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INTRODUCTION |
Chronic treatment with most classes of antidepressants leads to a
reduction in the number of central postsynaptic
-adrenoceptors (
AR)1 in vivo
and of surface
AR density in cell cultures (1-4). This effect is
reported to be specific for the
1-adrenoceptor subtype (5, 6).
AR down-regulation is accompanied by decreased receptor-stimulated cAMP formation (7). The two major effects at the
molecular level become apparent in vivo after 10-20 days of
drug administration and coincide with the onset of clinical antidepressant response in humans. Therefore
AR down-regulation and
diminished cAMP response to catecholamines may relate to the therapeutic action of antidepressants.
It has been proposed that the reduction in the number of functional
AR could be a regulatory response to the enhanced presence of
norepinephrine in the synaptic cleft after acute inhibition of
norepinephrine reuptake or of monoamine oxidase activity by antidepressants (1, 8). Some clinically effective antidepressants, however, neither influence norepinephrine reuptake nor inhibit monoamine oxidase activity but still cause a
AR down-regulation. Furthermore, this model fails to explain the observed time lag between
the rapid drug-induced increase in intrasynaptic neurotransmitter concentrations and the delayed receptor down-regulation.
Decreased
AR densities following antidepressant treatment can also
be seen in cell culture systems lacking a presynaptic input,
e.g. in cultured rat C6 glioblastoma cells (9, 10). Thus,
AR down-regulation may directly result from postsynaptic actions of
the antidepressants. Previous studies have shown that chronic treatment
of cultured cells with the tricyclic antidepressant desipramine (DMI)
induces phospholipidosis by inhibition of lysosomal phospholipid
degradation, leading to changes in membranous and total phospholipid
compositions (11). Altered membrane properties may directly influence
receptor function or have an effect on vesicular membrane traffic and
therefore on
AR endocytosis and recycling.
Desensitization of
AR after agonist exposure induces receptor
endocytosis, protecting cells from overstimulation. It is mediated by
uncoupling the activated
AR from the GS protein and
internalization of receptor from the plasma membrane to early endosomes
(12-15). While
2-adrenoceptor desensitization is well
studied, much less is known about the mechanism of
1-adrenoceptor desensitization (16, 17).
Within seconds of agonist binding,
1- and
2-adrenoceptors are phosphorylated by G protein-coupled
receptor kinases and cAMP-dependent protein kinases (18,
19). This phosphorylation promotes the binding of the cytosolic protein
-arrestin, which inhibits the ability of the
AR to couple to
GS (20).
-Arrestin then targets the phosphorylated
receptor to clathrin-coated pits for endocytosis (21). The modes of
internalization and recycling of
1-adrenoceptors (
1AR) are not known in detail.
2-Adrenoceptors
(
2AR) are internalized via a dynamin-dependent mechanism
through clathrin-coated pits similar to the transferrin receptor (22,
23). There is a significant difference in the rate of internalization
between the
1- and
2-adrenoceptor
subtypes.
2AR are efficiently endocytosed following agonist
stimulation, whereas
1AR undergo only slight internalization. The
low affinity of the activated
1AR for
-arrestin may provide an
explanation for the small extent of internalization (24). In early
endosomes,
AR are dephosphorylated and subsequently recycle back via
a perinuclear compartment to the plasma membrane in a fully
resensitized state (25-27). Prolonged agonist treatment for several
hours causes down-regulation of total receptor number (28).
The currently accepted model for
AR down-regulation postulates that
during chronic agonist exposure, endocytosed
AR do not recycle to
the plasma membrane but are sorted to lysosomes, where they are
degraded by proteases (29, 30). However, Jockers et al. (31)
have recently reported that
2AR down-regulation is fully maintained
upon inhibition of receptor endocytosis and blockade of the lysosomal
and ubiquitin proteasome pathway, suggesting that the primary
inactivation step may occur at the plasma membrane. There is evidence
in some cell lines that
AR mRNA levels are decreased during long
term exposure to agonist, resulting in a reduced receptor synthesis
(32, 33). Whether antidepressant-induced
1AR down-regulation
requires one of these molecular mechanisms is unknown.
The aim of the present study was to determine whether the reduction of
1AR cell surface density observed in cultured rat C6 glioblastoma
cells following chronic DMI treatment is caused by drug-induced changes
in receptor trafficking. Endocytosis and recycling of the
1AR fused
to green fluorescent protein (
1AR-GFP) were therefore compared in
untreated and chronically DMI-treated C6 cells using a complementary
approach of confocal fluorescence microscopy and ligand binding studies.
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EXPERIMENTAL PROCEDURES |
Materials--
Materials for cell culture were supplied by Sigma
and Invitrogen. Unless otherwise mentioned, all chemicals
(analytical grade), were from Sigma or from Merck.
Plasmid Construction--
Wild-type human
1AR DNA was
retrieved from pSP65-
1AR (a gift from Dr. Susanna Cotecchia,
University of Lausanne) with EcoRI and ApaI and
subcloned into pcDNA3 (Invitrogen) at the respective restriction sites.
Enhanced green fluorescent protein (EGFP; Clontech)
was fused to the carboxyl terminus of the human
1AR. The 3'-end
sequences of
1AR including the stop codon were removed with
TfiI. To restore the 3'-end of the coding sequence and to
create a suitable BamHI restriction site at the 3'-end for
fusion to EGFP, the synthetic complementary oligonucleotides
5'-AATCCAAGGTGGATCTGCAG-3' and 5'-GATCCTGCAGATCCACCTTGG-3' were used
for the ligation to pEGFP-N1 (Clontech). The
predicted nucleotide sequence of the fusion region was confirmed by
dideoxy sequencing of the resultant construct,
1AR-GFP. The
synthetic linker sequence between
1AR and EGFP encodes the amino
acids ESKVDLQ, whereby the amino acids ESKV restore the original
carboxyl terminus of the
1AR.
Cell Culture and Transfection--
Rat C6 glioblastoma cells
(European Collection of Animal Cell Cultures) were maintained in
Eagle's high glucose minimum essential medium supplemented with 10%
fetal bovine serum, 3.7 g/liter NaHCO3, 200 units/ml
penicillin G, and 10 µg/ml chlortetracycline at 37 °C in a
humidified atmosphere of 95% air and 5% CO2. Cells were passaged once a week and replated at a density of 2 × 106 cells/10-cm dish.
For transfection, cells were grown to 80% confluence in 10-cm dishes.
The
1AR-GFP plasmid (6 µg) was linearized with AflIII and mixed with 20 µl of LipofectAMINE reagent (Invitrogen) according to the manufacturer's instructions. Cells were exposed to the DNA-LipofectAMINE mixture for 8 h. Two days after transfection cells were trypsinized, diluted, and seeded into 96-well plates at a
density of 15,000 cells/well. Stably transfected cells were selected
with Geneticin G418 (500 µg/ml; Calbiochem) that was added to the
culture medium 3 days after transfection. Wells with cells that were
resistant to the antibiotic were identified 2 weeks after transfection.
Cells were then reseeded into 96-well plates at limiting dilutions to
establish clonal C6-
1AR-GFP cell lines. Cell lines with highest
expression levels of the receptor were identified by Western blot and
fluorescence microscopy.
COS-1 cells (American Type Culture Collection) were transiently
transfected with 5 µg of either wild-type
1AR or
1AR-GFP DNA,
essentially as described earlier (34). For the cAMP assay, COS-1 cells
were seeded into 24-well dishes at a density of 15,000 cells/well and
transiently transfected with 3 µg of
1AR or
1AR-GFP DNA per 24 wells. Experiments were performed 48 h after transfection.
Western Blot--
Untransfected and transiently transfected
COS-1 cells expressing either wild-type
1AR or
1AR-GFP as well as
untransfected rat C6 or stably transfected C6-
1AR-GFP cells were
washed with phosphate-buffered saline and scraped into ice-cold buffer
1 (137 mM NaCl, 20 mM Hepes, pH 7.2, 2 mM MgCl2, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 0.5 mM
dithiothreitol, 10 µg/ml leupeptin). Following two freeze/thaw cycles
to disrupt the cells, the resulting suspensions were centrifuged at
14,000 × g for 2 min. The cell pellets were resuspended in lysis buffer (20 mM Tris-HCl, 137 mM NaCl, 1 mM MgCl2, 1% Triton
X-100, pH 7.4, 1 mM phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol, 10 µg/ml leupeptin) and incubated for
30 min on ice. The lysates were centrifuged at 14,000 × g for 2 min, and the supernatants were collected. Proteins
from these membrane extracts were resolved by SDS-polyacrylamide (10%)
gel electrophoresis under reducing conditions and then transferred to
nitrocellulose membranes by electroblotting. The blots were then probed
with antibodies to the
1AR (Calbiochem) or GFP
(Clontech) and horseradish peroxidase-conjugated
secondary antibody (Sigma). Bound antibodies were visualized using
enhanced chemiluminescence detection (Roche Molecular Biochemicals).
Radioligand Binding Assay--
The number of cell surface
AR
was determined in intact C6 and COS-1 cells using the hydrophilic
AR
antagonist [3H]CGP-12177 (Amersham Biosciences). The
cells were gently scraped into Hanks' solution (1.25 mM
CaCl2·2H2O, 5.5 mM
D-(+)-glucose·H2O, 5.4 mM KCl,
0.44 mM KH2PO4, 0.8 mM
MgSO4·7H2O, 137 mM NaCl, 0.34 mM Na2HPO4·2H2O) at
room temperature. 200 µl of this cell suspension and 50 µl of
radioligand solution (final concentrations 0.5-10 nM
[3H]CGP-12177) were incubated at 37 °C for 60 min.
Nonspecific binding was determined in the presence of the antagonist
timolol (1 µM).
The number of total cellular
AR was determined using the
membrane-permeant radioligand [3H]dihydroalprenolol
(Amersham Biosciences). Cells were harvested in Hanks' solution,
pelleted, lysed in 5 mM Tris/HCl, 2 mM EDTA, 5 µg/ml leupeptin, 0.5 mM phenylmethylsulfonyl fluoride, pH
7.4, and subjected to two freeze/thaw cycles. This cell lysate was diluted 1:12 with binding buffer (75 mM Tris/HCl, 12 mM MgCl2, 2 mM EDTA, pH 7.4).
Triplicates (200 µl each) of the diluted lysate were incubated with
50 µl of radioligand solution (final plateau concentrations 10-14
nM [3H]dihydroalprenolol) for 30 min at
37 °C. Nonspecific binding was determined in the presence of 3 µM alprenolol (Novartis).
The binding reaction was terminated by adding 1 ml of ice-cold Hanks'
solution. 1 ml of the incubation mixture was immediately filtered
through presoaked glass fiber filters (Whatman GF/C) at constant vacuum
pressure. The filters were washed three times with 5 ml of 0.9% NaCl
solution, dried, and placed in counter vials. Cells were then digested
overnight using a tissue solubilizer. After neutralization and the
addition of 5 ml of liquid scintillation mixture (ULTIMA GOLD; Packard
Instruments), radioactivity was determined in a liquid scintillation
counter. Protein concentrations were determined using the method of
Lowry. Binding curves were fitted by nonlinear regression using
GraphPad Prism (version 3.0).
cAMP Assay--
Transiently transfected COS-1 cells were
serum-starved for 2 h and then incubated at 37 °C in 500 µl
of Hanks' solution containing 1 mM
1-methyl-3-isobutylxanthine and varying concentrations of isoproterenol
(10
11 to 10
6 M) or no agonist
(basal). The reaction was stopped after 10 min by exchanging the
Hanks' solution for 500 µl of ice-cold 0.1 N HCl.
Following a freeze/thaw cycle to lyse the cells, the cytosolic extracts
of each well were collected, lyophilized, resolved in 50 mM
Tris-HCl, 4 mM EDTA, pH 7.5, and assayed for cAMP using the
Amersham Biosciences cyclic AMP assay kit. The cAMP produced in
response to agonist exposure was calculated as the cAMP accumulation in stimulated cells minus the cAMP level in unstimulated cells from the
same 24-well dish. Protein concentrations were determined using the
method of Lowry.
-Adrenoceptor Endocytosis and Recycling--
COS-1 cells
transiently expressing either wild-type
1AR or
1AR-GFP and
C6-
1AR-GFP cells were stimulated with 10 µM
isoproterenol at 37 °C for the indicated times. Subsequently, the
medium was removed, cells were chilled on ice, washed 10 times with
ice-cold Hanks' solution followed by further incubation for 1 h
at 37 °C in fresh medium to allow receptor recycling. Cell surface
AR densities were determined in parallel cultures before agonist addition, after desensitization as well as at the end of the
resensitization period as described above (radioligand binding assay).
To prevent receptor internalization and recycling in the intact cells
during exposure to radioligand, the temperature was lowered to
13 °C. Triplicates (200 µl each) of the cell suspension were mixed
with 50 µl of radioligand solution and incubated for 3 h to
ensure equilibrium binding. Bmax values were
determined with plateau concentrations of the radioligand, which were
previously determined in complete saturation binding experiments.
Total cellular binding sites (surface and internal) were measured in
C6-
1AR-GFP cells before agonist addition and after resensitization using [3H]dihydroalprenolol as described above.
In some experiments, the protein synthesis inhibitor cycloheximide (200 µM) was used. C6-
1AR-GFP cells were preincubated with
the inhibitor for 60 min before agonist stimulation. Cycloheximide was
also present during all subsequent steps.
Desensitization Studies--
C6-
1AR-GFP cells at 90%
confluence were incubated at 37 °C for 30 min in Hanks' solution in
the absence or presence of 10 µM isoproterenol. Cells
were then washed five times and maintained in agonist-free Hanks'
solution containing 1 mM 1-methyl-3-isobutylxanthine for 10 min at 37 °C prior to stimulation for the same period of time with
10 µM isoproterenol. The cyclic AMP production in
response to the 10-min challenge with isoproterenol was determined as
described above (cAMP assay).
Effect of Chronic Treatment with Desipramine on
AR
Trafficking--
C6-
1AR-GFP cells (7 × 105 cells)
were seeded into 10-cm dishes 8 days prior to the experiment. 48 h
later, pharmacological treatment was started. The cells were exposed to
10 µM DMI (Novartis) for 6 days or left untreated. The
culture medium was changed 2, 4, and 5 days after the beginning of the treatment.
Confocal Laser-scanning Microscopy--
Cells were observed
using a laser-scanning confocal microscope (Zeiss Axiovert 100M; LSM
510) equipped with a Zeiss Plan-Apo 63 × 1.4 NA oil immersion
objective). GFP was excited at 488 nm (argon laser), and fluorescence
emission was detected using a 505-530-nm band pass filter.
Rhodamine-conjugated probes (transferrin, dextran) were excited at 543 nm (neon/helium laser). Red fluorescence was detected with a long pass
(560 nm) filter. The pinholes were set to 1.1 airy units.
C6-
1AR-GFP cells were grown on glass coverslips that were placed in
12-well dishes. Cells were seeded at a density of 4 × 104 cells/well. 48 h later, medium was removed, and
coverslips were rinsed with Hanks' solution and mounted on a
thermostated imaging chamber. To study receptor endocytosis, the cells
were stimulated with 10 µM isoproterenol at 37 °C in
Hanks' solution. Receptor recycling was then observed at 37 °C in
fresh Hanks' solution after agonist washout.
To study the effects of DMI on receptor trafficking, C6-
1AR-GFP
cells were seeded at a density of 1 × 104 cells/well.
24 h later, antidepressant treatment was started. To label
lysosomal compartments, cells were incubated for the final 24 h of
DMI treatment with rhodamine-dextran (1 mg/ml; Molecular Probes, Inc.,
Eugene, OR). Excess lysosomal marker was then removed, and the cells
were incubated in fresh medium in the absence or presence of
isoproterenol (10 µM) for 2 h at 37 °C before
imaging. For the transferrin experiments, cells were incubated with 10 µM isoproterenol for 2 h at 37 °C.
Rhodamine-labeled transferrin (200 µg/ml; Molecular Probes) was added
for the last 60 min of the incubation period. Before imaging, the cells
were thoroughly washed with Hanks' solution. To study
AR recycling
after agonist removal, cells were maintained in fresh medium, returned
to a 37 °C incubator for 60 min, and then viewed under the microscope.
For three-dimensional modeling of fluorescence distribution in single
live cells, stacks of confocal images were recorded before and 30 min
after stimulation with 10 µM isoproterenol. Sampling
distances were ~100 nm in the lateral and
150 nm in the axial
direction to meet the Nyquist criterion.
Three-dimensional Image Restoration and Image
Quantification--
Stacks of confocal fluorescence images were
subjected to image restoration using the iterative maximum likelihood
estimate algorithm of the Huygens System 2 (Scientific Volume Imaging, Hilversum, The Netherlands) and a theoretical point spread function. Subsequently, three-dimensional models of fluorescent objects were
generated with the Imaris 3.2 Surpass and Measurement Pro software
modules (Bitplane AG, Zürich, Switzerland) capable of separating
objects based on surfaces of equal intensities (isosurfaces). Appropriate threshold values for the generation of isosurfaces were
applied to separate intracellular objects and objects representing fluorescence at the cell surface. Quantification of the voxel densities
enclosed in these objects then provided a measure for the fluorescence
of intracellular and cell surface-associated objects.
 |
RESULTS |
Expression of
1AR-GFP Fusion Proteins in Cultured
Cells--
The
1AR was fused to the amino terminus of green
fluorescent protein to provide a tool that would allow us to follow
1AR trafficking by confocal fluorescence microscopy in live cells during agonist-induced internalization and the subsequent
resensitization phase.
First, plasmids encoding the fusion construct (
1AR-GFP) or the
wild-type
1AR were transiently transfected into COS-1 cells, and the
expressed proteins were analyzed by immunoblotting (Fig. 1). An antibody to the
1AR detected
two main bands with molecular masses of 50 and 70 kDa in cells
expressing wild-type
1AR (Fig. 1A, lane
2) but not in untransfected cells (Fig. 1A,
lane 1).
1AR are glycoproteins that contain
one N-glycosylation site in the receptor N terminus (35). It
is likely that these two bands detected by the antibody represented
nonglycosylated (50 kDa) and glycosylated (70-kDa)
1AR. As expected
from fusion to the 28-kDa GFP protein, the recombinant receptors showed
a lower mobility than wild-type
1AR (Fig. 1A,
lane 3). The 80-kDa band represented the
unglycosylated fusion protein with a predicted molecular mass of 79 kDa. Similar to the wild-type receptor, the band with the lower
mobility most likely represented a glycosylated form of the GFP-tagged
1AR, since the intensity of the 100-kDa band was strongly reduced by
treatment of the cells with the glycosylation inhibitor tunicamycin,
whereas the intensity of the 80-kDa band was augmented (not shown). The
bands with a molecular weight of about 20 kDa (lane
2) and 52 kDa (lanes 3 and
5) seem to represent a carboxyl-terminal receptor fragment,
since the difference in mobility shift of the two bands corresponds to
the molecular mass of GFP. An anti-GFP antibody detected the
receptor-GFP fusion proteins exclusively (Fig. 1A,
lane 5). Expression of the GFP-tagged
1AR was
also observed in membrane fractions of rat C6 glioblastoma cells stably
transfected with
1AR-GFP DNA (Fig. 1A, lane
6).

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Fig. 1.
Structural and functional validation of
the 1AR-GFP construct. COS-1 and rat C6
glioblastoma cells were transfected with wild-type 1AR or 1AR-GFP
DNA. A, membrane fractions of either untransfected
(lane 1) or of transiently transfected COS-1
cells expressing wild-type (lanes 2 and
4) and GFP-tagged 1AR (lanes 3 and
5) were subjected to SDS-polyacrylamide gel electrophoresis
and analyzed by immunoblotting using antibodies to 1AR or GFP.
Similarly, membrane fractions of untransfected C6 glioblastoma cells
(lane 7) or of stably transfected C6 cells
expressing GFP-tagged 1AR (lane 6) were probed
for GFP. B, COS-1 cells transiently expressing wild-type or
GFP-tagged 1AR were exposed to increasing concentrations of the
radioligand [3H]CGP-12177 to compare antagonist binding.
Results represent means ± S.D. of three independent experiments.
C, cAMP levels were measured in COS-1 cells transiently
transfected with either wild-type 1AR or 1AR-GFP DNA or with
empty vectors (pcDNA3 and pEGFP, respectively). 48 h later,
cells were exposed to increasing concentrations of isoproterenol for 10 min, and cAMP formation was determined. The results of each experiment
were normalized to the maximal cAMP accumulation measured in cells
expressing wild-type 1AR. Expression levels of wild-type and
GFP-tagged 1AR were comparable within the experiments. Data
(means ± S.D.) are from three experiments (except for pEGFP (two
experiments)). D, receptor internalization and recycling
were studied in COS-1 cells transiently expressing wild-type or
GFP-tagged 1AR. The cells were incubated for 30 min at 37 °C in
the presence of 10 µM isoproterenol, washed, and
maintained in agonist-free medium for 60 min at 37 °C. Cell surface
AR density was determined before the agonist addition
(unstimulated), immediately after agonist exposure
(agonist-stimulated), and at the end of the resensitization period
(resensitized). Data were normalized to the Bmax
value obtained in unstimulated cells. Results represent the mean ± S.D. of three different experiments.
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Functional Analysis of the
1AR-GFP Fusion Protein--
A number
of experiments were performed to demonstrate that the
1AR-GFP fusion
protein maintained the biochemical and signal transducing properties of
the native receptor.
[3H]CGP-12177 binding of the
1AR was not affected by
attaching GFP to its COOH-terminal tail. Binding isotherms were nearly identical for wild-type and GFP-tagged receptors, as shown in Fig.
1B, with equilibrium dissociation constants of 5.3 ± 1.3 nM for
1AR and 3.6 ± 1.0 nM for
1AR-GFP.
The ability of wild-type and GFP-tagged
1AR to couple to
GS proteins and to activate adenylyl cyclase upon the
addition of agonist was also examined. Transfected cells were
stimulated with varying concentrations of isoproterenol to initiate
second messenger formation. Isoproterenol-stimulated cAMP responses
were comparable in cells expressing wild-type
1AR or
1AR-GFP at
similar levels (Fig. 1C). Thus, both forms of the receptor
activated adenylyl cyclase with similar efficacy. Cells transfected
with empty vector alone also exhibited increased cAMP levels when
stimulated with high agonist concentrations. COS-1 cells appear to
express sufficient endogenous
AR to efficiently activate adenylyl
cyclase at high agonist concentrations.
The ability of the fusion construct to internalize upon agonist binding
was assessed in transiently transfected COS-1 cells. Incubation with
isoproterenol for 30 min led to a decrease in surface receptor density
of 9.9 ± 1.9%, as determined by radioligand binding (Fig.
1D). Under identical conditions, transiently expressed wild-type receptors internalized to a similar extent (13.5 ± 1.7%). Agonist was then removed to study receptor recycling. The return of the
1AR-GFP to the cell surface was quantitatively similar to that of
the wild-type
1AR. 60 min after agonist washout a complete
recovery of surface receptor numbers was detected in
1AR- and in
1AR-GFP-expressing cells.
Receptor trafficking was further investigated in C6-
1AR-GFP cells
using laser-scanning confocal microscopy (Fig.
2A). In the absence of
isoproterenol, GFP-tagged
1ARs were mainly localized in the plasma
membrane. No detectable receptor endocytosis was observed. Challenging
the cells with isoproterenol caused a profound change in receptor
distribution. Receptors were internalized and appeared as green
fluorescent speckles in the cytosol. The first intracellular receptors
could be observed as early as 3 min after agonist addition. Receptor
accumulation in intracellular structures became progressively more
pronounced during 30 min of agonist stimulation. The density of
endocytosed receptors sharply decreased following agonist washout and a
30-min incubation of cells in fresh medium at 37 °C (Fig.
2B), suggesting that receptors relocalized to the plasma
membrane upon removal of agonist. Radioligand binding studies revealed
that a 30-min exposure of C6-
1AR-GFP cells to isoproterenol prompted
a 21.9 ± 2.6% reduction in cell surface ligand binding sites.
Furthermore, rapid recovery of surface receptors was observed in these
cells following removal of agonist from the culture medium, confirming
the ability of internalized
1AR-GFP to recycle to the plasma
membrane (Fig. 3A). In order
to examine whether this reduction was indeed due to a concomitant
increase in intracellular receptor, the amount of internal fluorescence was quantified in complementary experiments using three-dimensional models reconstructed from stacks of confocal images (Fig.
3B). Fluorescence in intracellular objects that could be
clearly separated from the cell surface ranged from 6.9 to 12.5%
(9.2 ± 2.4%, mean ± S.D., n = 6) of total
fluorescence in single, isoproterenol-treated cells, compared with
0.3 ± 0.4% in control cells (Fig. 3C). These data
confirm that internalization of receptor occurs to an extent that is
consistent with the biochemically detected reduction of ligand binding
sites at the cell surface.

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Fig. 2.
Visualization of 1AR
endocytosis and recycling. A, C6- 1AR-GFP cells were
observed during stimulation with 10 µM isoproterenol at
37 °C using laser-scanning confocal microscopy. Confocal images are
from midsections of the cells. Below each image,
an enlarged region corresponding to the boxed
area is shown. Cells are shown before treatment
(unstimulated) or after 7 and 30 min of exposure to isoproterenol.
B, C6- 1AR-GFP cells were incubated with isoproterenol (10 µM) at 37 °C for 30 min. AR distribution was
examined immediately after agonist removal (left
panel) and after 10 or 20 min of incubation in the
agonist-free medium (center panel and
right panel, respectively). Confocal images from
midsections of the cells are shown. Selected regions (indicated by
boxes) were enlarged and are shown under each
panel. Representative images of three separate experiments
with similar results are shown.
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Fig. 3.
Quantitative analysis of
1AR internalization and recycling.
C6- 1AR-GFP cells were stimulated with 10 µM
isoproterenol for 30 min at 37 °C, washed thoroughly, and
subsequently incubated for 30 min at 37 °C in agonist-free medium.
A, cell surface AR densities were measured before agonist
exposure (unstimulated), after exposure to isoproterenol for 30 min
(agonist-stimulated), and 30 min after removal of agonist
(resensitized) using the hydrophilic radioligand
[3H]CGP-12177. Data were normalized to the
Bmax value obtained in unstimulated cells (590 fmol/mg of protein). Shown are the mean values ± S.D. from one
experiment out of three with similar results. *, p < 0.01 compared with unstimulated cells (one-way analysis of variance).
B, three-dimensional models of fluorescence distribution in
single cells were generated from restored stacks of confocal images
taken before and 30 min after stimulation with 10 µM
isoproterenol. The figure shows the fluorescence image from
a single plane ~2 µm above the glass surface
(green) and the distribution of cell
surface-associated (translucent blue) and
internalized (yellow) fluorescence represented as
isosurfaces with equal threshold. C, the amount of internal
fluorescence was determined in single cells before and 30 min after
agonist exposure. Voxel densities enclosed in intracellular and cell
surface-associated objects were quantified, and internal fluorescence
was expressed as a percentage of total fluorescence. The data represent
the mean ± S.D. obtained from the analysis of six single
cells.
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We then studied whether the reduced surface receptor density after
exposure of C6-
1AR-GFP cells to 10 µM isoproterenol
had functional consequences on receptor signaling. The cells were incubated for 30 min at 37 °C in the absence or presence of agonist, washed, and rechallenged with 10 µM isoproterenol for 10 min. Receptor-mediated cAMP production in response to the second
stimulus was then determined. Agonist pre-exposure led to a significant decline in the isoproterenol-stimulated cAMP response. The second messenger production in prestimulated cells was reduced by 31.8 ± 5.9% compared with cAMP levels in cells not pre-exposed to agonist. Thus, the observed reduction in cell surface receptor density results
in a more profound decrease of receptor-stimulated second messenger production.
Effects of Chronic Treatment with Desipramine on
-Adrenoceptor
Trafficking--
We next examined whether receptor endocytosis and
recycling were changed in C6-
1AR-GFP cells that were chronically
treated with the tricyclic antidepressant DMI in comparison with
untreated control cells using a radioligand binding assay (Fig.
4A). Isoproterenol stimulation
promoted endocytosis of about 15% of the receptor sites in control and
DMI-treated cells. Thus,
1AR endocytosis was not influenced by
chronic DMI treatment. 1 h after removal of agonist from the
culture medium, complete restoration of surface receptor numbers was
observed in control cells. The recovery of surface receptor density
resulted from the return of internalized
AR back to the plasma
membrane and not from the biosynthesis of new receptor protein, since
identical restoration occurred in cells incubated in the presence of
the protein synthesis inhibitor cycloheximide (not shown). In contrast,
in the plasma membrane of antidepressant-treated cells,
AR density
was still reduced at the end of the resensitization period, indicating
that under these conditions endocytosed
1AR-GFP failed to recycle
efficiently. The recycling block in DMI-treated cells appeared to be
irreversible, because even after a prolonged resensitization period
(180 min of incubation at 37 °C) cell surface receptor densities did
not recover (not shown).

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Fig. 4.
Chronic DMI treatment impairs recycling of
GFP-tagged 1AR. C6- 1AR-GFP cells were
grown for 6 days in the absence (control) or presence of 10 µM DMI (DMI-treated). Then AR endocytosis was induced
by stimulating the cells with 10 µM isoproterenol at
37 °C for 2 h. After removal of the agonist, cells were
incubated for 60 min in fresh medium at 37 °C to allow for receptor
recovery. A, internalization and recycling of 1AR-GFP as
assessed by radioligand binding. Surface AR densities were measured
using [3H]CGP-12177 before (unstimulated) and immediately
after the 2-h exposure to isoproterenol (agonist-stimulated) as well as
1 h after removal of agonist (resensitized). For each experiment,
the Bmax value of unstimulated cells was
designated 100%, and all other data were normalized to this value.
Results are presented as mean ± S.D. from five independent
experiments. *, p < 0.01 compared with the mean values
of unstimulated cells (one-way analysis of variance). B,
localization of intracellular 1AR-GFP was determined before agonist
stimulation (top row), after agonist exposure
(middle row), and at the end of the
resensitization period (bottom row).
Representative images of confocal midsections of cells are shown. Three
separate experiments produced similar results. Internalized 1AR-GFP
are indicated by arrows.
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To identify the step where the recycling process was interrupted by
chronic DMI treatment,
1AR trafficking was assessed by laser-scanning confocal microscopy. Isoproterenol exposure of untreated
and DMI-treated C6-
1AR-GFP cells caused a receptor redistribution
from the cell surface into intracellular compartments (compare Fig.
4B, top and center rows,
respectively). The extent of
1AR-GFP endocytosis was similar in
untreated and DMI-treated cells, and receptors appeared to accumulate
in perinuclear compartments. However, substantial differences in
1AR-GFP localization were detected between untreated and DMI-treated
cells after resensitization. Only a small number of internalized
receptors was detected in control cells at the end of the
resensitization period. By contrast, numerous intracellular structures
containing internalized GFP-tagged
1AR were observed in
antidepressant-treated cells (compare Fig. 4B,
bottom row, right and left
panels). These results suggest that in control cells
internalized
1AR-GFP returned back to the plasma membrane upon
removal of agonist, whereas in DMI-treated cells a large fraction of
endocytosed receptors was retained in intracellular compartments. These
findings are consistent with the data of the binding assay and indicate
that chronic DMI treatment inhibits recycling of endocytosed receptors
back to the plasma membrane.
One might assume that internalized receptors in DMI-treated cells
failed to recycle, because they were sorted to lysosomes for
degradation. To test this possibility, we performed double fluorescence
studies with rhodamine-dextran in combination with
1AR-GFP. Dextran
is known to specifically accumulate in late endosomes and lysosomes
(36). Chronically DMI-treated C6-
1AR-GFP cells were preincubated
with rhodamine-labeled dextran and subsequently stimulated with
isoproterenol to induce receptor endocytosis. Agonist exposure did not
result in detectable colocalization of
1AR-GFP with the lysosomal
marker. Internalized
1AR-GFP appeared in intracellular structures
that were completely devoid of dextran labeling (Fig.
5), suggesting that the receptors were
retained in early endosomes. The cells were then further incubated at
37 °C in agonist-free medium to allow receptor recycling. At the end
of the resensitization period, still no
1AR-GFP was detected in
lysosomes (not shown). To rule out the possibility that proteolytic receptor degradation prevented the detection of
1AR-GFP that might
have been diverted to lysosomes, the same experiment was performed in
the presence of the protease inhibitor leupeptin (100 µM). Even under these conditions, no
1AR-GFP could be
detected in lysosomes (not shown). Radioligand binding experiments with the membrane-permeant antagonist [3H]dihydroalprenolol
confirmed that in DMI-treated cells internalized
1AR were not
subject to degradation, since no decrease in the number of total
(surface and internal)
AR was observed after long term
desensitization (not shown).

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Fig. 5.
Chronic DMI treatment does not result in
lysosomal targeting of internalized
1AR-GFP. Chronically DMI-treated C6- 1AR-GFP
cells were incubated overnight with rhodamine-dextran to label
lysosomes. Subsequently, cells were stimulated with 10 µM
isoproterenol for 2 h at 37 °C. Agonist exposure did not result
in a colocalization of endocytosed 1AR-GFP (green) with
rhodamine-labeled dextran (red), since no overlapping
structures were detected in the merged
image.
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As an alternative explanation, we hypothesized that the
1AR-GFP of
DMI-treated cells were delivered to a nonrecycling, nonlysosomal compartment. Therefore, we compared the localization of internalized
1AR-GFP with that of transferrin receptors.
AR and transferrin receptors are internalized via distinct primary endocytic vesicles to
the same early endosomes (37). Transferrin receptors then return via a
recycling compartment back to the plasma membrane (38, 39). If, indeed,
internalized
1AR-GFP were delivered to another compartment in
DMI-treated cells, one would expect that in control cells internalized
1AR-GFP and transferrin receptors would colocalize in early
endosomes, whereas in DMI-treated cells no colocalization would occur.
Experimentally, the trafficking of the transferrin receptor may be
followed by rhodamine-labeled transferrin, because transferrin remains
associated with transferrin receptors during internalization and
constitutively recycles with the receptors back to the cell surface
(22, 40). As illustrated in Fig. 6
(central panel), in chronically DMI-treated cells
a significant portion of the internalized
1AR-GFP following
stimulation with isoproterenol colocalized with endocytosed transferrin
receptors (colocalization shown in yellow), confirming that
1AR-GFP were present in early endosomal compartments following a 2-h
exposure to agonist. Intracellular distribution of
1AR-GFP following
agonist stimulation was also studied in untreated cells (Fig. 6,
left panel). The extent of colocalization of
endocytosed
1AR-GFP and transferrin receptors in control cells was
comparable with that in DMI-treated cells, suggesting that chronic DMI
treatment did not affect the primary subcellular localization of
internalized
1AR-GFP. DMI-treated C6-
1AR-GFP cells labeled with
rhodamine transferrin were then allowed to recover in fresh medium at
37 °C for 1 h and were subsequently examined for receptor
distribution. Only a small number of internalized transferrin receptors
was detected at the end of this recovery period, whereas endocytosed
1AR-GFP remained intracellular (Fig. 6, right
panel). Thus, in DMI-treated cells transferrin receptors
were able to return to the plasma membrane but not
1AR-GFP. This
observation suggests that
1AR-GFP are internalized to an early
endosomal compartment like transferrin receptors from where recycling
to the cell surface is possible in principle. However, it seems that
DMI treatment causes posttranslational modifications of the
1AR
itself or of some accessory proteins, leading to an impaired receptor
recycling.

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Fig. 6.
Transferrin receptor recycling in DMI-treated
C6 cells. Untreated (control) and chronically DMI-treated
C6- 1AR-GFP cells were desensitized with 10 µM
isoproterenol for 2 h at 37 °C. During the second hour of the
stimulation period, cells were loaded with rhodamine-labeled
transferrin. DMI-treated cells were then washed and incubated for an
additional 1 h in fresh medium at 37 °C to allow for
resensitization. Localization of 1AR-GFP (green) and
transferrin receptors (red) was examined by laser-scanning
confocal microscopy after desensitization and at the end of the
resensitization period.
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DISCUSSION |
Tricyclic antidepressants are well known for their potential to
modulate the density of functional neurotransmitter receptors such as
1AR and serotonin receptors in the brain (1-3, 41, 42) as well as
in cultured cells (4, 9). The mechanisms for this reduction in receptor
numbers are not completely understood. Importantly, the onset of
down-regulation and clinical effectiveness requires 10-20 days of
antidepressant treatment (43). We have previously shown that in
cultured C6 cells chronic exposure to DMI results in a progressive
reduction in
1AR surface receptor density within similar periods of
time (10). C6 cells thus provide a valid model system to examine
potential mechanisms that might underlie the clinical and experimental
long term observations.
A chimeric protein of GFP fused to the carboxyl terminus of the
1AR
was used as a tool to visualize the processes of receptor internalization and recycling in live cells and to combine these observations with the biochemical assessment of
1AR on the cell surface and the total receptor numbers in whole cells. The feasibility of such an approach depends largely on the compatibility of the fusion
partner at the carboxyl-terminus with the functional integrity of the
receptor. Several studies with carboxyl-terminal fusions of G
protein-coupled receptors (GPCR), such as the cholecystokinin A
receptor (44), the thyrotropin-releasing hormone receptor (45), the A1
adenosine receptor (46), and the
2AR (31, 47), demonstrated that
ligand binding affinities, G protein coupling, and downstream effector
activation are not significantly affected. Similarly, fusing GFP to the
carboxyl terminus of
1- and
2-adrenoceptors only marginally
affects ligand binding (48). It may result in quantitative but not in
qualitative changes in receptor internalization and recycling
properties. However, recent studies have shown that the carboxyl
terminus of the
2AR interacts with a number of proteins that seem to
play an important role in determining the fate of endocytosed receptors
(49, 50). Elimination or alteration of a single amino acid at the
extreme carboxyl terminus of the
2AR has been reported to disrupt
such interaction and to influence receptor sorting (49-52). Whether the addition of GFP to the intact COOH-terminal tail of GPCR also prevents these protein-protein interactions has not been explored. In
this study, ligand binding, activation of adenylyl cyclase, and
receptor internalization as well as recycling properties were similar
for wild-type and for the chimeric receptors. Thus, there is no
evidence that the addition of GFP affects receptor function or
trafficking. However, our results cannot exclude the possibility that
fusion of GFP to the carboxyl terminus of the receptor might modulate
other aspects of
1AR regulation.
Studying agonist induced trafficking of
1AR in C6-
1AR-GFP cells,
significant differences were found between cells that were chronically
exposed to DMI and untreated control cells. Whereas chronic exposure to
DMI had no effect on agonist-induced receptor internalization or on the
localization of internalized receptors, the fate of the internalized
receptors seemed to be quite different under these conditions. In
antidepressant-treated cells, internalized receptors after agonist
stimulation remained trapped intracellularly, whereas in control cells
receptors recycled to the cell surface within the first hour after
agonist removal. It appeared that receptors were internalized normally
but were then redirected to a slowly recycling or even nonrecycling
compartment. Chronic application of tricyclic antidepressants may thus
modulate the fate of the internalized receptors. The long lasting
intracellular receptor accumulation would be consistent with
trafficking along a "long cycle." Such a trafficking route had been
postulated for the V2 vasopressin receptor (53).
Contrary to most other GPCR, stimulation of the
1AR results only in
partial receptor internalization, possibly as a consequence of a weak
association with
-arrestins (24, 54, 55). In C6-
1AR-GFP cells, a
small but reproducible, agonist-induced reduction of the plasma
membrane-associated receptors of ~15% was observed in ligand binding
experiments (Fig. 4A). These properties of the model system
provided the basis for studying cellular receptor trafficking after
antidepressant treatment. Accumulation of internalized receptors could
be readily observed by confocal fluorescence microscopy and was
quantified in three-dimensional models of single cells that were
calculated from restored image stacks. The lower estimate for
internalized receptors obtained from the model data could be due to the
difficulty of separating internalized from plasma membrane-associated
material in cases where internalized material was located in close
proximity to the cell surface or in budding vesicles. This led to the
unavoidable exclusion of some material from the estimate of
internalized receptor. However, we cannot exclude the possibility that
a subpopulation of stimulated receptor is unavailable for ligand
binding after agonist washout but may still be present at the cell
surface. The reduction of receptors in the plasma membrane could not be
visualized, but it could be derived from ligand binding assays, which
demonstrated the initial decrease and the subsequent reappearance of
cell surface receptors after a resensitization period. The combination
of the two approaches in this study proved to provide a powerful method
to study limited receptor trafficking and revealed a defect in receptor
recycling to the cell surface in DMI-treated cells.
Our desensitization studies showed that exposure of C6-
1AR-GFP
cells to isoproterenol led to a marked decline in
receptor-dependent cAMP production, although ligand binding
to
AR at the cell surface was reduced by only 15%. A large portion
of lost binding sites can be attributed to internalization of
1AR-GFP as our estimates from the three-dimensional models show.
Therefore, reduced signaling in desensitized cells may be explained at
least in part by receptor internalization. Chronic treatment of cells
with DMI leads to a comparable reduction in surface
AR density.
Hence, our results suggest that the inability of internalized
1AR-GFP in DMI-treated cells to recycle back to the plasma membrane
is likely to cause a substantial decrease in receptor signaling.
Interestingly, chronic exposure of
1AR-GFP cells to DMI in the
absence of agonist stimulation also led to reduced
AR numbers in a
majority of cases. In contrast to cells that were challenged with
agonist, intracellular receptor accumulation could not be found under
these basal conditions. In agonist-treated cells, no indication for
proteolytic degradation was obtained within a resensitization period of
up to 3 h. This does not exclude the possibility that receptors
could still be subject to lysosomal degradation, albeit using a slow
cycling pathway. As shown for mannose 6-phosphate receptors by
Gonzalez-Noriega et al. (56), definitive fate decisions may
be made as late as 3-4 h after internalization, because receptors
could still be salvaged within this time period, whereas intervention
at later time points could not prevent degradation in the lysosomal
compartment anymore.
We previously reported that chronic DMI exposure results in alterations
of the cellular and membranous phospholipid patterns (11) and promotes
lysosomal phospholipid accumulation (57). The modification of the
physicochemical membrane characteristics does not interfere with
receptor binding and internalization. However, it may be the reason for
an inefficient membrane recycling and thus for incomplete receptor
recycling. Slowing down receptor recycling may lead to a new steady
state distribution in the absence of any changes in the rate of
de novo synthesis of receptors. Since the recycling and its
modification only relate to a fraction of the surface receptors, one
would predict that such a mechanism would take considerable time for a
new steady state of total receptor distribution to be reached. This and
the prolonged time required for changes in the phospholipid patterns
might also explain the observed lag in receptor down-regulation and
possibly in the onset of the clinical effectiveness in the treatment of
depressive states. Future experiments will address the question whether
DMI-induced changes in phospholipid composition could provide a
molecular basis for the altered receptor recycling.
The antidepressant-induced switch redirecting a GPCR from a fast
recycling pathway to a slowly recycling or degradative pathway presents
a new mode for drug action. It appears to engage a cellular machinery
that may divergently sort receptors of different families after their
endocytosis (58). As the underlying molecular mechanisms, two major
principles may be envisioned. Antidepressant-induced changes in the
phosphorylation pattern of specific carboxyl-terminal regions of the
receptor may induce strong retention of endocytosed receptors in
intracellular compartments. Such a
phosphorylation-dependent mechanism is exemplified by the
vasopressin 2 receptor (59). Alternatively, association of receptors
with cellular targets may be regulated by drug-induced changes of
cellular components other than the receptor. In such a model, the
receptor would not be the direct target of the chronic drug treatment.
Rather, the cellular handling of the internalized receptor would differ
in accordance with the drug treatment. In that respect, it should be
noted that a substantial collection of receptor-associated proteins,
including
-arrestins, scaffold proteins, and protein kinases,
co-migrate with the receptor during internalization and recycling (60,
61). These components represent attractive candidates for the
antidepressant-induced switch of receptor fate. The present data would
be consistent with either of the two potential mechanisms. The
identification of the molecular correlate(s) responsible for the
divergence from the normal recycling pathway is likely to link well
known signaling components to the action of antidepressants.
In summary, our results demonstrate that chronic exposure to the
tricyclic antidepressant DMI impairs
1AR recycling in rat C6
glioblastoma cells. Agonist-stimulated
1AR are internalized normally
but are then redirected to a slowly recycling or even nonrecycling
compartment. Antidepressant-induced reduction of
1AR density may
thus be caused by a modulation of receptor trafficking routes.