described previously by our laboratory (Maurer and McKay, 1994). DMEM and N2+ media were supplemented with 250 ng/ml amphotericin B, 100 U/ml penicillin, 100 e蘥/ml streptomycin, 2 mM L-glutamine, and 10 e 5-fluoro-2'-deoxyuridine. One day before experimentation, the culture medium was removed and replaced with medium free of amphotericin B and 5-fluoro-2'-deoxyuridine. Cells were used 4 to 7 days after isolation.
Catecholamine Secretion Studies. A [3H]NE assay was used to monitor catecholamine release from cultured cells (McKay and Schneider, 1984). The amount of radioactivity released after a 5-min incubation with nicotine (stimulated release) or without nicotine (basal release) was determined using liquid scintillation spectroscopy. The radioactivity remaining in the cells was extracted with 8% trichloroacetic acid and also quantified. Results are expressed as either a percentage of the total incorporated [3H]NE released under the treatment conditions or as a percentage of net release, which is equal to percentage of the total minus percentage of basal (nonstimulated) release. When 56 mM KCl was used to stimulate catecholamine release, the sodium concentration of the buffer was reduced to maintain isotonicity.
Turnover Studies. For nAChR turnover studies, two methods were used to examine nAChR turnover. First, adrenal nAChRs were irreversibly inactivated via alkylation with bromoacetylcholine using techniques previously published by our laboratory (Gu et al., 1996; Wenger et al., 1997; Free and McKay, 2001). In brief, cells were treated with 1 mM dithiothreitol for 15 min at 37°C to reduce receptor disulfide bonds. Cells were then washed with a physiological salt solution for 15 min and then treated with 100 e bromoacetylcholine for 6 min at room temperature. After washing (15 min), the disulfide bonds were reoxidized via treatment with 1 mM DTNB for 15 min at 37°C. After a 5-min wash, cells were placed back into N2+ media for long-term studies. Second, adrenal nAChRs were treated with the monoclonal antibody mAb35 (50 nM) by adding it to the culture media for 24 h. Cells were washed and placed back in N2+ media to allow for recovery.
[3H]Epibatidine Binding to Intact Bovine Adrenal Chromaffin Cells in Culture. Binding to adrenal nicotinic receptors in intact cells was performed using techniques described previously by our laboratory (Free and McKay, 2003). In brief, cells were incubated for 60 min at room temperature in binding buffer containing 2 nM [3H]epibatidine and 1 e -bungarotoxin to eliminate binding to -bungarotoxin binding sites. After the 60-min incubation, the binding buffer was aspirated, and the cells were rapidly washed. Cells were extracted in 1 M NaOH, and the cells scraped from the plates. The cellular extracts were neutralized and counted using liquid scintillation spectroscopy. Nonspecific binding was determined in the presence of 300 e nicotine and typically represented 50 to 55% of the total binding. When binding to only surface nAChRs was investigated, nonspecific binding was determined in the presence of 5 mM carbachol, an impermeant cholinergic receptor agonist. Under these conditions, nonspecific binding usually represented 65 to 75% of total binding. This higher value probably represents a combination of nonspecific binding and specific intracellular binding (Free and McKay, 2003).
Production of Peptide Conjugates and an Anti-Bovine 4 Polyclonal Antibody. Amino acid sequences of the intracellular loops of bovine 3, 5, 7, and 4 receptors were aligned for homology, and sequences unique to 4 were identified by inspection. Peptides were synthesized with a carboxyl-terminal cysteine for conjugation via the thiol group, and purity was assessed using mass spectroscopy and capillary electrophoresis. The 22-mer 4 peptide (RPRQQPSRAPQSSLARLTKSEC) was conjugated with keyhole limpet hemocyanin (Calbiochem, San Diego, CA) using sulfo-succinimidyl 4-[N-maleimidomethyl] cyclohexane-1-carboxylate (Pierce Chemical, Rockford, IL) as described previously (Hermanson, 1996). The conjugates were dialyzed against phosphate buffer, pH 7.3, and frozen before use at a concentration of 1.4 to 2.25 mg/ml. Peptide synthesis, analysis, and conjugation were done in the Protein Core facility (Institute for Cell and Molecular Biology, University of Texas at Austin, Austin, TX). Immunizations were performed by Cocalico Biologicals, Inc. (Reamstown, PA). Each conjugate was thawed, agitated to resuspend any precipitated protein, emulsified with complete Freund's adjuvant, and injected into multiple sites on the backs of two rabbits. Subsequent injections were given as emulsions with incomplete Freund's adjuvant at approximately 2-wk intervals, and bleeds were tested for activity and specificity by Western blot against endogenous and recombinant receptor proteins.
Western Blot Analyses. Equivalent amounts of protein were separated in 10% SDS-polyacrylamide gels and then electroblotted onto Hybond membranes (Amersham Biosciences UK, Ltd., Little Chalfont, Buckinghamshire, UK). The blots were probed overnight at 4°C with a 1:2500 dilution of polyclonal antibody to the bovine 4 peptide as described above. Bound antibodies were detected using a 1:2500 dilution of an anti-rabbit IgG linked to horseradish peroxidase (Amersham Biosciences Inc.). Western blots were probed and detected according to the manufacturer's instructions, using enhanced chemiluminescence (ECL; Amersham Biosciences UK, Ltd.). Blots were visualized using a Biochemi imaging system (UVP, Inc., Upland, CA). Molecular weights were determined using LabWorks imaging software (UVP, Inc.).
Northern Blot Analyses. Chromaffin cells grown on 60-mm dishes either were not treated, alkylated with bromoacetylcholine, or were alkylated and allowed to recover for 24 or 48 h. RNA was isolated by TRIzol (Invitrogen). Northern blot analysis was performed using 1% (w/v) agarose gels containing 7.4% (v/v) formaldehyde in 20 mM MOPS, 1 mM EDTA, and 5 mM sodium acetate at pH 7.0. Equal amounts of RNA were run on each lane of the gel. After electrophoresis, the RNA was transferred to GeneScreen Plus (PerkinElmer Life and Analytical Sciences) in 10x standard saline citrate according to the manufacturer's instructions. Bovine 3, 5, and 4 and mouse GAPDH (Ambion, Austin, TX) cDNAs were labeled with [-32P]dCTP using the Prime-It Rm T random primer labeling kit (Stratagene, La Jolla, CA). The 32P-labeled probes were hybridized to the GeneScreen Plus membrane in 5x SSPE, 50% deionized formamide, 5x Denhardt's solution, 1% SDS, 10% dextran sulfate, and 100 mg/ml salmon sperm DNA at 42°C. The filters were washed in 2x SSPE at room temperature, 2x SSPE, 2% SDS at 65°C, 0.1x SSPE for 45 min, and 0.1% SDS at room temperature for 15 min. The blots were exposed to X-ray (XAR-5; Eastman Kodak, Rochester, NY) film at eC70°C with an intensifying screen. An Amersham Biosciences PhosphorImager was used to quantify the signal intensities in each lane. The nAChR signals were normalized to the GAPDH signals to measure relative changes in mRNA levels.
Data Analyses. Results were calculated from the number of observations (n) performed in duplicate (intact cell binding studies) or triplicate (catecholamine secretion studies). All experiments were performed on two to four different cell isolations. Experimental values were compared using the t test (p < 0.05) or Dunnett's multiple comparison test (p < 0.05), as indicated.
Results
Paradigms to Investigate nAChR Turnover. Two distinct paradigms were used to investigate nAChR turnover processes. The first paradigm involves irreversible alkylation of surface nAChRs via treatment with the cell impermeant alkylating agent bromoacetylcholine. Previous studies from our laboratory have demonstrated that nAChR alkylation with bromoacetylcholine results in a rapid and complete loss of nAChR-mediated catecholamine release from bovine adrenal chromaffin cells (Gu et al., 1996; Wenger et al., 1997). The second paradigm involves antigenic modulation via treatment with the anti-nAChR antibody mAb35 and results in a partial loss of nAChR function (Gu et al., 1996).
[3H]Epibatidine binding experiments (using 2 nM epibatidine, 80% receptor occupancy) on intact adrenal chromaffin cells were designed to directly investigate the ability of alkylation to eliminate surface nAChRs. Intact cell binding experiments allow for the examination of changes to 1) total nAChRs (Rt, surface plus intracellular nAChRs), 2) surface nAChRs (Rs), and 3) intracellular nAChRs (Ri). Previous studies from our laboratory document the feasibility of this approach (Free and McKay, 2003). As seen in Fig. 1, alkylation produced a 40% loss of binding to Rt and a nearly complete loss of binding to Rs. Alkylation had no effect on binding to Ri (data not shown). When the cells were allowed to recover for 48 h after alkylation, binding to both Rt and Rs significantly increased to near prealkylation levels (Fig. 1). These binding studies document the nearly complete loss of surface adrenal nAChRs after alkylation and their recovery from alkylation within 48 h. Treatment with mAb35 resulted in a partial loss of surface receptor expression (data not shown), consistent with functional data (Gu et al., 1996; Wenger et al., 1997).
Transcriptional Consequences of the Loss of Functional Surface nAChRs. We investigated the transcriptional consequences of the loss of functional surface nAChRs via alkylation on nAChR mRNA levels. mRNA was isolated from cultured adrenal chromaffin cells immediately after nAChR alkylation (recovery 0 h) and 24 and 48 h after alkylation (i.e., recovery from alkylation). As demonstrated in Fig. 2, no significant changes were observed in 3, 5, and 4 nAChR subunit mRNA levels either immediately after nAChR alkylation and during the recovery period (24 and 48 h). Untreated cells maintained an unchanging level of nAChR subunit mRNAs expression during this time period (data not shown). These data demonstrate that mRNA levels are not likely to be regulated after the loss of functional nAChRs via alkylation, consistent with a constant rate of 34 nAChR subunit protein synthesis.
Effects of Puromycin on Recovery of nAChR-Mediated Functional Responses: Translational Effects. To investigate the importance of protein synthesis in the recovery of nAChRs after the loss of functional nAChRs via alkylation, the protein synthesis inhibitor puromycin (10 e蘥/ml) was included in the media during recovery from nAChR alkylation. As seen in Fig. 3A, alkylation of cultured chromaffin cells resulted in a complete loss of functional nAChRs, as documented by a decrease in nAChR-mediated catecholamine release. Significant recovery of the functional response is observed after allowing cells to incubate in media alone for 24 h after receptor alkylation. This functional recovery is prevented by inclusion of the protein synthesis inhibitor puromycin in the recovery media (Fig. 3A), implicating the need for protein synthesis.
To investigate whether puromycin's effects were specific for alkylation-induced functional recovery, similar experiments were conducted after functional down-regulation via antigenic modulation using the anti-nAChR antibody mAb35. As seen in Fig. 3B, treatment of cultured chromaffin cells with mAb35 resulted in a partial loss of secretory function that significantly recovered 24 h after removal of the antibody. With puromycin present during the recovery period, the return of function was completely inhibited (Fig. 3B). It is important to note that catecholamine release stimulated by depolarizing concentrations of KCl was not affected by treatment (24 h) with puromycin (15.2 ± 1.7 and 15.5 ± 2.3%, respectively). These data provide evidence for the effects of puromycin on nAChR recovery and not on a distal step in the stimulus-secretion pathway. These studies support the need for de novo protein synthesis in recovery of nAChR-mediated catecholamine release following two mechanistically distinct paradigms that eliminate functional surface nAChRs.
Involvement of Constitutive nAChR Turnover in Recovery Processes. In addition to their sensitivity to protein synthesis blockade, the two paradigms show similar recovery rates (Gu et al., 1996; Wenger et al., 1997). The above-mentioned findings indicate that recovery of nAChR-mediated neurosecretion may be caused by constitutive nAChR turnover, rather than stimulated nAChR synthesis. Thus, inhibition of new protein synthesis should decrease the number of functional surface nAChRs. To address this hypothesis, the effects of puromycin treatment on nicotine-stimulated catecholamine release were investigated. As seen in Fig. 4A, when cells were treated with 10 e蘥/ml puromycin for 24 and 48 h, there was a time-dependent, rightward shift, followed by a downward shift of the nicotine concentration-response curves. These shifts of agonist concentration-response curves are typically seen when receptors are lost in cells expressing spare receptors, as we have demonstrated previously in adrenal chromaffin cells (Wenger et al., 1997), and they do not represent actual changes in receptor affinity (for review, see Ruffolo, 1982). These effects of puromycin were also reversible. Figure 4B demonstrates a leftward and upward shift of the nicotine concentration-response curves after puromycin was removed, until full recovery of secretory function returned after 48 h. These data are consistent with a loss of surface receptors followed by a return of surface receptors. These data demonstrate the necessity of de novo protein synthesis for the maintenance of functional 34 nAChRs and support constitutive nAChR turnover.
Effects of Puromycin Treatment on [3H]Epibatidine Binding Sites. To directly address the importance of de novo protein synthesis in maintaining both the surface and intracellular pools of nAChRs, [3H]epibatidine binding experiments (2 nM, 80% receptor occupancy) were performed on intact cells. Puromycin treatment for 24 h significantly decreased the number of [3H]epibatidine binding to both Rt and Rs (Fig. 5A). Binding to Ri was also significantly decreased with puromycin treatment (Fig. 5B). These effects of puromycin were reversible. After a 48-h recovery period, both Rs and Ri sites significantly recovered (Fig. 5), albeit to apparently slightly different degrees. These data indicate that de novo protein synthesis is necessary for the maintenance of both surface and intracellular nAChRs.
Effects of Puromycin Treatment on 4 nAChR Protein Levels. Binding and functional studies confirm that puromycin treatment decreases the expression of Rs. However, these studies do not rule out the potential that puromycin treatment is affecting a protein independent of the nAChR protein, such as a protein needed for nAChR assembly or trafficking. To address this issue, the ability of puromycin to inhibit the synthesis of nAChR protein was investigated.
To examine changes in nAChR protein, a novel anti-bovine 4 nAChR polyclonal antibody was created for use in Western blots to measure bovine 4 nAChR subunit levels. To ensure that specificity of this polyclonal antibody, it was tested using HEK 293 cells heterologously expressing individual, (3, 5, and 4) bovine nAChR subunits. As demonstrated in Fig. 6, the antibody bound to a single band in the HEK 293 cell line transfected with the bovine 4 nAChR subunit protein. No proteins were detected in either untransfected, control HEK 293 cell lines, or those expressing either the bovine 5 or 3 nAChR subunit protein.
In native bovine adrenal chromaffin cells the anti-bovine 4 polyclonal antibody detected three proteins (52.5, 51, and 48 kDa) in the range of predicted molecular mass for the 4 subunit (Fig. 7, left-most lane). The observation of differences in apparent stability of the three proteins is unknown, but it may be caused by differences in specific degradation rates of the proteins or differences in susceptibility to proteases. Differences in size of the band seen when the 4 subunit was expressed in HEK cells compared with the multiple bands observed in chromaffin cells may be caused by differences in cell-specific subunit protein modifications. To determine the ability of puromycin treatment to inhibit the synthesis 4 protein, Western analysis was conducted on protein isolated from cells treated with puromycin for either 24 or 48 h. Figure 7 shows a representative blot of cultured cell extract, demonstrating significantly decreased 4 nAChR subunit levels after treatment of the cells with puromycin for 24 and 48 h. These data are consistent with puromycin acting to reduce the synthesis of adrenal nAChRs.
Discussion
Despite the importance of nAChR turnover and regulation in a number of pathological conditions, very few studies have examined such mechanisms in native systems. We hypothesized that perturbation of surface nAChRs results in a compensatory increase in the surface expression of nAChRs. Contrary to our hypothesis, we found that nAChR antigenic nAChR modulation or loss of functional nAChRs do not produce compensatory increases in transcriptional or translational processes and that constitutive turnover accounts for replacement of surface nAChRs. We also found that de novo protein synthesis is necessary for the replacement of surface nAChRs and have no evidence that intracellular pools of nAChRs play a role in surface expression under our conditions of nAChR down-regulation.
These studies are important because very little is known about processes mediating surface expression of functional neuronal nAChRs. In addition, gaining a better understanding of mechanisms that regulate expression of functional nAChRs receptors may be relevant to ameliorating the loss of nAChRs that is seen with Alzheimer's disease, where nAChR receptor loss or down-regulation is occurring. The majority of neuronal nAChR regulation studies investigated nAChR up-regulation in response to long-term agonist treatment. These studies have resulted in a better understanding of nAChR subtype-specific sensitivity to up-regulation (Flores et al., 1997; Olale et al., 1997) and up-regulatory mechanisms (Peng et al., 1994; Fenster et al., 1999). However, they have not clearly addressed the underlying mechanisms of nAChR turnover and how cells respond to receptor down-regulation. One important observation from long-term agonist treatment studies is that different nAChR subtypes may be differentially regulated both in terms of response to long-term agonist treatment (Olale et al., 1997; Wang et al., 1998) and native versus recombinant systems (Sweileh et al., 2000). Paradoxical results for up-regulation studies using 34 nAChRs have been reported in recombinant systems (Wang et al., 1998; Meyer et al., 2001). These findings highlight the difficulties of studying 34 nAChR turnover using up-regulation paradigms and indicate the necessity of additional methods for investigating 34 nAChR turnover. The current studies seek to define approaches for studying nAChR regulation in native systems and to identify regulatory events for 34 nAChRs.
To address questions regarding turnover of native 34 nAChRs, two paradigms were used: nAChR alkylation and nAChR antigenic modulation. Our laboratory has previously characterized these paradigms using functional (neurosecretion) assays (Gu et al., 1996; Wenger et al., 1997) We have now coupled these paradigms to direct radioligand binding assays to define changes in both surface and intracellular nAChRs for the study of nAChR turnover (Free and McKay, 2003). In the studies presented here, both alkylation and mAb35 treatment produced decreases in surface nAChR binding, which recovered with time to control levels. These results parallel previously published functional data (Gu et al., 1996; Wenger et al., 1997). Receptor alkylation is a useful paradigm for studies involving expression of 34 nAChR subtypes because alkylation results in a complete loss of nAChR-mediated function and [3H]epibatidine binding. This contrasts with the antigenic modulation paradigm that results in an incomplete reduction in nAChR-mediated neurosecretion (Gu et al., 1996; Wenger et al., 1997) and a correspondingly smaller reduction in [3H]epibatidine binding to surface nAChRs. These findings support the use of these mechanistically distinct paradigms to investigate 34nAChR turnover and indicate that they may be useful to differentiate populations of nAChRs.
Because receptor alkylation caused a loss of surface receptors, we hypothesized that chromaffin cells might respond by increasing transcription of neuronal nAChR subunit genes. When adult muscle is denervated, increased synthesis of extrajunctional nAChRs occurs with an increase in subunit RNAs (Linden and Fambrough, 1979; Goldman et al., 1988). During myogenesis, increased expression of surface nAChRs coincides with increases in nAChR subunit mRNAs (Evans et al., 1987). In contrast to what is observed for muscle nAChRs, no increases in transcript levels for 3, 4, or 5 subunits were observed over the 48-h period. Although it seems that transcriptional regulation does play a vital role in both developmental and basal levels of nAChR expression, very little evidence supports transcriptional regulation playing a role in changes in receptor expression during disease. Most nAChR mRNA levels are not altered in Alzheimer's disease or by nicotine exposure (Marks et al., 1992; Nordberg, 2001). These findings suggest that the primary mechanism of change in nAChR expression is probably via post-transcriptional or trafficking modifications. Our findings are consistent with this in that we see no changes in mRNA levels after loss of functional nAChRs, supporting a lack of transcriptional regulation.
We found that inhibition of protein synthesis was able to prevent recovery of nAChR-mediated catecholamine secretion. When protein synthesis was inhibited in these cells, a reduction in surface nAChR binding sites was observed. In addition, puromycin treatment caused loss of nAChR-mediated function and a time-dependent shift in the nicotine concentration-response curve. These effects were not caused by a loss of cells or a generalized toxicity because, when the inhibitor was removed, both nAChR function and binding returned to pretreatment levels. Previous studies have also shown that in the continued presence (48 h) of the protein synthesis inhibitors actinomycin D or cycloheximide, cell survival for cultured chromaffin cells is at least 70 to 80% (Cardenas et al., 1995). In addition, puromycin treatment did not affect secretion stimulated by depolarizing concentrations of KCl. These findings support the necessity for de novo protein synthesis in the return of functional receptors to the surface after down-regulation, implying a lack of receptor recycling and the need for continual replacement as a result of nAChR turnover on the cell surface.
In addition, our results using puromycin treatment alone indicate that constitutive de novo protein synthesis is also required to maintain normal levels of nAChRs. This is interesting in that adrenal chromaffin cells have a large pool of intracellular nAChRs, 2 to 3 times the number of surface nAChRs (Free and McKay, 2003), which might be expected to move to the surface in response to down-regulation or as part of the normal turnover/cycling process. The data presented here suggest that the intracellular pool of receptors does not play a primary role in recovery after alkylation or in normal turnover because new receptor synthesis is required in both cases. These findings are however consistent with previous studies that suggest only a small percentage of the intracellular binding sites in chick ciliary ganglion neuron (Stollberg and Berg, 1987) or muscle cells (Pestronk, 1985) are destined for surface expression. The precise physiological implications of intracellular nAChRs remain unclear. These intracellular nAChRs may consist of two distinct pools: nAChRs destined for immediate surface expression and nAChRs stored as an independent pool. Intracellular nAChR pools in other tissues (Stollberg and Berg, 1987) and recombinant cell lines (Whiteaker et al., 1998) can be modulated (Rothhut et al., 1996) and may be involved with the receptor turnover process. It is also possible that these intracellular nAChRs may represent internalized receptors that were once on the receptor surface. This is unlikely, however, because they maintain a high affinity for [3H]epibatidine (Free and McKay, 2003). Internalized receptors would be expected to degrade more rapidly when they are internalized (Xu and Salpeter, 1999) and therefore would not be likely to represent a significant amount of high-affinity binding. Furthermore, it is not likely that newly internalized old receptors would be sensitive to puromycin, as is the 34 intracellular nAChR pool. Another intriguing possibility is that the intracellular receptors lack the necessary signals to be targeted for surface expression and are therefore shunted away from the trafficking pathway. This contention is supported by studies showing muscle nAChRs must interact with certain regulatory elements in the endoplasmic reticulum to be directed to the cell surface (Keller et al., 2001). Regardless, our data suggest that intracellular 34 nAChRs do not contribute to normal surface nAChR turnover and probably are an independent pool.
Constitutive nAChR turnover is supported by puromycin's effects on both surface and intracellular nAChRs and on secretion. However, in addition to its effects on the synthesis of nAChR protein, puromycin treatment may also affect the synthesis of other proteins possibly involved with trafficking, insertion, or internalization of adrenal 34 nAChRs. Our data do not indicate that puromycin significantly affects proteins involved with receptor internalization because we have previously documented an internalization rate for mAb35-induced turnover (Wenger et al., 1997) that followed a similar time course as loss of surface receptors after puromycin treatment. These studies, however, do not rule out puromycin having effects on synthesis of a trafficking/chaperone or assembly proteins, because several studies have demonstrated that nAChRs interact with chaperone proteins (Keller et al., 1996; Jeanclos et al., 2001). We would hypothesize that because the cell makes many more fully assembled nAChRs than are inserted on the cell surface (Free and McKay, 2003), a chaperone/trafficking protein probably exists and may represent the limiting regulatory factor for shunting nAChRs to the cell surface. Regardless of any effects on other proteins, our Western analyses document that puromycin treatment lowers the levels of 4 subunit protein under our treatment conditions.
Based on our findings, we propose a model for surface expression of 34 nAChRs (Fig. 8). Surface expression of nAChRs is dependent on constitutive turnover. Adrenal cells contain a large intracellular population of nAChRs that bind epibatidine with high affinity, whose function is not understood (Free and McKay, 2003) (Fig. 8, dotted box). This intracellular nAChR pool, as well as the nAChRs expressed on the cell's surface, depends on protein synthesis (step 2). The predominant route of nAChR surface expression is via step 3 (Fig. 8) and is dependent on de novo protein synthesis. We found no evidence that preformed, intracellular pools of nAChRs are involved with surface expression (step 5); however, we cannot definitively rule this possibility out. Based on data in muscle nAChRs, it is not likely that endocytosis of surface nAChRs (step 6) contributes significantly to the intracellular pool of nAChRs.
In summary, our findings highlight the importance of continual turnover of 34 nAChRs and demonstrates that lack of receptor recycling and the necessity of de novo protein synthesis in the maintenance on surface nAChRs. Because of the involvement of nAChRs in nicotine addiction and their possible roles in a number of disease states, a more thorough understanding of cellular and molecular mechanisms involved with nAChR expression, down-regulation, and turnover are of critical importance. These studies significantly add to the current understanding of how neuronal cells regulate nAChR number and turnover. Further investigation of this pathway will probably provide novel targets for the manipulation of receptor number such as chaperone proteins or other post-translational modulators that underlie receptor trafficking.
Acknowledgements
Paul D. Gottlieb died during the preparation of this manuscript after a brief illness. He was a valued friend and mentor and will be sorely missed.
This project was supported by National Institutes of Health grant DA10569 (to D.B.M.).
doi:10.1124/mol.104.009282.
1 Current address: Molecular Neuropharmacology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892-9405.
References
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