Dopamine D₂ Receptor Activation Causes Mitogenesis via p44/42 Mitogen-Activated Protein Kinase in Opossum Kidney Cells

Cell Culture

OK cells (American Type Culture Collection, Rockville, MD) were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (FCS), 3% gentamicin, and 0.4% amphotericin. The cells were maintained in monolayers at 37°C in 90% air/10% CO₂. All experiments were performed with cells of passages 37 to 46. Cell culture media and related chemicals were purchased from Hyclone (Logan, UT).

Radioligand Binding

Cells were seeded in 150-mm dishes and allowed to reach confluence (6 to 7 d). Confluent cells were FCS-starved for 24 h before experiments. OK cell membranes were prepared by scraping and suspending the cells from 10 or 11 dishes in ice-cold phosphate-buffered saline (140 mM NaCl, 3 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄, pH 7.4). The suspension was centrifuged at 3000 × g for 6 min at 4°C. The pellet was resuspended in hypotonic buffer (5 mM Tris-HCl, 5 mM ethylenediaminetetraacetate, pH 7.4) containing a protease inhibitor cocktail (Boehringer Mannheim, Mannheim, Germany). The suspension was homogenized using a tissue homogenizer at setting 6, with three pulses of 5 s each. The homogenate was then centrifuged at 200 × g for 5 min at 4°C. The supernatant was separated and further centrifuged at 37,000 × g for 20 min at 4°C, to yield the crude membrane pellet. The pellet was resuspended in binding buffer (50 mM Tris-HCl, 1 mM ethylenediaminetetraacetate, 5 mM KCl, 1.5 mM CaCl₂, 4 mM MgCl₂, 120 mM NaCl, pH 7.4) at a protein concentration of 1 mg/ml. For saturation curves, 25 μg of membrane preparation was incubated with increasing concentrations (0.1 to 4 nM) of [³H]spiperone (Amersham, Elk Grove, IL), in 250 μl (final volume) of binding buffer, at 25°C for 1 h. Nonspecific binding was defined using 10 μM butaclamol.

Dopamine D₂-Like Receptor Subtypes

Approximately 1 × 10⁶ cells/well were seeded in six-well plates and grown to confluence (3 to 4 d). Before the experiments, the cells were maintained in FCS-free medium for 24 h. On the day of the experiment, the cells were rinsed twice with FCS-free medium and dissolved in sodium dodecyl sulfate (SDS)-Laemmli buffer. The protein content in each cell extract was assayed using a bicinchoninic acid protein assay kit, and the protein concentration was adjusted to 2 mg/ml. Approximately 45 μg/lane (170 μg/lane for D₃ receptors) of protein was resolved by SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA). The membrane was probed with D₂-, D₃-, or D₄-specific antibodies (1:500), followed by horseradish peroxidase-conjugated secondary antibody (1:15,000; Santa Cruz Biotechnology, Santa Cruz, CA) for detection of the presence of the receptor subtype. Finally, the PVDF membrane was incubated with enhanced chemiluminescence substrate (Santa Cruz Biotechnology) and exposed to x-ray film (Kodak, Rochester, NY). To ensure the specificity of the primary antibody for the receptor protein, control experiments in which the primary antibody was preincubated with its corresponding blocking peptide, to block the receptor binding site, were performed in parallel. Preincubation was performed at room temperature for 2 h, according to the instructions provided by the manufacturer (Santa Cruz Biotechnology).

[³H]Thymidine Incorporation

Mitogenesis was measured as a function of [³H]thymidine incorporation. Approximately 1 × 10⁵ cells/well were seeded in 24-well plates and incubated at 37°C until they reached approximately 90% confluence. Before the experiments, cells were washed twice with FCS-free medium and then fresh FCS-free medium (495 μl) was added to each well. For establishment of the concentration-response curve, cells were treated with vehicle (basal) or different concentrations of D₂-like, D₂, D₃, or D₄ receptor agonist (5 μl, 0.0001 μM to 1 μM) for 16 h. After 16 h, 1 μCi/well [³H]thymidine (NEN Life Sciences, Boston, MA) was added and the cells were incubated for an additional 2 h. [³H]Thymidine incorporation was stopped by removal of the medium and washing of the cells three times with ice-cold physiologic saline solution, to ensure complete removal of free [³H]thymidine. Furthermore, cells were treated three times with 10% TCA for 10 min at 4°C. Finally, the cells were dissolved in 0.5 M NaOH for 30 min at 37°C. The samples were transferred to 5 ml of Beckman Ready Safe cocktail (Beckman, Fullerton, CA). The radioactivity of the samples was measured using a Beckman liquid scintillation counter. Values were expressed as percent increases in [³H]thymidine incorporation in agonist-treated OK cells, compared with cells treated with the vehicle (basal values).

Phospho-p44/42 MAPK Measurements

For these experiments, OK cells were prepared as described for [³H]thymidine incorporation experiments. The FCS-starved cells were then rinsed twice with FCS-free medium, and 495 μl of fresh medium was added to each well. Vehicle (basal) or bromocriptine (a D₂-like receptor agonist) (5 μl) was added to each well to yield the appropriate concentration, and the cells were incubated at 37°C for the indicated times. To stop the reaction, the medium was aspirated from each well and the cells were solubilized in SDS-Laemmli buffer. The cell lysate from each well was sonicated (45 s), boiled (5 min), and centrifuged (14,000 rpm, 5 min). The supernatant was assayed for protein content, and the protein concentration of each extract was adjusted to 1 mg/ml. The protein samples (10 μl) were then resolved by SDS-polyacrylamide gel electrophoresis and transferred to a PVDF membrane. The phospho-p44/42 MAPK bands on the PVDF membrane were detected using an anti-phospho-p44/42 MAPK antibody kit (New England Biolabs, Beverly, MA), according to the instructions provided by the manufacturer. Densitometric analysis of the phospho-p44/42 MAPK bands was performed, and values were expressed as fold increases in the density of the agonist-treated bands, compared with vehicle-treated bands (basal values). Total p44/42 MAPK levels in the same blots was also determined, to confirm that the same amounts of protein were used.

For all inhibition studies, the cells were preincubated without (control/untreated) or with (treated) inhibitors [1 μM domperidone, 1 μM U-101958 maleate (1-benzyl-4-aminomethyl-N-[(3′-isopropoxy)-2′-pyridyl]piperidine maleate), 10 μM PD 98059, 100 μM papaverine, 1 μM forskolin, 1 to 100 μM genistein, 0.1 to 10 μM AG 1478, 0.1 to 10 μM wortmannin, or 0.1 to 10 μM staurosporine] for 30 min (except 200 ng/ml PTX for 20 h and papaverine plus forskolin for 15 min) at 37°C before the treatment with agonist. All other reagents were obtained from Sigma-Aldrich (St. Louis, MO), Research Biochemicals International (Natick, MA), Bio-Rad (Hercules, CA), or Fisher Scientific (Fair Lawn, NJ) and were of the highest grade obtainable.

Statistical Analysis

Where applicable, data are presented as means ± SEM of the indicated number of experiments. Statistical analysis was performed using the unpaired t test for comparisons between vehicle-treated (basal) and agonist-treated groups. One-way ANOVA was used for comparisons between groups not treated (control) or pretreated with inhibitors. Differences were considered statistically significant at P < 0.05.

Data from saturation experiments were analyzed using the computer program GraphPad Prism (GraphPad Software, San Diego, CA). In saturation studies, data were subjected to nonlinear, least-squares, regression analysis for one-site binding, for determination of the receptor density and the affinity for [³H]spiperone.

Dopamine D₂-Like Receptor Binding Sites in OK Cells

We first performed saturation radioligand binding experiments in OK cell membranes, for detection of D₂-like receptor sites. Saturation binding experiments with [³H]spiperone (a D₂-like receptor antagonist) revealed a single binding site, with a receptor density of 45.6 ± 6.3 fmol/mg protein and an affinity of 1.3 ± 0.2 nM (Figure 1), suggesting the presence of low-density/high-affinity D₂-like receptor sites in OK cell membranes.

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Figure 1.

Representative saturation curve for D₂-like receptors in opossum kidney (OK) cells. Saturation analyses were performed in OK cell membranes using [³H]spiperone (0.01 to 4 nM) as a ligand and butaclamol (10 μM) for nonspecific binding. The binding data from each experiment were analyzed by nonlinear, least-squares analysis for one-site binding, for calculation of the receptor density and affinity for [³H]spiperone. The saturation curve shown represents data from one experiment. Inset, Scatchard plot of bound/free versus bound, calculated from the representative experiment. All experiments were performed in triplicate. Data from five such experiments yielded an average receptor density of 45.6 ± 6.3 fmol/mg protein and an affinity for [³H]spiperone of 1.3 ± 0.2 nM.

Dopamine D₂-Like Receptor Subtypes in OK Cells

Furthermore, we determined the D₂-like receptor subtypes in OK cells using specific polyclonal antibodies for D₂, D₃, and D₄ receptors. Western blot analysis revealed D₂, D₃, and D₄ receptors in OK cells. The dopamine D₂ receptor-specific antibody immunoreacted with a D₂ receptor protein (approximately 80 kD) on the blots. The antibody failed to react when it was preincubated with a blocking peptide, confirming that the protein was a D₂ receptor (Figure 2A). The dopamine D₃ receptor-specific antibody immunoreacted with a D₃ receptor protein (approximately 40 kD) on the blots. The antibody failed to react when it was preincubated with a blocking peptide, confirming that the protein was a D₃ receptor (Figure 2B). Similarly, the D₄ receptor-specific antibody detected a D₄ receptor protein (approximately 50 kD) on the blots (Figure 2C) and failed to do so when preincubated with a blocking peptide. Therefore, OK cells express all types of D₂-like receptors (D₂, D₃, and D₄).

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Figure 2.

Dopamine D₂-like receptor subtypes in OK cells. Whole-cell lysates of OK cells (45 μg/lane for D₂ and D₄ receptors and 170 μg/lane for D₃ receptors) were resolved by gel electrophoresis and then transferred to polyvinylidene difluoride (PVDF) membranes. The PVDF membranes were treated with D₂, D₃, or D₄ receptor-specific antibodies, as described in the Materials and Methods section. (A) The dopamine D₂ receptor-specific antibody, not treated or treated with blocking peptide, did or did not detect D₂ receptors in the OK cell lysates, respectively. (B) The dopamine D₃ receptor-specific antibody, not treated or treated with blocking peptide, did or did not detect D₃ receptors in the OK cell lysates, respectively. (C) The dopamine D₄ receptor-specific antibody, not treated or treated with blocking peptide, did or did not detect D₄ receptors in the OK cell lysates, respectively.

Dopamine D₂ Receptor Activation-Induced Increases in [³H]Thymidine Incorporation in OK Cells

We measured mitogenesis as a function of [³H]thymidine incorporation in OK cells. Both bromocriptine (a D₂-like receptor agonist) and (±)-2-(N-phenylethyl-N-propyl)amino-5-hydroxytetralin hydrochloride [(±)-PPHT-HCl] (a D₂ receptor agonist) increased [³H]thymidine incorporation in OK cells (Figure 3A). However, (R)-(+)-2-dipropylamino-7-hydroxy-1,2,3,4-tetrahydronaphthalene hydrobromide (7-OH-DPAT) (a D₃ receptor agonist) and PD 168,077 maleate (N-[[4-(2-cyanophenyl)-1-piperazinyl]methyl]-3-methylbenzamide, a D₄ receptor agonist) failed to increase [³H]thymidine incorporation in OK cells (data not shown). Whereas domperidone (a D₂-like receptor antagonist, at 1 μM) blocked the bromocriptine-mediated increase in [³H]thymidine incorporation (Figure 3B), U-101958 maleate (a D₄ receptor antagonist, at 1 μM) failed to do so (data not shown). In addition, simultaneous treatment of OK cells with (±)-PPHT-HCl (10^-8 M) and 7-OH-DPAT (10^-8 M) did not yield an additive response or a response greater than that obtained with bromocriptine (10^-8 M) (data not shown). These results indicate that activation of D₂ receptors, but not D₃ or D₄ receptors, causes mitogenesis in OK cells.

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Figure 3.

(A) Effects of various D₂-like receptor agonists on [³H]thymidine incorporation in OK cells. OK cells (approximately 90% confluent) were treated with vehicle (basal) or increasing concentrations (10^-10 to 10^-6 M) of bromocriptine (a D₂-like receptor agonist; —[UNK]—) or (±)-2-(N-phenylethyl-N-propyl)amino-5-hydroxytetralin hydrochloride [(±)-PPHT-HCl] (a D₂ receptor agonist; ---○---) for 16 h at 37°C. The cells were then pulsed with [³H]thymidine (1 μCi) for 2 h. After 2 h, the incorporated [³H]thymidine was measured as described in the Materials and Methods section. (B) Effects of bromocriptine on [³H]thymidine incorporation in OK cells not treated (control; —[UNK]—) or treated with 1 μM domperidone (a D₂-like receptor antagonist; ---○---). Data are expressed as percent increases in [³H]thymidine incorporation, compared with corresponding basal values for the respective agonist. Data are presented as mean ± SEM of three or four experiments, each performed in triplicate. ^*, P < 0.05, significantly different from the corresponding basal value for the respective agonist (t test). #, P < 0.05, significant difference between untreated cells and cells treated with domperidone (ANOVA).

Evidence that Dopamine D₂ Receptor Activation Increases [³H]Thymidine Incorporation in OK Cells via G_i or G_o Protein Coupling and ERK Kinase

When the OK cells were pretreated with PTX (200 ng/ml), bromocriptine (0.1 μM) failed to increase [³H]thymidine incorporation, suggesting the coupling of D₂ receptors to G_i or G_o proteins (Figure 4). Also, when the OK cells were pretreated with PD 98059 [a specific inhibitor of ERK kinase (MEK1/2), at 10 μM], bromocriptine (0.1 μM) failed to increase [³H]thymidine incorporation, indicating the involvement of MEK1/2 in this response (Figure 4). PD 98059 also reduced basal [³H]thymidine incorporation. Activation of D₂ receptors thus increases [³H]thymidine incorporation via G_i or G_o protein coupling and MEK1/2.

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Figure 4.

Effects of bromocriptine on [³H]thymidine incorporation in the absence or presence of pertussis toxin (PTX) (a G_i or G_o inactivator) or PD 98059 [an extracellular signal-regulated kinase (MEK1/2) inhibitor]. OK cells were incubated with vehicle (□), 200 ng/ml PTX (20 h) ([UNK]), or 10 μM PD 98059 (▪) for 30 min at 37°C, followed by stimulation without or with bromocriptine (0.1 μM). [³H]Thymidine incorporation was measured in cells as described in the Materials and Methods section. Data are presented as mean ± SEM of three to six experiments. ^***, P < 0.0001, significantly different from the corresponding basal value (t test). ###, P < 0.0001, significant difference between untreated cells and cells treated with inhibitors (ANOVA).

Dopamine D₂ Receptor Activation-Induced Increases in the Phosphorylation of p44/42 MAPK in OK Cells

MEK1/2 is known to phosphorylate p44/42 MAPK. Because activation of D₂ receptors by bromocriptine increased [³H]thymidine incorporation via MEK1/2, we further examined whether bromocriptine would produce an increase in p44/42 MAPK phosphorylation. Bromocriptine (1 μM) caused a timedependent increase in p44/42 MAPK phosphorylation. Phosphorylation was maximal at 5 min and decreased thereafter (Figure 5A). Therefore, all further agonist treatments were performed for 5 min. The total amounts of p44/42 MAPK in each lane were similar (Figure 5B). Pretreatment of OK cells with domperidone (1 μM) attenuated bromocriptine (1 μM)-mediated phosphorylation of p44/42 MAPK (Figure 5C). Activation of D₂ receptors thus increases phosphorylation of p44/42 MAPK in OK cells.

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Figure 5.

Phosphorylation of p44/42 mitogen-activated protein kinase (MAPK) by bromocriptine via D₂ receptors. (A) For determination of the time course, fetal calf serum (FCS)-starved OK cells were treated with bromocriptine (1 μM) for various times (0 to 30 min). Phospho-p44/42 MAPK was detected by Western blotting, as described in the Materials and Methods section. Maximal phosphorylation of p44/42 MAPK occurred with 5-min treatment with bromocriptine. The experiment was repeated three times and similar results were obtained, as in the representative immunoblot. (B) Total p44/42 MAPK levels were also measured in the same blots, to ensure the use of similar amounts of protein in each lane. (C) FCS-starved OK cells were incubated with vehicle or domperidone (1 μM) for 30 min. The untreated and treated cells were then incubated without or with bromocriptine for 5 min (□, basal; [UNK], 1 μM bromocriptine; ▪, 1 μM domperidone; [UNK], domperidone plus bromocriptine). After Western blotting, phospho-p44/42 MAPK bands were subjected to densitometric analysis, and the effect of bromocriptine was expressed as the fold increase in p44/42 MAPK phosphorylation, compared with basal levels. Data are mean ± SEM of four experiments. ^*, P < 0.05, significantly different from the corresponding basal value (t test). #, P < 0.05, significant difference between untreated cells and cells treated with domperidone (ANOVA).

Evidence that Dopamine D₂ Receptor Activation Increases Phosphorylation of p44/42 MAPK in OK Cells via G_i or G_o Protein Coupling

As in the case of [³H]thymidine incorporation, D₂ receptor activation increased phosphorylation of p44/42 MAPK via coupling to G_i or G_o proteins. This was evident in OK cells pretreated with PTX (200 ng/ml), in which D₂ receptor activation by bromocriptine failed to increase the phosphorylation of p44/42 MAPK (Figure 6A, upper). Total p44/42 MAPK levels were the same in all treatment groups (Figure 6A, lower).

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Figure 6.

Phosphorylation of p44/42 MAPK by bromocriptine via G_i or G_o protein and MEK1/2. FCS-starved OK cells were incubated with vehicle (untreated) or treated with PTX (200 ng/ml) or PD 98059 (10 μM) for 20 h or 30 min, respectively. The untreated and treated cells were further incubated without or with bromocriptine (1 μM), and phospho-p44/42 MAPK and total p44/42 MAPK were detected as described. (A) (Upper) Representative immunoblot of phospho-p44/42 MAPK from cells treated with bromocriptine and PTX as indicated. (Lower) Total p44/42 MAPK detected in the same blots, to ensure similar amounts of protein in each lane. The experiment was repeated three times, with similar results. (B) (Upper) Representative immunoblot of phospho-p44/42 MAPK from cells treated with bromocriptine and PD 98059 as indicated. (Lower) Total p44/42 MAPK detected in the same blots, to ensure similar amounts of protein in each lane. The experiment was repeated three times, with similar results.

PD 98059 Blockade of p44/42 MAPK Phosphorylation in OK Cells

Because we observed that the MEK1/2 inhibitor PD 98059 blocked bromocriptine-mediated [³H]thymidine incorporation, we examined whether PD 98059 would prevent bromocriptine-mediated phosphorylation of p44/42 MAPK. PD 98059 (10 μM) blocked phosphorylation of p44/42 MAPK in both non-bromocriptine-treated and bromocriptine-treated OK cells (Figure 6B, upper). Total p44/42 MAPK levels were same in all treatment groups (Figure 6B, lower). These findings confirm the involvement of phospho-p44/42 MAPK in D₂ receptor-mediated increases in [³H]thymidine incorporation.

Effects of cAMP Accumulation on D₂ Receptor-Mediated Phosphorylation of p44/42 MAPK

We used activators or inhibitors of several signaling components that are usually involved in the p44/42 MAPK pathway to identify the components involved in D₂ receptor-mediated phosphorylation of p44/42 MAPK. Simultaneous pretreatment of OK cells with papaverine (a phosphodiesterase inhibitor, at 100 μM) and forskolin (an adenylate cyclase activator, at 1 μM) attenuated D₂ receptor-mediated phosphorylation of p44/42 MAPK (Figure 7A, upper). Furthermore, papaverine and forskolin, individually or in combination, also decreased basal p44/42 MAPK phosphorylation (Figure 7A, upper). Total p44/42 MAPK levels were similar in all treatment groups (Figure 7A, lower). Increases in cAMP accumulation thus cause a decrease in p44/42 MAPK phosphorylation, and D₂ receptors may increase p44/42 MAPK phosphorylation by decreasing cAMP accumulation.

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Figure 7.

Effects of different activators or inhibitors on bromocriptine-mediated phosphorylation of p44/42 MAPK. FCS-starved OK cells were incubated with vehicle (Control) or activators/inhibitors for 30 min (15 min in the case of papaverine and forskolin) at 37°C, followed by stimulation without or with bromocriptine (1 μM). Phospho-p44/42 MAPK (upper) and total p44/42 MAPK (lower) were detected as described. (A) FCS-starved cells were treated with papaverine (100 μM) and/or forskolin (1 μM), followed by bromocriptine, as indicated. (B) FCS-starved cells were treated with genistein (1 to 100 μM) or AG 1478 (0.1 to 10 μM), followed by bromocriptine, as indicated. (C) FCS-starved cells were treated with wortmannin (1 to 100 μM) or staurosporine (0.1 to 10 μM), followed by bromocriptine, as indicated. All experiments were repeated three times, and similar results were obtained.

Effects of Genistein and AG 1478 on D₂ Receptor-Mediated Phosphorylation of p44/42 MAPK

Pretreatment of OK cells with genistein (a tyrosine kinase inhibitor, at 1 to 100 μM) or AG 1478 (an epidermal growth factor [EGF] receptor kinase inhibitor, at 0.1 to 10 μM) attenuated D₂ receptor-mediated phosphorylation of p44/42 MAPK (Figure 7B, upper). The total amounts of p44/42 MAPK were similar in all treatment groups (Figure 7B, lower). Therefore, D₂ receptors may increase phosphorylation of p44/42 MAPK via activation of a tyrosine kinase and transactivation of EGF receptors.

Effects of Wortmannin and Staurosporine on D₂ Receptor-Mediated Phosphorylation of p44/42 MAPK

Pretreatment of OK cells with wortmannin [a phosphatidylinositol-3-kinase (PI3K) inhibitor, at 0.1 to 10 μM] or staurosporine [a protein kinase C (PKC) inhibitor, at 0.1 to 10 μM] blocked D₂ receptor-mediated phosphorylation of p44/42 MAPK (Figure 7C, upper). Total p44/42 MAPK levels were similar in all treatment groups (Figure 7C, lower). Therefore, D₂ receptor-mediated phosphorylation of p44/42 MAPK may involve PI3K and PKC.