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INTRODUCTION |
It has been shown that the binding characteristics of one type of
G protein-coupled receptor can be altered by the stimulation of another
type of G protein-coupled receptor in crude membrane preparations (1).
Such intramembrane interactions have been postulated to represent
direct interactions between the receptor molecules and/or to involve G
proteins or other mobile molecules associated with the membrane (1).
There is increasing evidence suggesting that antagonistic intramembrane
interactions between specific subtypes of adenosine and dopamine
receptors constitute an important integrative mechanism in the basal
ganglia (2, 3). Adenosine A1 and A2A receptors
antagonistically and specifically modulate the binding characteristics
of dopamine D1 and D2 receptors, respectively
(2, 3). In membrane preparations from rat striatum, the stimulation of
A2A receptors decreases the affinity of D2 receptors for agonists (4). On the other hand, the stimulation of
A1 receptors was shown to decrease the proportion of
D1 receptors in the high affinity state, without modifying
the dissociation constants of high and low affinity D1
agonist-binding sites (5). Thus, the A1 receptor agonist
had the same effect as that induced by the GTP analogue
Gpp(NH)p.1 It was
hypothesized that A1 receptor stimulation might uncouple the striatal D1 receptor from the G protein (5). There is
evidence that the antagonistic A2A-D2 and
A1-D1 intramembrane interactions are involved
in the motor depressant effects of adenosine receptor agonists and the
motor stimulant effects of adenosine receptor antagonists, such as
caffeine (2-5).
The same changes in the binding characteristics of striatal
D2 receptors after A2A receptor stimulation
have been obtained in membrane preparations from a mouse fibroblast
cell line (Ltk
) stably cotransfected with the dog
A2A receptor and human D2 (long-form) receptor
cDNAs (6). In these transfection studies, it was also found that
activation of adenylyl cyclase was not involved in the intramembrane
A2A-D2 interaction (6). Altogether, these
results showed that stably cotransfected cell lines constitute a
valuable model to study the mechanistic aspects involved in the
intramembrane receptor-receptor interactions. In the present work, this
methodology has been applied to study the antagonistic interaction
between A1 and D1 receptors. The first aim of
the study was to demonstrate the existence of an antagonistic
A1-D1 intramembrane interaction in mammalian
cells stably cotransfected with A1 receptor and
D1 receptor cDNAs. The second aim was to demonstrate
the existence of a functional antagonistic interaction between
A1 and D1 receptors in the cotransfected cells
by means of cAMP accumulation experiments. Finally, the third aim of
the study was to find a functional significance of the antagonistic A1-D1 intramembrane interaction.
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EXPERIMENTAL PROCEDURES |
Transfection and Maintenance of Fibroblast Ltk
Cells--
Cells from the mouse fibroblast Ltk
cell line
previously transfected with the human D1 receptor cDNA
(7) were used. The expression vector pZEM-3 (8) containing the full
coding sequence of the human D1 receptor in front of mouse
metallothionein promoter I had been cotransfected with the plasmid
pRSV-neo, which confers resistance to neomycin and Geneticin (G418).
Metallothionein promoter I allows transcriptional induction by
including zinc sulfate in the cell culture (8). Nevertheless, a clone
expressing a relatively high level of D1 receptor mRNA
and protein was obtained (D1 cells) without zinc-mediated
induction (7). The D1 cells were cotransfected with the
human adenosine A1 receptor cDNA
(A1D1 cells). The expression vector pcDNA3
(Invitrogen) containing the full coding sequence of the human
A1 receptor (gift from M. Lohse) (9, 10) in front of
enhancer-promoter sequences from the immediate-early gene of the human
cytomegalovirus was cotransfected with a hygromycin resistance plasmid
(pHyg; gift from G. Vassart) with the calcium phosphate precipitation
method as described in detail (6). The expression of the A1
receptor was verified by Northern blot and radioligand binding
techniques (see below), and a clone expressing similar levels of
A1 and D1 antagonist-binding sites
(A1D1 cells) was chosen for further
experiments. A1D1 cells were cultured routinely at 37 °C with 5% CO2 in Dulbecco's minimal essential
medium with 4.5 mg/ml glucose and 0.11 mg/ml sodium pyruvate
supplemented with 10% fetal calf serum, 2 mM glutamine,
100 units/ml penicillin, 100 units/ml streptomycin, 200 µg/ml G418,
and 300 µg/ml hygromycin in plastic Petri dishes. D1
cells were cultured as described for A1D1
cells, but without hygromycin. The splitting of cell cultures was
performed by replacing the medium with a modified Puck's saline containing trypsin (0.5 mg/ml) and EDTA (0.2 mg/ml). The experiments were performed at a cell confluence of ~80%.
Analysis of RNA--
Isolation of RNA from the Ltk
cells was carried out according to the method of Chomcyznski and Sacchi
(11). For preparation of the Northern blots, 20 µg of total RNA/lane
was denatured in a 2.1 M formaldehyde and 50% formamide
solution by heating for 2 min at 95 °C, separated by electrophoresis
on a 2.2 M formaldehyde and 1.0% agarose gel, and
transferred to a nitrocellulose membrane. Blots were hybridized with
32P-labeled adenosine A1 receptor cDNA by
nick translation. Following hybridization, the membrane was washed and
exposed to Kodak XAR-5 film with an intensifying screen at
70 °C.
The optical density of the bands was measured by computer-assisted
densitometric analysis (IBAS image analyzer).
Membrane Preparation--
The D1 and
A1D1 cells were lifted from Petri dishes with a
cell scraper. Harvested cells were washed twice with ice-cold
phosphate-buffered saline and centrifuged at 2000 rpm for 5 min at
4 °C. The cell pellet was sonicated (30 s) and resuspended in the
incubation buffer containing adenosine deaminase (Boehringer Mannheim;
10 units/ml). The homogenate was centrifuged at 3000 rpm for 10 min at
4 °C; the precipitated nucleic fraction was discarded; and the
supernatant was incubated for 30 min at 37 °C (to activate adenosine
deaminase and to remove endogenous adenosine) and centrifuged at 20,000 rpm for 40 min at 4 °C. The membrane pellet was then resuspended by
sonication in the incubation buffer without adenosine deaminase (final
protein concentration of ~0.2 mg/ml). In the experiments with
[3H]SCH 23390 (NEN Life Science Products), the incubation
buffer was 50 mM Tris-HCl (pH 7.4) containing 120 mM NaCl, 5 mM KCl, 2 mM
CaCl2, and 1 mM MgCl2. In the
experiments with 1,3-[3H]dipropyl-8-cyclopentylxanthine
([3H]DPCPX; NEN Life Science Products), the incubation
buffer was 50 mM Tris-HCl (pH 7.4) containing 2 mM MgCl2.
Pertussis Toxin Pretreatment and
[32P]ADP-ribosylation of Membranes--
Experiments with
pertussis toxin (PTX) were performed with A1D1
cells exposed to PTX (200 ng/ml) for 4 h before membrane
preparation for radioligand binding experiments or cAMP accumulation
experiments. The effectiveness of PTX pretreatment and the consequent
inactivation of Gi proteins were evaluated in membrane
preparations by subsequent [32P]ADP-ribosylation in the
presence of [32P]NAD and PTX (12). PTX was preactivated
in 50 mM 1,4-dithiothreitol for 1 h at 25 °C. The
reaction mixture (100 µl) contained 0.5-1 mg/ml protein, 1 mM ATP, 10 mM thymidine, 250 µM
GTP, 1 µM [32P]NAD (0.5-1 Ci/mmol), and 20 µg/ml PTX in a buffer containing 50 mM Tris-HCl (pH 7.4).
At the end of a 45-min incubation period at 30 °C, the reaction was
stopped by adding 10 µl of 1% sodium deoxycholate followed by 12 µl of 4 M perchloric acid. The samples were kept on ice
for 20 min and centrifuged for 5 min at 20,000 rpm. The pellet was
neutralized with 1 M NaOH; 30 µl of Laemmli buffer was
added; and the proteins were separated by SDS-polyacrylamide gel
electrophoresis with 10% acrylamide. Autoradiography was performed by
exposure of dried gels to Fuji RXNIF film for 2-4 days.
Radioligand Binding Experiments--
Saturation experiments with
the D1 receptor antagonist [3H]SCH 23390 were
carried out with 10 concentrations (0.2-6.0 nM) of [3H]SCH 23390 (70.3 Ci/mmol) in the presence or absence
of the A1 receptor agonist
N6-cyclopentyladenosine (CPA; 10 nM) by incubation for 15 min at 37 °C. Nonspecific
binding is defined as the binding in the presence of 100 µM dopamine. Saturation experiments with the
A1 receptor antagonist [3H]DPCPX were carried
out with 10 concentrations (0.6-27.7 nM) of
[3H]DPCPX (120.0 Ci/mmol) by incubation for 2 h at
room temperature. Nonspecific binding is defined as that occurring in
the presence of the adenosine A1 receptor agonist
N6-cyclohexyladenosine (40 µM). Competition experiments of dopamine (1 nM to 10 mM) versus the dopamine
D1 antagonist [3H]SCH 23390 (~2
nM) were performed by incubation for 15 min at 37 °C in
the presence or absence of CPA or the nonhydrolyzable GTP analogue
Gpp(NH)p. Competition experiments of CPA (10 pM to 10 µM) versus [3H]DPCPX (~1
nM) were performed by incubation for 2 h at room
temperature. The incubation was stopped by fast filtration through
glass-fiber filters (GF/B, Whatman) by washing three times with 5 ml of
50 mM ice-cold Tris-HCl (pH 7.4) with an automatic cell
harvester (Brandel). The radioactivity content of the filters was
detected by liquid scintillation spectrometry. To avoid the variability of the binding parameters associated with the assay conditions, such as
cell confluency (~80%) and number of passages (up to 10), the same
membrane preparation was used to study the effect of different drugs on
the binding characteristics of dopamine D1 receptors, and
each experiment was independently analyzed. Data from saturation
experiments were analyzed by nonlinear regression analysis (GraphPad)
for the determination of dissociation constants (KD)
and the number of receptors (Bmax). Data from
competition experiments were also analyzed by nonlinear regression
analysis, and the fitting for either one or two binding sites was
statistically compared (F test). For a two-binding site fit,
the dissociation constants for the high (KH) and
low affinity (KL) binding sites and for the
proportion of binding sites in the high affinity state (RH) were determined. For a one-binding site
fit, the concentration of agonist that displaced 50% of the labeled
antagonist (IC50) was determined. The amount of nonspecific
binding was calculated by extrapolation of the displacement curve.
Protein determinations were performed using bovine serum albumin as a
standard. The Kruskal-Wallis test and Mann-Whitney's U test
were used to analyze differences in KD,
Bmax, KH, and
KL values.
cAMP Accumulation Experiments--
After scraping the cells off
the culture plates, they were washed twice with phosphate-buffered
saline and resuspended in serum-free medium at a concentration of
0.5-1.2 × 106 cells/ml. Aliquots of 0.2 ml were
transferred to test tubes along with the phosphodiesterase inhibitor
rolipram (30 µM) added to a final volume of 0.3 ml. The
reaction was terminated with 50 µM perchloric acid to a
final concentration of 0.1 M after a 10-min incubation at
37 °C. Samples were neutralized with 60 µl of KOH, and the cAMP
content in the supernatants was determined with a protein binding assay
(13). The following experiments were performed: first, the effect of
different concentrations of CPA (0.3-300 nM) on the cAMP
accumulation induced by forskolin (30 µM) and dopamine
(0.1 µM); second, the effect of CPA (30 nM)
and DPCPX (30 nM) on the cAMP accumulation induced by
different concentrations of dopamine (10 nM to 30 µM); and third, the effect of different concentrations of
CPA (0.01-10 µM) on the cAMP accumulation induced by
dopamine (10 µM) after PTX pretreatment (200 ng/ml for
4 h). Student's t test and one-way and bifactorial
ANOVA (followed by post hoc protected least square difference method)
were used for statistical analysis.
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RESULTS |
Northern Blotting and Saturation Experiments with the Adenosine
A1 Antagonist [3H]DPCPX--
Different
levels of A1 receptor mRNA were obtained with the
Northern blot analysis of the different hygromycin-resistant clones. A1 receptor mRNA was not detected in cells not
transfected with A1 receptor cDNA (D1
cells) (Fig. 1). A significant
correlation (linear regression analysis, r2 = 0.93 and p = 0.0001) was found between the relative
A1 receptor mRNA content (optical density) obtained
from the Northern blot analysis and the A1 receptor density
(log Bmax values) obtained from the saturation
experiments with [3H]DPCPX in membrane preparations from
the different clones (Fig. 1). The nonspecific binding was <5% of the
total binding. The D1 cells did not show any significant
[3H]DPCPX-specific binding. A clone with similar
concentrations of A1 and D1 binding sites
(A1D1 cells) was chosen for the subsequent experiments. The Bmax and KD
values for [3H] DPCPX binding in the
A1D1 cells were 4.0 ± 0.4 pmol/mg of
protein and 2.0 ± 0.1 nM (means ± S.E.,
n = 4), respectively (Fig.
2). The determined KD
value is in close agreement with the values previously reported for
membrane preparations from Chinese hamster ovary cells and
Escherichia coli cells expressing human A1
receptors (9, 10, 14). For comparison, the Bmax
and KD values for [3H]DPCPX binding in
membrane preparations from rat striatum have been reported to be ~1
pmol/mg of protein and 1 nM, respectively (15).

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Fig. 1.
Northern blot analysis of different clones
transfected with adenosine A1 receptor cDNA. A
significant correlation between the relative A1 receptor
mRNA content (optical density), obtained from Northern blot
analysis, and the total concentration of A1 receptors (log
pmol/mg of protein), analyzed in radioligand binding experiments, was
obtained in different clones of cells cotransfected with human
adenosine A1 and dopamine D1 receptor
cDNAs. Clone 7 was selected as
A1D1 cells. Lane C, control
D1 cells.
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Fig. 2.
Representative saturation curve of specific
binding of the adenosine A1 receptor antagonist
[3H]DPCPX in membrane preparations from
A1D1 cells. The
Bmax and KD values obtained
by nonlinear regression analysis were 4.9 pmol/mg of protein and 1.8 nM, respectively.
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Competition Experiments of the Adenosine A1 Agonist CPA
Versus the Adenosine A1 Antagonist
[3H]DPCPX--
Competition experiments of the adenosine
A1 agonist CPA versus the adenosine
A1 antagonist [3H]DPCPX in membrane
preparations from A1D1 cells showed a
significantly better fit for two binding sites than for one binding
site (F test, p < 0.05).
KH, KL, and
RH values (in medians and, in parentheses, the
interquartile range) were estimated to 4.2 (22.1) nM, 163.0 (145.2) nM, and 8.0% (5.8), respectively. The determined
KL value is similar to the Ki
value obtained from competition experiments of CPA versus
[3H]DPCPX in membrane preparations from E. coli cells expressing human A1 receptors (14) (the
absence of G proteins in E. coli membranes precludes the
analysis of the high affinity state of a G protein-linked
receptor).
Saturation Experiments with the Dopamine D1 Antagonist
[3H]SCH 23390--
No significant differences were
obtained between the D1 and A1D1
cells regarding the Bmax and
KD values for the D1 binding sites
labeled with [3H]SCH 23390. The
Bmax values for the D1 and
A1D1 cells were 4.2 ± 0.2 and 4.6 ± 0.3 pmol/mg of protein (means ± S.E.), respectively. The
KD values for the D1 and
A1D1 cells were 2.6 ± 0.3 and 2.4 ± 0.2 nM (means ± S.E., n = 4),
respectively. The nonspecific binding was <5% of the total binding.
The A1 agonist CPA did not significantly alter
[3H]SCH 23390 binding in A1D1
cell membranes (Bmax = 4.4 ± 0.1 pmol/mg of protein and KD = 2.2 ± 0.2 nM
(means ± S.E.)) (Fig. 3).

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Fig. 3.
Representative saturation curves of specific
binding of the dopamine D1 receptor antagonist
[3H]SCH 23390 in membrane preparations from
A1D1 cells. The Bmax and KD values obtained
by nonlinear regression analysis were 4.5 pmol/mg of protein and 2.5 nM, respectively, under control conditions and 4.6 pmol/mg
of protein and 2.6 nM, respectively, in the presence of the
A1 agonist CPA (10 nM).
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Competition Experiments of Dopamine Versus the Dopamine
D1 Antagonist [3H]SCH 23390--
Competition
experiments of dopamine versus the dopamine D1
antagonist [3H]SCH 23390 in membrane preparations from
both D1 and A1D1 cells showed a
significantly better fit for two binding sites than for one binding
site (F test, p < 0.05). Similar
KH and KL values were
obtained in membrane preparations from D1 and
A1D1 cells, and the proportion of
D1 receptors in the high affinity state (RH values) was ~10% in both cases (Fig.
4 and Table
I). In the presence of Gpp(NH)p (100 µM), a significantly better fit for one binding site
(RH = 0) was obtained in most of the membrane preparations from either D1 or A1D1
cells, with IC50 values very similar to the
KL values obtained in the absence of Gpp(NH)p. The same effect as that induced by Gpp(NH)p was obtained in the presence of the A1 agonist CPA (1-100 nM) in
membrane preparations from A1D1 cells. On the
other hand, CPA (0.1 and 10 µM) was ineffective in
membrane preparations from D1 cells (Fig. 4 and Table I). Pretreatment of the A1D1 cells with PTX
counteracted the effect of a low concentration of CPA (10 nM), but it did not counteract the effect of 10 µM CPA or 100 µM Gpp(NH)p (Fig.
5 and Table I). The degree of PTX-induced
[32P]ADP-ribosylation was markedly reduced in membrane
preparations from PTX-pretreated A1D1 cells
compared with nonpretreated cells (Fig.
6).

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Fig. 4.
Representative competitive inhibition curves
of dopamine versus the D1 receptor antagonist
[3H]SCH 23390 in membrane preparations from
D1 (a and b) and
A1D1 (c and d)
cells. In both D1 (a) and
A1D1 cells (c), the GTP analogue
Gpp(NH)p (100 µM) induced a shift to the right of the competitive inhibition curves of dopamine versus
[3H]SCH 23390. Nonlinear regression analysis indicated
that this effect is due to the disappearance of the dopamine
D1 receptors in a high affinity state. The same effect was
obtained in A1D1 cells (d) with the
A1 agonist CPA (10 nM), but CPA (100 nM) was ineffective in D1 cells
(b).
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Table I
Competition-inhibition experiments of dopamine versus the dopamine
D1 antagonist [3H]SCH 23390 in membrane
preparations from A1D1 and D1 cells
RH, KH, and
KL/IC50 values are expressed as medians, and
the interquartile ranges are given in parentheses (n = 4-8/experiment).
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Fig. 5.
Representative competitive inhibition curves
of dopamine versus the D1 receptor antagonist
[3H]SCH 23390 in membrane preparations from
A1D1 cells after PTX pretreatment. In
A1D1 cells, PTX pretreatment counteracted the
shift to the right of the competitive inhibition curves of dopamine
versus [3H]SCH 23390 induced by a low
concentration of the A1 agonist CPA (10 nM). On
the other hand, the effects of the GTP analogue Gpp(NH)p (100 µM) or a high concentration of CPA (10 µM)
were not counteracted by PTX.
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Fig. 6.
Effects of PTX on the
[32P]ADP-ribosylation of G proteins in membrane
preparations from A1D1 cells. PTX
ADP-ribosylates G proteins with molecular masses of of 39-41 kDa.
[32P]ADP-ribosylation is markedly reduced in
A1D1 cells previously exposed to PTX compared
with controls. The positive control (+) contained whole rat brain; the
negative control ( ) contained buffer. PTX and C
(control) represent A1D1 cells previously
pretreated or not with PTX, respectively.
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cAMP Accumulation Experiments--
In A1D1
cells, but not in D1 cells (data not shown), CPA induced a
significant concentration-dependent inhibition of cAMP accumulation induced by 30 µM forskolin and 0.1 µM dopamine (one-way ANOVA, p < 0.001 in
both cases), with IC50 values (95% confidence intervals in
parentheses) of 0.9 (0.2-2.8) nM and 0.8 (0.3-1.8) nM, respectively (Fig. 7). In
A1D1 cells, dopamine induced a significant concentration-dependent increase in cAMP accumulation (Fig.
8). The effect of dopamine was
significantly antagonized by CPA (30 nM) (bifactorial
ANOVA, p < 0.001 for the factors dopamine and CPA)
(Fig. 8). The EC50 values (95% confidence intervals in
parentheses) of the concentration-response curves of dopamine in the
absence and presence of CPA, obtained by nonlinear regression analysis, were 0.2 (0.03-1.6) µM and 0.6 (0.02-14.9)
µM, respectively. The maximal response values (95%
confidence intervals in parentheses) in the absence and presence of CPA
were 0.5 (0.4-0.6) pmol/50 µl and 0.3 (0.2-0.4) pmol/50 µl,
respectively. On the other hand, the effect of dopamine was
significantly potentiated by DPCPX (30 nM) (bifactorial
ANOVA, p < 0.001 for the factors dopamine and DPCPX)
(Fig. 8). The EC50 values (95% confidence intervals in
parentheses) of the concentration-response curves of dopamine in the
absence and presence of DPCPX were 0.4 (0.2-0.9) µM and 0.3 (0.09-0.8) µM, respectively. The maximal response
values (95% confidence intervals in parentheses) in the absence and
presence of DPCPX were 0.9 (0.8-0.9) pmol/50 µl and 1.2 (1.0-1.3)
pmol/50 µl, respectively. Since they were independently analyzed, the basal levels of cAMP (dopamine concentration = 0) were not
included in the ANOVA. cAMP basal levels were significantly increased
by DPCPX (Student's t test, p < 0.05), and
they were not modified by CPA (Fig. 8). In PTX-pretreated
A1D1 cells, the basal levels of cAMP were
significantly higher than in the control experiment, without PTX
pretreatment (0.41 ± 0.01 and 0.23 ± 0.01 pmol/50 µl
(means ± S.E.), respectively). CPA also induced a significant concentration-dependent inhibition of cAMP accumulation
induced by a high concentration of dopamine (10 µM), and
the cAMP accumulation induced by dopamine (10 µM) was
significantly higher in PTX-pretreated cells (bifactorial ANOVA,
p < 0.001 for the factors CPA and PTX) (Fig.
9). However, CPA was more effective in
control cells. In PTX-pretreated cells, only a high concentration of
CPA (10 µM) significantly antagonized cAMP accumulation
induced by dopamine (10 µM) (post hoc one-way ANOVA,
p < 0.05) (Fig. 9).

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Fig. 7.
Counteracting effects of the adenosine
A1 agonist CPA on the cAMP accumulation induced by
forskolin (30 µM) and dopamine (0.1 µM) in
A1D1 cells. Results are expressed as
means ± S.E. (n = 6, in triplicate/experiment).
Basal cAMP accumulation levels for forskolin and dopamine experiments
were 1.2 ± 0.1 and 1.3 ± 0.1 pmol/50 µl (means ± S.E.), respectively.
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Fig. 8.
Counteracting effect of the adenosine
A1 agonist CPA (30 nM) and potentiating effect
of the A1 antagonist DPCPX (30 nM) on the cAMP
accumulation induced by dopamine in A1D1
cells. Results are expressed as means ± S.E.
(n = 6, in triplicate/experiment).
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Fig. 9.
Counteracting effects of the adenosine
A1 agonist CPA on the cAMP accumulation induced by dopamine
(10 µM) in A1D1 cells after PTX
pretreatment. Results are expressed as means ± S.E. (n = 3, in triplicate/experiment). Basal cAMP
accumulation levels for control and PTX-pretreated cells were 0.2 ± 0.02 and 0.4 ± 0.04 pmol/50 µl (means ± S.E.),
respectively. * and **, significantly different compared with 0 CPA
(p < 0.05 and p < 0.01, respectively).
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DISCUSSION |
A mouse fibroblast Ltk
cell line stably
cotransfected with human A1 and D1 receptor
cDNAs was obtained, and a clone containing similar amounts of both
receptors (~4 pmol/mg of protein) was chosen
(A1D1 cells). Both receptors were shown to be
functional in cAMP accumulation experiments. It has been previously
shown that dopamine induces a concentration-dependent
increase in cAMP accumulation in Ltk
cells transfected
with the human D1 receptor cDNA (D1 cells), but not in nontransfected cells (7). It was also shown that the effect
of dopamine is mediated by D1 receptors since it was selectively counteracted by a D1 but not a D2
receptor antagonist (7). In the present experiments, dopamine-induced
cAMP accumulation was also demonstrated in the
A1D1 cells, with an EC50 value very similar to the KH value obtained in the
[3H]SCH 23390-dopamine competition experiments (~0.3
µM). Furthermore, in A1D1 cells,
the A1 agonist CPA counteracted the cAMP accumulation induced by dopamine or forskolin, with an IC50 value
similar to the KH value shown in
[3H]DPCPX-CPA competition experiments (1 nM
range). In addition, the A1 antagonist DPCPX was found to
significantly increase the basal levels of cAMP and to potentiate
dopamine-induced cAMP accumulation. Altogether, these results show the
existence of a functional antagonistic interaction between
A1 and D1 receptors in
A1D1 cells. Nonlinear regression analysis
indicated that the CPA- and DPCPX-mediated effects on dopamine-induced
cAMP accumulation were mainly due to changes in the maximal stimulation
without changes in EC50. This suggests that, in agreement
with the results obtained from radioligand binding experiments, the
A1 receptor-mediated modulation of D1 receptors
does not involve changes in the affinity of D1 receptors
for agonists. This is in contrast to the A2A-D2
interaction (see the Introduction), where A2A receptor
stimulation induces a decrease in the affinity of D2
receptors for agonists (4, 6). The effects of the A1
antagonist also suggest that adenosine released by these cells exerts a
tonic inhibition of D1 receptor-mediated function through
the A1-D1 interaction.
The radioligand binding experiments carried out with membrane
preparations from the cotransfected A1D1 cells
showed results very similar to those obtained with rat striatal
membrane preparations (5). The only difference was the lower proportion
of D1 receptors in the high affinity state
(D1H) in the A1D1 cells (~10%)
as compared with the rat striatal membranes (~30%) (5). Since it has
been previously shown that the density of D1H correlates
with the G protein content and with the endogenous dopamine levels
(16), this difference might reflect either a low content of G proteins or the absence of a previous exposure of the D1 receptors
to dopamine. Both the GTP analogue Gpp(NH)p and CPA induced a
significant reduction in the proportion of D1H. Gpp(NH)p,
but not CPA, was also effective in membrane preparations from control
cells containing D1 but not A1 receptors, which
demonstrates that the A1 receptors are required for CPA to
have an effect. Since D1H represents the D1 receptors coupled to the G protein, these results can be interpreted as
an uncoupling of the D1 receptor from its G protein induced by A1 receptor stimulation.
PTX induces an uncoupling of the A1 receptor from its G
protein by inducing an ADP-ribosylation of the G
subunit
of the Gi (and Go) protein family. This results
in a reduction of the number of A1 receptors in the high
affinity state and in a blockade of A1 receptor signal
transduction (17-19). A1D1 cells were exposed to PTX to study the possible involvement of Gi proteins in
the A1 receptor-mediated uncoupling of the D1
receptor from the Gs protein. As with the blockade of
A1 receptors with the A1 antagonist DPCPX, PTX
induced a significant increase in the basal levels of cAMP and
potentiated dopamine-induced cAMP accumulation. This gives functional
support for the blockade of A1 receptor signal transduction
by PTX in these experiments. It was found that PTX counteracted the
effect of CPA (10 nM), but not of Gpp(NH)p, on D1 receptor binding characteristics, suggesting that the
Gi protein was, in fact, necessary for the intramembrane
A1-D1 interaction. However, a higher
concentration of CPA (10 µM), which is sufficient to bind
to the A1 receptor in the low affinity state in the
A1D1 cells (see competitive inhibition curves
of CPA versus [3H]DPCPX), could still uncouple
D1 receptors from the Gs protein after PTX
pretreatment. This effect of CPA was not reproduced in D1
cells, which shows that it is not a nonspecific effect, but is
A1 receptor-mediated. Furthermore, in agreement with the radioligand binding experiments, a high concentration of CPA (10 µM) was still able to significantly decrease
dopamine-induced cAMP accumulation after PTX pretreatment. The
PTX-induced ribosylation, although very distinct, was not complete (see
the SDS-polyacrylamide gel in Fig. 6). Therefore, it is still possible
that stimulation of the low amount of A1 receptors in the
high affinity state left after PTX pretreatment with the high
concentration of CPA would be able to reproduce the same effect as the
low concentration of CPA in PTX-nonpretreated cells. Nevertheless, the
clear correlation between the results obtained with the radioligand
binding and cAMP accumulation experiments suggests that the
intramembrane A1-D1 interaction involved in the
binding experiments is related to the A1-D1
interaction found at the adenylyl cyclase level.
In summary, three main findings have been obtained in this work. The
first finding is that in membrane preparations from stably cotransfected A1D1 cells, the stimulation of
A1 receptors induces an uncoupling of the D1
receptor from its G protein. This intramembrane A1-D1 interaction has the same characteristics
as that previously found in rat striatum (5). The demonstration of this
interaction in an artificial and very different cellular type and
cellular environment strongly suggests that these kind of intramembrane receptor-receptor interactions (1) represent a generalized functionally
important mechanism in mammalian cells. The second finding is a
functional antagonistic A1-D1 interaction at
the adenylyl cyclase level. Although previously shown in homogenates of
rat striatum (20), this is the first time that such an interaction has
been demonstrated at the cellular level. Finally, the third finding is
the correlation between the results obtained with the radioligand
binding and cAMP accumulation experiments, suggesting that the
intramembrane A1-D1 interaction involved in the
binding experiments is related to the A1-D1
interaction found at the adenylyl cyclase level. Similar interactions
are likely to occur in nerve cells that express both A1 and
D1 receptors, such as the
-aminobutyric acidergic
strionigral-strioentopeducular neurons (21).