Adenosine A1 Receptor-mediated Modulation of Dopamine D1 Receptors in Stably Cotransfected Fibroblast Cells*

Sergi FerréDagger §, Maria TorvinenDagger , Katerina AntoniouDagger , Eva Irenius, Olivier Civellipar , Ernest Arenas**, Bertil B. Fredholm, and Kjell FuxeDagger

From the Departments of Dagger  Neuroscience,  Physiology and Pharmacology, and ** Molecular Biochemistry and Biophysics, Karolinska Institute, S-171 77 Stockholm, Sweden and the par  Department of Pharmacology, University of California, Irvine, California 92697-4625

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The antagonistic interactions between adenosine A1 and dopamine D1 receptors were studied in a mouse Ltk- cell line stably cotransfected with human adenosine A1 receptor and dopamine D1 receptor cDNAs. In membrane preparations, both the adenosine A1 receptor agonist N6-cyclopentyladenosine and the GTP analogue guanyl-5'-yl imidodiphospate induced a decrease in the proportion of dopamine D1 receptors in a high affinity state. In the cotransfected cells, the adenosine A1 agonist induced a concentration-dependent inhibition of dopamine-induced cAMP accumulation. Blockade of adenosine A1 receptor signal transduction with the adenosine A1 receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine or with pertussis toxin pretreatment increased both basal and dopamine-stimulated cAMP levels, indicating the existence of tonic adenosine A1 receptor activation. Pretreatment with pertussis toxin also counteracted the effects of low concentrations of the A1 agonist on D1 receptor-agonist binding. The results suggest that adenosine A1 receptors antagonistically modulate dopamine D1 receptors at the level of receptor binding and the generation of second messengers.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

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.

    EXPERIMENTAL PROCEDURES
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Introduction
Procedures
Results
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References

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.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

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.

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).

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.

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).

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

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 Galpha 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 gamma -aminobutyric acidergic strionigral-strioentopeducular neurons (21).

    FOOTNOTES

* This work was supported by grants from the Swedish Research Council, the Marianne and Marcus Walenberg Foundation, CIRIT (Generalitat de Catalunya), and Åke Wibergs Stiftelse and by BIOMED 2 Program BMH4-CT96-0238.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed. Tel.: 46-8-7287081; Fax: 46-8-3379411.

1 The abbreviations used are: Gpp(NH)p, guanyl-5'-yl imidodiphospate; DPCPX, 1,3-dipropyl-8-cyclopentylxanthine; PTX, pertussis toxin; CPA, N6-cyclopentyladenosine; ANOVA, analysis of variance.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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