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INTRODUCTION |
Nicotinic acetylcholine receptors
(nAChRs)1 containing 
3
subunits are found in autonomic ganglia where they have been found in
various combinations with 
2, 4, and 5 subunits (1-3). They have also been found in brain (4), adrenal gland (5), thymus (6),
respiratory epithelial cells (7), and keratinocytes (8, 9). In ganglia,
3 AChRs play a postsynaptic role similar to that of muscle AChRs (1,
2). 3 AChRs also have been found in various presynaptic locations in
rat brain and implicated in promoting neurotransmitter release
(e.g. in dopaminergic and noradrenalinergic pathways)
(10-15). We have previously studied the subunit compositions and the
pharmacological properties of human 3 AChR subtypes using the
Xenopus oocyte expression system and the human peripheral
neuroblastoma cell line SH-SY5Y (16-19). We describe here our study of
human 3 AChRs stably expressed in a derivative of human embryonic
kidney (HEK293) cells. Compared with the Xenopus oocyte
expression system, stable mammalian cell lines provide several
advantages. 1) We can obtain more reproducible and long lasting levels
of AChR expression that are required for detailed single channel
studies without the seasonal vagaries of transient expression in
Xenopus oocytes. 2) We can better study the effects of
chronic exposure to ligands such as nicotine than is convenient
in a transient expression system like Xenopus oocytes. 3)
Cell lines expressing various AChR subtypes provide a potential for
drug discovery by high-throughput screening. Previously, others have
reported in article or abstract form permanently transfected cell lines
expressing rat 34 (20, 21) or human 32 (20, 21), 34
(23), and 325 AChRs (24). This is the first detailed report of
a matched set of four cell lines expressing human 32,
325, 34, or 345 AChRs.
One important application of human 3 AChR cell lines is to study the
effect of chronic nicotine exposure. Addiction to nicotine is
characterized by up-regulation of nAChRs in brain (25-27). The number
of high affinity nicotine-binding sites in the brains of tobacco
smokers and animals chronically given nicotine is increased up to
2-fold (26-30). Most of this increase is in 42 AChRs (30), but
the amount of increase varies between brain regions and probably involves several AChR types (28, 29). It has been hypothesized that
smoking a cigarette results in a rapid bolus of nicotine that activates
the mesolimbic dopaminergic system producing pleasure and reward and
that nicotine in the smoker's brain slowly builds to a low steady
concentration which causes both reversible desensitization and long
term inactivation of some AChR subtypes as well as increases in the
amount of some AChR subtypes (19, 31-33). An understanding of the
effects of chronic exposure to nicotine on the amount and functional
activity of various AChR subtypes might provide better insight into
mechanisms of nicotine dependence, tolerance, and withdrawal, as well
as the effects of medication with nicotinic drugs.
Chronic exposure to nicotine has been shown to differentially affect
both the amount and function of neuronal AChR subtypes. 42 AChRs
expressed in Xenopus oocytes or a permanently transfected cell line were shown to double in amount when chronically exposed to
submicromolar concentrations of nicotine (34). These concentrations of
nicotine eliminated most 42 AChR function due to permanent desensitization (19, 34). On the other hand, a mixture of 3 AChRs
expressed by the human neuroblastoma cell line SH-SY5Y increased by
600% in response to chronic exposure to very high concentrations of
nicotine (18). Micromolar concentrations of nicotine blocked only a
small part of 3 AChR function (19). It has been suggested that some
of the behavioral effects of nicotine are likely to depend on the
inactivation of both 42 and 7 AChRs while leaving 3 AChRs
and other subtypes available to respond to endogenous ACh-mediated
signaling and to the micromolar boluses of nicotine that occur after
inhaling smoke (19, 35). Like mammalian cell lines expressing 42
AChRs (34, 36) or 7 AChRs (37), cell lines stably expressing various
subtypes of 3 AChRs provide excellent tools for studying the
mechanisms and physiological significance of nicotine up-regulation of
human 3 AChRs. Here we provide an initial description of these cell lines, including pharmacological and electrophysiological
characterization of their AChRs, and study the effects of chronic
nicotine exposure on the amount of these AChRs.
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EXPERIMENTAL PROCEDURES |
cDNAs, mAbs, and Antisera--
The cDNAs for human 3,
2, and 4 subunits were cloned in this laboratory and described in
our previous report (16). The cDNA for human 5 was kindly
provided by Dr. Francesco Clementi (see Ref. 38). The cDNA for the
human 3 subunit was subcloned into the selective mammalian
expression vector pcDNA3.1/Zeo(+)(Invitrogen), which carries the
ZeocinTM resistance gene. The cDNAs for human 2 and
4 subunits were subcloned into the expression vector pRc/CMV
(Invitrogen), which carries the neomycin resistance gene. The cDNA
for the human 5 subunit was subcloned into the expression vector
pCEP4 (Invitrogen), which carries the hygromycin resistance gene.
Monoclonal antibodies mAb210 to 1, 3, and 5 subunits (39),
mAb290 to 2 subunits (40), mAb268 to 5 subunits (41, 42), rabbit
antiserum 3709 to a synthetic peptide corresponding to amino acids
348-387 of human 3 subunits, and rabbit antiserum 3724 to a
synthetic peptide corresponding to amino acids 387-401 of human 2
subunits have been described previously (16). Mouse antiserum Hub4.4 to
human 4 subunits was raised against a fusion protein of human 4
subunit large cytoplasmic domain (amino acids 305-419) with bacterial glutathione S-transferase. The fusion protein was
constructed in the pGEX-4T-2 vector (Amersham Pharmacia Biotech) and
expressed in bacteria. It was affinity purified with a
glutathione-Sepharose 4B (Amersham Pharmacia Biotech) column before
being used to immunize mice. Hub4.4 does not cross-react with human
AChR subunits 3, 2, or 5 on Western blots (data not shown).
Rat antiserum to human 5 subunits was raised against a fusion
protein of human 5 subunit extracellular domain and large
cytoplasmic domain (43). The fusion protein was constructed in the
pET-26b(+) vector (Novagen) and expressed in bacteria. It was purified
by gel exclusion chromatography before being used to immunize rats. The
antiserum to 5 was absorbed with a resin-coupled fusion protein of
human 3 AChR subunit extracellular and cytoplasmic domains to remove
antibodies to conserved epitopes shared with 3 and other
subunits.
Cell Culture and Stable Transfection of Human tsA201
Cells--
Human tsA201 cells, a derivative of the human embryonic
kidney cell line 293 (44), were maintained in Dulbecco's modified Eagle's medium (high glucose) (Life Technologies, Inc.) supplemented with 10% fetal bovine serum (Hyclone), 100 units/ml penicillin, 100 µg/ml streptomycin (Life Technologies, Inc.), and 2 mM
L-glutamine (Life Technologies, Inc.) in a CO2
(5%) incubator at saturating humidity. The 32 cell lines were
established by co-transfecting tsA201 cells with
Hu3/pcDNA3.1/Zeo(+) and Hu2/pRc/CMV using LipofectAMINE
(Life Technologies, Inc.) following the manufacture's instructions.
The 34 cell lines were developed by co-transfecting tsA201 cells
with Hu3/pcDNA3.1/Zeo(+) and human4/pRc/CMV following the
same procedure. The 325 and 345 cell lines were
obtained by transfecting the established 32 and 34 cell
lines with Hu5/pCEP4. For the cloning of stably transfected cell
lines, 72 h after transfection, selection medium containing
Geneticin (600 µg/ml, Life Technologies, Inc.) and Zeocin (500 µg/ml, Invitrogen) was used for the 32 and 34 cell lines.
For the 325 and 345 cell lines, the third antibiotic
drug hygromycin (200 µg/ml, Boehringer Mannheim) was added to the
above selection medium. The initial screening for cell colonies
expressing functional 32 and 34 AChRs was based on a solid
phase radioimmunoassay (RIA) with [3H]epibatidine on
mAb210-coated Immulon 4 (Dynatech) microwells. Screening for
325 and 345 cell lines was performed by both RIA and
Western blot analysis with mAb268 (16) to detect the presence of the
5 subunit. Untransfected tsA201 cells were used as a negative
control for these assays. Chronic exposure of cells to nicotine and/or
other ligands (all purchased from Sigma) was performed by adding the
ligand(s) into the culture medium at the relevant concentration and
incubating the cells in the medium for the indicated duration of time
before harvesting the cells. Effects of the protein synthesis inhibitor
cycloheximide (Sigma) on nicotine-induced up-regulation were studied by
adding the chemical into the culture medium with or without the
presence of nicotine and incubating the cells in the medium for a
certain period of time before harvesting.
Isolation, Purification, and Immunoprecipitation of AChRs from
Cells--
Cells were suspended from 35-mm Petri dishes with buffer A
(in mM:
Na2HPO4·NaH2PO4, 50;
pH 7.5; NaCl, 50; EDTA, 5; EGTA, 5; benzamidine, 5; iodoacetamide, 15;
phenylmethylsulfonyl fluoride, 2) and pelleted by centrifugation. The
pellets were rinsed 3 times with buffer A before buffer C (buffer A
containing 2% Triton X-100) was used to solubilize the AChRs in the
cells (4 °C, 1 h). After removing cellular debris by
centrifugation, the cleaned extracts were incubated in mAb-coated
microtiter wells for solid phase RIA, or with mAb-coupled Actigel
(Sterogen) for purifying AChRs for use in immunoblot assays, or loaded
directly onto 5-ml sucrose gradients (5-20% sucrose, w/w) for
sedimentation analysis (16). For immunoprecipitation of AChRs with
subunit-specific antibodies, the extract was incubated with mAb or
antiserum in the presence of [3H]epibatidine (5 nM) for 12 h. The AChR-antibody complexes were immunoprecipitated with Zysorbin (Zymed Laboratories
Inc.) for mouse antibodies or sheep anti-rat IgG for rat
antibodies. [3H]Epibatidine-labeled AChRs in the pellet
were quantified using liquid scintillation counting. Nonspecific
precipitation was measured using either normal mouse serum or normal
rat serum. For filter binding assays, the AChRs in the extract (100 µl) were labeled with [3H]epibatidine (5 nM) at 4 °C for 12 h. Then the extract was diluted with 4 ml of 10 mM Tris-HCl, pH 7.5, 0.5% Triton X-100
before it was loaded onto Whatman GF/B filters (presoaked with 0.5%
polyethyleneimine). The filters were rinsed three times with 4 ml of 10 mM Tris-HCl, pH 7.5, 0.5% Triton X-100 to remove free
[3H]epibatidine. [3H]Epibatidine-labeled
AChRs on the filter were quantified using liquid scintillation
counting. Nonspecific binding was measured using extracts of
non-transfected tsA201 cells.
Cell-surface Labeling with 125I-mAb210 or mAb210 Plus
Fluorescein-labeled Goat Anti-rat IgG (F-GART)--
For surface
labeling with 125I-mAb210, cells in 35-mm Petri dishes were
rinsed with phosphate-buffered saline (PBS) before
125I-mAb210 (2 nM, 0.7-2.6 × 1018 dpm/mol, diluted in PBS, 10% fetal bovine serum) was
added into the dish. Labeling took place at 25 °C for 1 h.
Cells were then suspended from the dish and collected in
microcentrifuge tubes by centrifugation. The pellets were washed three
times with PBS to remove nonspecifically bound mAbs. The bound
125I-mAb210 was measured by 
-counting. For surface
labeling with mAb210 followed by F-GART (Kirkegaard & Perry
Laboratories, Inc.), cells were cultured on collagen-coated glass
coverslips. Cells were rinsed once with PBS before fixation in 2%
paraformaldehyde/PBS for 20 min at 25 °C. After fixation, cells were
rinsed 3 times with PBS before being incubated with mAb210 (30 nM) diluted in PBS containing 1% bovine serum albumin for
1 h at 25 °C. This was followed by 3 washes with PBS and
incubation for 1 h at 25 °C with F-GART (50 µg/ml). The cells
were then washed three times with PBS before being mounted in Pro-Long
(Molecular Probes). Digital photomicrographs were taken with a Leica
TCS 4D confocal microscope.
Electrophysiology--
Transfected cells were plated onto glass
coverslips coated with rat tail collagen (Type 1; Collaborative
Biomedical Products, Bedford, MA) at least 2 days prior to use.
Currents were monitored by standard whole cell patch clamp techniques
(45). Agonist-containing solutions were applied to the cells by gravity
fed fused glass tubing that was connected to multiple reservoirs
mounted above the recording chamber. The recording solution was
composed of the following (in mM): NaCl, 150; KCl, 5;
MgCl2, 1; CaCl2, 2; HEPES, 5; pH 7.3. The
recording electrodes were formed from borosilicate glass tubing and had
resistances of typically 5-8 M
and were filled with a solution
having the following composition (in mM): cesium gluconate,
150; EGTA, 10; HEPES, 10; pH 7.2 (CsOH). Cell access resistances were
typically 8-12 M and were compensated only when peak currents were
in excess of 2 nA. Cells transfected with 32 or 352
AChRs were treated for 12 h with 100 µM nicotine to
increase the levels of functional AChRs followed by a minimum of a 1-h
wash with normal media prior to recording. Currents were evoked by
application of agonist flowing from one of the two glass perfusion
tubes. Prior to application of agonist, the cell was isolated in a
continuously flowing control solution from the other tube. Agonists
were selected via two 6-way valves in series. Currents were activated
by a 2-s application of agonist flowing from the glass tubing attached
to a piezo-electric application system that was activated by an
isolated pulse stimulator (A-M Systems model 2100). The speed of
solution exchange was determined by clamping an open recording
electrode at 0 mV and then measuring the change in the solution
junction potential when moving from a normal recording solution to one
that had been diluted 5-fold with deionized water. The time constant
for the change in clamping current during the solution exchange was
approximately 1 ms at the solution flow rates used for activating
currents in these cells. Recordings were obtained with an Axopatch 1-D
amplifier (Axon Instruments, Inc., Foster City, CA) with the filtered
output at 2 kHz and sampled with pClamp 6.0.3 (Axon Instruments) onto a
personal computer using a TL-1 DMA interface (Axon Instruments) and a
Labmaster A-D converter (Axon Instruments) at 2 kHz. All currents were
normalized to the peak current obtained for 300 µM ACh,
and concentration/response curves were fitted to a logistic equation in
Origin (Version 4.1; Microcal Software, Inc., Northampton, MA). In some
cases, the concentration/response relationship would peak and then
decline with increasing concentrations of agonist. In the cases where the decline caused an obviously inferior fit, the reduced amplitude responses at higher concentrations were not included in the fit, i.e. for 325 both 3 mM ACh and 1 mM nicotine were not included in the fits but are shown in
the figures. Representative traces were constructed by opening data
files in Axograph 3.55 (Axon Instruments) and exporting data to Canvas
5.0 (Daneba Software, Inc., Miami, FL).
Nicotine Treatment and Electrophysiological Recordings in Xenopus
Oocytes--
Oocytes were obtained from Xenopus laevis
(Nasco). Stage V-VI oocytes were selected and injected with
combinations of 3 and 2 or 3 and 4 subunit cRNAs (15 ng of
each subunit in a total volume of 55 nl). Currents were measured using
a standard two-microelectrode voltage clamp amplifier (Oocyte Clamp
OC-725; Warner Instrument Corp., Hamden, CT) as described previously
(17). All recordings were digitized using MacLab software and hardware
(AD Instruments, Castle Hill, Australia). Data were analyzed using
Kaleidagraph (Synergy Software, Reading, PA). For nicotine treatment,
the oocytes were incubated overnight at 18 °C in Petri dishes
containing the relevant concentration of nicotine. After overnight
nicotine treatment, oocytes were placed in dishes of control saline for
at least 1 h before electrophysiological recordings.
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RESULTS |
Expression of AChR Subunit Combinations in Stably Transfected
tsA201 Cells--
Human embryonic kidney tsA201 cells expressing the
AChR subunit combinations 32 or 34 were initially grown in
culture medium containing Zeocin to select for the 3 containing
plasmid and neomycin to select for the 2 or 4 containing plasmid.
Cell colonies that survived for 3 weeks in the selection medium were tested for AChR expression using a solid phase RIA with
[3H]epibatidine as ligand (Fig.
1A). Most (>90%) of the
selected colonies contained AChRs that bound
[3H]epibatidine. Expression levels varied. Colonies
expressing higher levels of AChRs (>200 fmol/mg protein for 32
cell lines, and >800 fmol/mg protein for 34 cell lines) were
recloned by limiting dilution in 96-well plates. Expression of 3
AChRs in 32 and 34 cell lines was stable with respect to
[3H]epibatidine binding for at least 3 months in
continuous culture.
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Fig. 1.
Expression of 3 AChRs in permanently
transfected tsA201 cell lines. A, the expression levels
of detergent-solubilized 3 AChRs in different cell lines were
measured by solid phase RIA of Triton X-100-solubilized AChRs in
mAb210-coated microtiter wells with [3H]epibatidine as
ligand. For each assay, cells from one 35-mm Petri dish (containing
approximately 1 × 106 cells with a protein content of
0.1 mg) were collected for each assay. Values represent the mean ± S.E. from four dishes of each AChR subtype. B, Western
blots detect 5 subunits in 325 and 345 cell lines.
3 AChRs were affinity purified with mAb290 specific for 2
subunits or antiserum specific for 4 subunits as appropriate. The
immunoblot was probed with mAb268 which is specific for denatured 5
subunits. C, the fraction of 3 AChRs containing 5
subunits in 325, and 345 cell lines
(upper), and the SH-SY5Y cell line (lower) were
measured by immunoprecipitation analysis with antiserum to 5
subunits. [3H]Epibatidine (5 nM) was used to
label the AChRs for quantification. Data were normalized to the maximum
amount of 3 AChRs precipitated with a saturating concentration of
mAb210 (which recognizes both 3 and 5 subunits). A maximum of 49, 14, or <9% of the total 3 AChRs were bound in the presence of an
excess of antiserum to 5 in the three lines. Values represent
mean ± S.E. of triplicate determinations.
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Cell lines expressing 325 and 345 AChRs were
established by transfecting lines expressing 32 or 34 AChRs
with a cDNA encoding 5 and using hygromycin in the culture
medium for selection of clones containing 5 subunits. Expression of
5 subunits in the 325 or 335 cell lines was
monitored by immunoblots using mAb268 which is specific for denatured
5 subunits (Fig. 1B).
Our previous studies using the Xenopus oocyte
expression system and immunoprecipitation of 3 AChRs incorporating
epitope-tagged 5 subunits indicated that co-expression of equal
amounts of cRNAs for 3, 2 or 4, and 5 subunits resulted in
efficient incorporation of 5 subunits into >55% of AChRs (16).
In order to determine how efficiently 5 was incorporated in the 3
AChRs of our cell lines, we performed immunoprecipitation assays with
mAb210 (which binds both native 3 and 5 subunits) and an
antiserum to bacterially expressed 5 (which binds to both native and
denatured 5 subunits) to measure the fraction of 325 AChRs
in the cell line. Data in Fig. 1C show that 49% of 3
AChRs in the cell line contained 5 subunits. In a similar manner we determined that only 14% of the 3 AChRs in the 345 cell
line contained 5 subunits. In order to determine whether inefficient assembly with 5 subunits was an artifact of expression in
transfected lines, the fraction of 5-containing AChRs in the human
neuroblastoma cell line SH-SY5Y was also measured in the same way. Only
about 9% of the 3 AChRs in the native neuronal cell line appeared
to contain 5 subunits (Fig. 1C). Thus, it seems that in
both transfected cell lines and neuroblastoma cell lines 5 subunits
are incorporated into 3 AChRs less efficiently than is the case in
Xenopus oocytes.
We also studied the sedimentation behavior of 3 AChRs expressed in
the stably transfected cell lines. By comparing their sedimentation
properties with those of native 3 AChRs from SH-SY5Y and those of
human 32 AChRs expressed in Xenopus oocytes, we found
that in all four cell lines, the AChR complexes (indicated by the
binding of [3H]epibatidine) co-sedimented with native
3 AChRs in the 11 S region, which corresponded to fully assembled
pentamers (Fig. 2). There was no evidence
of partially assembled 32 dimers, for example, which would have
been expected to form an epibatidine-binding site at their interface
(46) but would have sedimented much more slowly than the
32322 arrangement of five subunits around the central
ion channel expected of native AChRs.
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Fig. 2.
Sucrose gradient sedimentation analysis of
human 3 AChRs expressed in cell lines. AChRs from different
cell lines were solubilized with Triton X-100 and were sedimented on
5-20% sucrose gradients. Fractions are numbered from the bottom of
the gradients. 3 AChRs were quantitated by
[3H]epibatidine binding (4 nM) in a solid
phase RIA on mAb210-coated microwells. 3 AChRs isolated from the
human neuroblastoma cell line SH-SY5Y were sedimented on a parallel
gradient as a size standard for the native AChR.
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Transfected Cells Express Functional AChRs--
All
transfected cell lines that exhibited binding of
[3H]epibatidine responded with currents to
applications of nicotine or ACh, although with differing current
amplitudes and differing percentages of cells responding (Figs.
3 and 4).
The subunit combination 34 gave the most robust responses,
typically reaching maximal currents of 1-5 nA, with some cells
responding with currents as large as 15-20 nA. The EC50
values for activation by ACh and nicotine for 34 AChRs were
79 ± 8 and 56 ± 15 µM, respectively. The Hill coefficients were 1.5 and 1.6, respectively. ACh and nicotine had
equivalent efficacies in activating 34 AChRs in these cells. The
currents of the 34 cells exhibited a slow decay during agonist application. 32 transfected cells had insufficient expression to
characterize their functional properties without nicotine-induced up-regulation (see below). Therefore, for functional studies, these
cells were incubated for 12 h with 100 µM nicotine
followed by a minimum of a 1-h wash in normal culture media prior to
recording. All functional studies for 2-containing AChRs expressed
in tsA201 cells were preceded by nicotine exposure in this manner. The
32 AChRs generally had smaller responses and were undetectable in a greater number of cells. The largest currents for these cells were
between 1 and 1.5 nA. The EC50 values for activation by ACh or nicotine were 209 ± 26 and 70 ± 6 µM,
respectively. The Hill coefficients were 1.7 and 1.3, respectively. For
32 AChRs, nicotine appears to have only 60% efficacy compared
with ACh. In stark contrast to the 34 AChR responses, the decay
of 32 currents was extremely rapid and usually complete within
0.5 s, indicating a rapid desensitization rate. The need to expose
the 2-containing cell lines to nicotine for functional studies
raises the concern that the functional properties of the AChRs might be
altered by nicotine exposure. To address this concern we performed a
parallel series of experiments in oocytes expressing 32 and
34 AChRs, which demonstrated that overnight incubation of the
oocytes in 100 µM nicotine had no substantial effect on
the rates of desensitization (which is also more rapid for 32
AChRs expressed in oocytes (16, 49)), EC50 values for
activation of the 32 or 34 AChRs by ACh and nicotine, or
the efficacy of nicotine (which also is a partial agonist for 32
AChRs expressed in oocytes (16, 49)) (data not shown).
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Fig. 3.
Functional properties of 3 AChRs.
Representative currents recorded for ACh concentration/response
analysis of the four 3 cell lines. All currents were recorded at a
holding potential of  60 mV, with the exception of the 34 cells
which were recorded at 30 mV to avoid the excessive current required
for maintaining adequate clamp at 60 mV. Agonist application
durations are indicated by the solid bars above each series
of traces. AChR expression was up-regulated before this analysis by
exposure of the 32 and 325 cell lines to 100 µM nicotine for 12 h followed by 1 h of rinsing
before analysis. As in Xenopus oocytes expressing cRNAs (16,
49), 32 AChR responses desensitize more rapidly than do 34
AChRs, but in both cases the kinetics are more rapid in transfected
cells.
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Fig. 4.
Concentration/response curves for the four
3 cell lines. Concentration/response curves for nicotine and
ACh are shown for the four cell lines. EC50 values are
given along with their Hill coefficients as determined by fitting the
data to a logistic equation as described under "Experimental
Procedures." Holding potentials were 30 mV for the 34 and
345 cells, whereas 32 and 325 cells were held
at 60 mV. As in Xenopus oocytes expressing cRNAs (16, 49),
nicotine is a partial agonist on 32 AChRs and nearly a full
agonist on 325 AChRs. However, the potency of agonists on
32 AChRs in transfected cells is lower than in oocytes, and the
potency on 34 AChRs is higher.
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The EC50 for activation of 345 AChRs by ACh was
81 ± 15 µM and by nicotine was 42 ± 5 µM. The Hill coefficients were 1.6 and 1.7, respectively.
ACh and nicotine had equivalent efficacies. Thus, the pharmacological
properties were essentially identical to 34 cells. This may not
be surprising given that Fig. 1 shows that only 14% of these AChRs
incorporated 5 subunits. There was no clear evidence for a dual
population of AChRs in the concentration/response relationships. The
decay of the currents was indistinguishable from that observed for
34 AChR currents. For 32 cells transfected with the 5
subunit, the cells were also incubated with 100 µM nicotine to increase AChR expression followed by at least a 1-h wash.
The maximal currents were about 1 nA and bore a striking resemblance in
time course to the parent 32 AChR cell line responses. Despite
the 49% incorporation of 5 subunits shown for these AChRs in Fig.
1, there was no evidence of two populations of AChRs greatly differing
in their responses. The EC50 for activation by ACh was 121 ± 18 µM and by nicotine was 83 ± 12 µM. The concentration/response relationships had Hill
coefficients of 1.6 and 1.3, respectively. The efficacy of nicotine
increased in the presence of 5 but remained less than that of ACh,
probably reflecting the somewhat lower incorporation of 5 (49%) in
the cells compared with Xenopus oocytes (72%) (16).
Although the EC50 for activation by ACh was somewhat lower
than that found for the 32 cells, there were diminishing responses at the saturation end of the concentration/response curves
suggestive of agonist-mediated channel blockade that would attenuate
the responses leading to an underestimation of the true EC50 value and obscuring the relative efficacies of these
agonists. Therefore, it would appear that the 5 subunit does not
alter the apparent affinity of this AChR for activation by ACh when compared with the 32 AChR, but the 5 subunit might increase the susceptibility to channel blockade by ACh.
Chronic Treatment with Nicotine Up-regulates 32 but Not
34 AChRs in Cell Lines--
Chronic exposure of the 32
cell line to nicotine increased the amount of 32 AChRs up to
24-fold as measured by [3H]epibatidine binding to
immunoisolated solubilized AChRs (Fig. 5). The EC50 for
nicotine-induced up-regulation of 32 AChRs was 2 ± 0.3 µM (n = 4). This concentration is
30-40-fold lower than the EC50 for activation of the
32 AChRs by nicotine (Fig. 4) but 10-fold higher than the
EC50 for up-regulation of 42 AChRs (34) or the
typical serum concentration of nicotine in a tobacco user (35).
Although the maximum effect of nicotine was seen at 1000 µM, at 0.2 µM, which is very close to the
serum concentration of nicotine for smokers (47), nicotine was able to
up-regulate 32 AChRs more than 3-fold in 7 h (Fig.
5). At concentrations as high as 1 mM, nicotine did not affect cell proliferation. The
up-regulation could be seen as early as 15 min after nicotine (100 µM) exposure and reached a maximum by 8 h. Nicotine
also up-regulated 3 AChRs in 325 cell lines to the same
extent (up to 22-fold) as AChRs in the 32 cell line (Fig. 5).
Considering that 32 AChRs can be up-regulated 24-fold by the same
concentration of nicotine (100 µM) and that 325
AChRs represent 49% of 3 AChRs in the cells before (see Fig.
1C) and after chronic exposure to nicotine (data not shown),
we conclude that nicotine-induced up-regulation is the same on
325 AChRs as on 32 AChRs.
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Fig. 5.
Nicotine-induced up-regulation of 32
and 325 AChRs in cell lines. After treating with nicotine
as indicated, 3 AChRs were solubilized using Triton X-100 and
measured by solid phase RIA on mAb210-coated microtiter wells with
[3H]epibatidine as ligand. Values represent means ± S.E. of triplicate determinations.
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Up-regulation of 32 AChRs was also assayed by measuring 3
AChRs on the cell surface using 125I-mAb210, which binds to
3 and 5 subunits. Exposing cells to 10 µM nicotine
for 12 h increased surface 32 AChRs more than 20-fold, from
7.6 ± 0.3 fmol/dish to 158.5 ± 7.6 fmol/dish
(n = 3). In contrast, for 34 cell lines, the
amount of AChRs on the surface was not changed by nicotine (108.7 ± 1.6 fmol/dish and 102.6 ± 2.3 fmol/dish, before and after
nicotine treatment, n = 3).
AChRs were visualized by labeling fixed cells with mAb210 followed by
F-GART (Fig. 6). Without exposure to
nicotine, 32 AChRs were virtually undetectable on the cell
surface, but 32 AChRs were detectable inside permeabilized cells.
Nicotine added to the culture medium at 10 µM for 12 h dramatically increased 32 AChRs on the cell surface and inside
the cells. Some clusters of 32 AChRs were formed on the cell
surface. The average diameter of the clusters was about 1 µm. Most
34 AChRs were present on the surface of the cells, and incubation
in nicotine did not change their amount or distribution. The clustering
of 34 AChRs on the surface membrane was striking. Their clusters
were bigger (1-3 µm in diameter) and much more frequent than those
of 32 AChRs.
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Fig. 6.
Nicotine up-regulates surface 32 AChRs
in cell lines. 3 AChRs on the cell surface were visualized by
immunofluorescent staining of fixed cells with mAb210 (30 nM) for 1 h at 4 °C followed by F-GART. To
visualize total 3AChRs, cells were permeabilized with 0.1% saponin
for 15 min before labeling with mAb210. Scale bars: 1, 2, 5, and 10 µm from top to bottom.
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Up-regulation by nicotine of 32 AChRs was studied by immunoblot
assay to demonstrate the increase of both 3 and 2 subunits in
assembled form. 32 AChRs were purified with mAb290-Actigel, which
binds specifically to 2 subunits. Blots of purified 32 AChRs
were labeled with an antiserum to an 3-specific oligopeptide (Fig.
7, lanes 1 and 2).
An increase in 3 subunits was detected as early as 15 min after
nicotine exposure. The maximum effect was seen after about 8 h.
32 AChRs were also immunopurified using mAb210-Actigel, which
recognized 3 subunits, and the Western blots labeled with an
antiserum to a 2-specific oligopeptide. The amount of 2 subunits
also increased from an early stage of nicotine exposure as well (Fig.
7, lanes 3 and 4). Doublet bands of 3 or 2
subunits were seen on the blots more obviously when cells were treated
with nicotine. This probably arises from variable glycosylation of the
subunit, because only one band of 3 or 2 was seen on the blots if
the purified AChRs were deglycosylated before they were loaded on the
SDS-polyacrylamide gel (data not shown).
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Fig. 7.
Prolonged nicotine exposure causes increased
32 but not 34 AChRs. AChRs were solubilized with
Triton X-100 and isolated with one set of subunit-specific antibodies
to obtain assembled subunits. The blots were then probed with another
set of antibodies to visualize the isolated subunits. Nicotine
treatment increased the amount of 3 and 2 subunits assembled into
AChR complexes in 32 cell lines. No change was found for the
amount of 3 and 4 subunits from assembled AChRs in 34 cell
lines.
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The up-regulation effect of nicotine was subunit-specific. We compared
the amount of 34 AChRs from the cell line before and after
nicotine exposure by surface binding assay (Fig. 6), immunoblot assay
(Fig. 7, lanes 5-8), and solid phase RIA (Fig. 11A). None of these assays detected a change even 48 h
after nicotine was added to the culture medium. 345 AChRs,
like 34 AChRs, were not up-regulated by nicotine (data not
shown). In the neuroblastoma cell line SH-SY5Y, there are both 32
and 34 AChRs (16). It is known that nicotine can up-regulate 3
AChRs in SH-SY5Y cells (18), but it was not clear which subtypes of
3 AChRs were up-regulated. In order to resolve this, we used
antibodies specific for 2 or 4 subunits separately to
immunoprecipitate 3 AChR subtypes in SH-SY5Y cells. After chronic
exposure to nicotine, the amount of 2-containing AChRs in SH-SY5Y
cells increased 3-fold, whereas the amount of 4-containing AChRs was
not changed (Fig. 8). These observations
strongly suggest that 2 subunits play an important role in
nicotine-induced up-regulation of 3 AChRs in neurons.
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Fig. 8.
Nicotine up-regulates 32 but not
34 AChRs in the human neuroblastoma cell line SH-SY5Y.
SH-SY5Y cells were exposed to 10 µM nicotine for 48 h. AChRs in the cells were then solubilized with Triton X-100, labeled
with [3H]epibatidine (5 nM), and
immunoprecipitated with mAb290 (specific for 2 subunits) or
antiserum to the bacterially expressed cytoplasmic domain of 4
subunits. Nonspecific precipitation was determined by using in the
assay mixture normal rat serum instead of mAb290 or normal mouse serum
instead of antiserum to 4. Values represent mean ± S.E. of
triplicate determinations.
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Nicotine-induced Up-regulation of 32 AChRs Results from
Enhanced Subunit Assembly and Decreased Turnover Rate of AChRs--
We
tested the possibility that nicotine treatment increased the affinity
of 3 AChRs for [3H]epibatidine by comparing the
binding to 32 AChRs before and after exposure to 100 µM nicotine. The KD value was not changed (107 ± 18 nM versus 126 ± 10 nM, n = 4), which means that nicotine did
not increase the affinity of detergent-solubilized immunoisolated
32 AChRs for [3H]epibatidine. We also excluded the
possibility that nicotine exposure increased the affinity of 32
AChRs for mAb210 and mAb290 by showing that nicotine-induced
up-regulation could also be demonstrated by employing a filter binding
assay using solubilized AChRs labeled with
[3H]epibatidine that did not require use of mAbs (data
not shown). It was clear that the up-regulation resulted from
increasing the amount of AChRs rather than from increasing their
affinity for both mAbs.
Since up-regulation of 32 AChRs started as early as 15 min after
the cells were exposed to nicotine, it is reasonable to suggest that
the proper folding and assembly of 3 and 2 subunits were enhanced
by nicotine. We tested this possibility by probing for 2 subunits on
immunoblots of 32 cell extracts, both affinity purified by
mAb210-Actigel and applied directly as crude extract (Fig.
9, upper panel). Comparing the
specific signal for 2 subunits on the blot with and without exposing
the cells to nicotine for 30 min, we found only a small increase in the
total amount of 2 subunits in the cell, but the amount of 2
subunits that was assembled with 3 subunits greatly increased (more
than 5-fold). Since the affinity of 32 AChRs for mAb210 was not
changed, the large increase in 2 subunits purified by the
3-specific mAb210-Actigel a short time after exposure to nicotine
(before the total amount of 2 subunits increased greatly) suggests
that the rate of subunit assembly into 32 AChRs increased upon
nicotine exposure.
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Fig. 9.
Nicotine enhances subunit assembly of
32 AChRs. Upper panel, immunoblots of 2
subunits either affinity purified with mAb210-Actigel to bind AChRs
containing 3 subunits or in a crude extract from 32 cells were
probed with rabbit antiserum to a human 2 subunit synthetic peptide.
This showed that nicotine caused an increase of 2 subunits assembled
with 3, much more than that of total 2 subunits. Lower
panel, protein synthesis in 32 cells was blocked with
cycloheximide (35 µM) for 3 h before nicotine (100 µM) was added to the culture medium. This showed that
nicotine could still cause some up-regulation of 32 AChRs in the
cells without synthesis of more subunits.
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To test whether up-regulation of 32 AChRs requires protein
synthesis, cycloheximide, a protein synthesis inhibitor, was used to
block the synthesis of 3 and 2 subunits for 3 h before nicotine was added into the culture medium. After 90 min, the total
amount of 32 AChRs in the cells was measured by RIA (Fig. 9,
lower panel). Compared with control cells, nicotine was
still able to up-regulate 32 AChRs 6-fold without new protein
synthesis. This suggests that at least 25% of the up-regulation in
32 AChRs (6-fold up-regulation without increased synthesis
versus 24-fold otherwise) is due to enhanced assembly and
perhaps the reduced degradation of pre-existing subunits.
Previous studies in our laboratory showed that nicotine was able to
decrease the turnover rate of avian 42 AChRs stably expressed in
mammalian fibroblast cells (34). To see if the up-regulation effect of
nicotine for human 32 AChRs could also be partially attributed to
the stabilization effect of nicotine on 32 AChRs in tsA201 cells,
we blocked protein synthesis in the cell using cycloheximide and
followed the influence of nicotine on 32 AChRs in the cells for
24 h (Fig. 10). In the absence of nicotine, the addition of cycloheximide prevented replacement of
32 AChRs, and the total amount decreased with a
t1/2 of around 30 h. When nicotine
and cycloheximide were added together, there was a rapid initial
increase in 32 AChRs due to assembly of pre-existing subunits.
Western blots like those in Fig. 7 using mAb210 to 3 to isolate
assembled AChRs and antiserum to 2 to reveal 2 on blots showed
about half-maximal assembly by 30 min, about twice this amount by
3 h, and a slight further increase by 24 h (data not shown).
Fig. 10 shows that by 3 h there was a 13-fold increase in 32
AChRs. This exceeds the 5-fold increase caused by nicotine after 3 h in cycloheximide shown in Fig. 9, presumably due to turnover of about
half of the unassembled 3 and 2 subunits during the 3 h
after cycloheximide was added and before nicotine was introduced. Both
Fig. 9 and Fig. 10 show that after nicotine is added, and the initial
burst of enhanced assembly of 32 AChRs is complete, the amounts
of 32 AChRs remain virtually constant for the duration of the
experiments. Thus, in the presence of nicotine the rate of turnover of
assembled AChRs is
3
2 but Not
,
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Abstract
Introduction
