Hormones, Growth Factors, and Cell Signaling

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

VIHANG A. NARKAR, TAHIR HUSSAIN, CARLOS PEDEMONTE and MUSTAFA F. LOKHANDWALA
JASN September 2001, 12 (9) 1844-1852;

Abstract

Abstract. This study was conducted to determine the expression of dopamine D2-like receptors in opossum kidney (OK) cells and to examine the potential role of these receptors in mitogenesis. First, the presence of D2-like receptor binding sites in OK cell membranes was demonstrated by radioligand binding, using [3H]spiperone. The D2-like receptor subtypes expressed in OK cells were subsequently demonstrated, by Western blotting, to be D2, D3, and D4 receptors. OK cells were stimulated with bromocriptine, (±)-2-(N-phenylethyl-N-propyl)amino-5-hydroxytetralin hydrochloride, (R)-(+)-2-dipropylamino-7-hydroxy-1,2,3,4-tetrahydronaphthalene hydrobromide, or PD 168,077 maleate (D2-like, D2, D3, and D4 receptor agonists, respectively), and mitogenesis was measured as a function of [3H]thymidine incorporation. It was observed that, whereas bromocriptine and (±)-2-(N-phenylethyl-N-propyl)amino-5-hydroxytetralin hydrochloride produced increases in [3H]thymidine incorporation, (R)-(+)-2-dipropylamino-7-hydroxy-1,2,3,4-tetrahydronaphthalene hydrobromide and PD 168,077 maleate did not produce such an effect, indicating the involvement of D2 receptors in the mitogenic response. Pertussis toxin and PD 98059 blocked the mitogenesis caused by bromocriptine, suggesting a role for Gi or Go proteins and p44/42 mitogen-activated protein kinase (MAPK), respectively. Furthermore, it was observed that bromocriptine produced a time-dependent increase in the phosphorylation (activation) of p44/42 MAPK, which was blocked by domperidone, pertussis toxin, or PD 98059. Therefore, this study demonstrates that, although OK cells express D2, D3, and D4 receptors, activation of only D2 receptors causes mitogenesis via phosphorylation of p44/42 MAPK. Furthermore, the cellular mechanisms contributing to D2 receptor-mediated phosphorylation of p44/42 MAPK seem to involve the tyrosine kinase, phosphatidylinositol-3-kinase, and protein kinase C pathways. It is likely that bromocriptine and other preferential D2 receptor agonists might provide protection against ischemic reperfusion injury in renal proximal tubular cells, by increasing the survival rates for ischemic cells.

Dopamine plays a significant role in the regulation of sodium and water excretion by the kidney. At higher concentrations, dopamine increases GFR and renal blood flow, thus causing sodium and water excretion (1,2). At lower concentrations, however, dopamine decreases the reabsorption of sodium and water via a direct action at the level of the proximal tubules, medullary thick ascending limb (loop of Henle), and collecting duct of the kidney (1,2,3).

At the cellular level, the actions of dopamine are mediated via the activation of specific receptors coupled to different G proteins. Dopamine receptors are classified into D1-like and D2-like receptor subtypes (4). The inhibitory effect of dopamine on sodium and water reabsorption in the proximal tubules of the kidney is predominantly a D1-like receptor effect (1). However, the proximal tubules of the kidney express a comparable population of D2-like receptors (5,6). Although the role of D1-like receptors in the proximal tubules of the kidney has been well studied and documented, no such progress has been made in the case of D2-like receptors.

Dopamine D2-like receptors are the class of receptors coupled to pertussis toxin (PTX)-sensitive Gi or Go proteins. These receptors are further classified into D2, D3, and D4 receptor subtypes (4). Dopamine D2-like receptors, via their Gi or Go protein coupling, are known to modulate the functions of various cellular proteins, such as adenylyl cyclase (7,8), potassium channels, and calcium channels (9,10). Therefore, identifying other cellular proteins affected by D2-like receptors in the proximal tubules of the kidney may allow us to understand the role of these receptors in the proximal tubules.

Recently, G protein-coupled receptors have been reported to regulate gene expression and subsequent cell proliferation (mitogenesis) or differentiation in various cell types. These effects usually involve activation (phosphorylation) of an intracellular class of proteins called the mitogen-activated protein kinases (MAPK) (11). MAPK are classified into three subclasses, i.e., (1) extracellular signal-regulated kinase (ERK) 1 and 2, also known as p44 and p42, respectively; (2) p38 kinases; and (3) stress-activated protein kinases, also known as c-Jun amino terminus kinases (JNK). Whereas p44/42 MAPK promote mitogenesis, p38 and stress-activated protein kinases promote apoptosis (12).

Dopamine D2-like receptors, when transfected into C6 glioma or Chinese hamster ovary cells, increase mitogenesis via p44/42 MAPK in these cell lines (13,14,15,16). However, there are no reports describing a role for D2-like receptors in mitogenesis in the proximal tubular cells of the kidney. Therefore, this study was designed to determine whether activation of D2-like receptor subtypes causes mitogenesis by increasing the phosphorylation of p44/42 MAPK in the proximal tubules of the kidney. For this study, we used opossum kidney (OK) cells, which are derived from the proximal tubules of a kidney from a North American opossum (17). These cells have been used to study the effects of dopamine on various cellular proteins (18,19). However, there are no reports on the expression of D2-like receptors in this cell system. Therefore, in our study, we first determined the D2-like receptor subtypes endogenously expressed in OK cells, using the techniques of radioligand binding and Western blotting. Next, we determined whether one or more D2-like receptor subtypes were involved in mitogenesis via phosphorylation of p44/42 MAPK. Furthermore, we investigated the signaling components involved in the D2-like receptor-mediated phosphorylation of p44/42 MAPK.

Materials and Methods

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.

Results

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.

Figure 1.
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).

Figure 2.
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.

Figure 3.
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 Gi or Go 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 Gi or Go 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 Gi or Go protein coupling and MEK1/2.

Figure 4.
Figure 4.

Effects of bromocriptine on [3H]thymidine incorporation in the absence or presence of pertussis toxin (PTX) (a Gi or Go 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.

Figure 5.
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 Gi or Go Protein Coupling

As in the case of [3H]thymidine incorporation, D2 receptor activation increased phosphorylation of p44/42 MAPK via coupling to Gi or Go 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).

Figure 6.
Figure 6.

Phosphorylation of p44/42 MAPK by bromocriptine via Gi or Go 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.

Figure 7.
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.

Discussion

In this study, we demonstrated that OK cells express the D2, D3, and D4 subtypes of dopamine D2-like receptors and that activation of D2 receptors causes mitogenesis via phosphorylation of p44/42 MAPK. The mitogenesis observed after D2 receptor activation involves receptor coupling to Gi or Go proteins. Although we have identified the presence of D3 and D4 receptors in OK cells, they do not seem to be involved in the mitogenic response in these cells. We further demonstrated that D2 receptor activation may increase p44/42 MAPK phosphorylation via multiple pathways, including decreases in cAMP accumulation, increases in tyrosine kinase, PI3K, and PKC activity, and transactivation of EGF receptors (Figure 8).

Figure 8.
Figure 8.

Dopamine D2 receptor (D2)-mediated phosphorylation of p44/42 MAPK, involving multiple pathways, in OK cells. Activation of D2 receptors leads to adenylate cyclase (AC) inhibition, decrease in cAMP accumulation, and protein kinase A (PKA) inhibition or activation of phosphatidylinositol-3-kinase (PI3K), protein kinase C (PKC), epidermal growth factor receptor (EGF-R), and tyrosine kinase (TK) in OK cells. These components may increase p44/42 MAPK activity individually or in concert in OK cells. Thick arrows, activation; dotted arrows, inhibition.

We demonstrated the expression of D2-like receptors in OK cells by using radioligand binding and Western blotting. In radioligand binding experiments with [3H]spiperone, we observed that [3H]spiperone binding was saturable and involved a single low-density/high-affinity site in OK cell membranes, suggesting the presence of D2-like receptors in these membranes. In Western blot analyses, specific antibodies raised against the D2, D3, and D4 receptors immunoreacted with proteins of approximately 80, 40, and 50 kD, respectively, in OK cell lysates. The molecular masses for D2 and D4 receptors were comparable to those reported previously (20,21). However, the molecular mass of 40 kD for D3 receptors has not been reported, to our knowledge. It is quite possible that the receptor we detected by Western blotting is a truncated or deglycosylated form of the D3 receptor.

We observed that, of the D2, D3, and D4 receptors expressed in OK cells, activation of only D2 receptors produced mitogenesis. This was evident from the observation that (±)-PPHT-HCl (a D2 receptor agonist), but not 7-OH-DPAT (a D3 receptor agonist) or PD 168,077 maleate (a D4 receptor agonist), caused an increase in mitogenesis in OK cells. This is an interesting finding, because all subtypes of D2-like receptors have been demonstrated to cause mitogenesis in C6 glioma or Chinese hamster ovary cells transfected with a single D2-like receptor subtype (13,14,15,16). However, this is not the case in OK proximal tubular cells, where the endogenous D2-like receptor subtypes coexist, as indicated by our results. Furthermore, bromocriptine produced a greater increase in mitogenesis than did (±)-PPHT-HCl. We conclude that this effect of bromocriptine is via D2 receptors, for three reasons. First, 7-OH-DPAT (a D3 receptor agonist) and PD 168,077 maleate (a D4 receptor agonist) did not increase [3H]thymidine incorporation. Second, U-101958 maleate (a D4 receptor antagonist) failed to block bromocriptine-mediated [3H]thymidine incorporation. Third, simultaneous treatment of OK cells with (±)-PPHT-HCl and 7-OH-DPAT did not elicit an additive response, compared with treatment with (±)-PPHT-HCl or 7-OH-DPAT. The greater stimulation of mitogenesis by bromocriptine, compared with (±)-PPHT-HCl, suggests greater efficacy of bromocriptine at D2 receptors. It is also possible that (±)-PPHT-HCl is a partial agonist at D2 receptors in OK cells.

Our results demonstrate that the increase in mitogenesis produced via D2 receptors in OK cells requires phosphorylation of p44/42 MAPK. Consistent with this, bromocriptine caused a time-dependent increase in p44/42 MAPK phosphorylation. Furthermore, by using PD 98059, we demonstrated that phosphorylated p44/42 MAPK is involved in D2 receptor-mediated mitogenesis in OK cells. It has been reported that PD 98059 inhibits MEK1/2 and thus prevents phosphorylation of p44/42 MAPK (22). In our study, PD 98059 decreased basal as well as bromocriptine-stimulated mitogenesis in OK cells, indicating the involvement of p44/42 MAPK. The decrease in basal mitogenic activity may be caused by inhibition of the basal activity (phosphorylation) of p44/42 MAPK by PD 98059. It should be noted that the increase in both mitogenesis and p44/42 MAPK phosphorylation via D2 receptor activation in OK cells requires receptor coupling to Gi or Go proteins. This is evident from the sensitivity of both responses to PTX, which is a Gi and Go protein inactivator.

To elucidate the cellular mechanisms, we investigated the signaling components that might be involved between the D2 receptors and the phosphorylation of p44/42 MAPK. We performed this investigation by using activators or inhibitors of several signaling components that are commonly involved in p44/42 MAPK activation. First, we observed that increasing intracellular cAMP levels by using forskolin and papaverine decreased basal and D2 receptor-mediated phosphorylation of p44/42 MAPK. It has been reported that increases in cAMP levels (and protein kinase A activity) may inhibit p44/42 MAPK (23). Furthermore, activation of D2-like receptors has been shown to decrease cAMP accumulation in kidney proximal tubules (7,8). Therefore, D2 receptor agonists may activate p44/42 MAPK by decreasing intracellular cAMP levels (and protein kinase A activity) in OK cells.

We also discovered that genistein (a tyrosine kinase inhibitor) and AG 1478 (an EGF receptor kinase inhibitor) attenuated D2 receptor-mediated phosphorylation of p44/42 MAPK. EGF receptors are linked to the activation of p44/42 MAPK via a tyrosine kinase pathway (24). Recently, G protein-coupled receptors were demonstrated to increase p44/42 MAPK activity via transactivation of EGF receptors (24). Therefore, D2 receptors may also activate p44/42 MAPK via transactivation of EGF receptors in OK cells. Interestingly, increases in intracellular cAMP levels inhibit EGF receptor activity and thus p44/42 MAPK (25). Therefore, it is likely that transactivation of EGF receptors via D2 receptors may involve decreases in intracellular cAMP levels in OK cells.

Additional experiments revealed that wortmannin (a PI3K inhibitor) and staurosporine (a PKC inhibitor) also blocked D2 receptor-mediated activation of p44/42 MAPK. These findings are in agreement with the previously reported role of the PI3K-PKC pathway in the activation of p44/42 MAPK via Gi or Go protein-coupled receptors (16). Therefore, D2 receptor-mediated activation of p44/42 MAPK involves several components in OK cells.

Although experiments with inhibitors of various cell signaling pathways indicated a role for each of the pathways in D2 receptor-mediated activation of p44/42 MAPK, the findings do not allow us to ascertain whether these pathways act independently or in an integrated manner to lead to the activation of p44/42 MAPK. However, closer examination of the effects of various blockers on the activation of p44/42 MAPK by bromocriptine revealed that papaverine, forskolin, genistein, AG 1478, and wortmannin did not completely block the activation of p44/42 MAPK by bromocriptine, whereas staurosporine completely blocked the activation of p44/42 MAPK by bromocriptine. As shown in a hypothetical scheme (Figure 8), which takes into consideration the roles of these pathways, it can be speculated that the various signaling pathways converge on PKC, which subsequently activates MEK1/2 and p44/42 MAPK.

The physiologic relevance of mitogenesis via D2 receptors in proximal tubular cells remains to be determined. Although dopamine promotes sodium and water excretion at the level of the proximal tubules via activation of D1-like receptors, it is likely that simultaneous activation of D2 receptors may play a role in protecting the epithelial cell lining of the proximal tubules against high-salt insults. However, ischemia/reperfusion injuries are known to induce differential activation of various MAPK in different segments of the nephron. Selective activation of JNK leads to apoptosis of tubular cells, whereas activation of p44/42 MAPK keeps the cells intact (26). It has been suggested that ischemia/reperfusion injuries lead to activation of JNK and not p44/42 MAPK in proximal tubules, making these cells susceptible to apoptosis (26). We speculate that preferential D2 receptor agonists may activate p44/42 MAPK in proximal tubular cells, which would provide protection against ischemic injuries to the kidney.

In conclusion, our study demonstrates that OK cells express D2, D3, and D4 receptor subtypes from the D2-like receptor family. Furthermore, activation of only D2 and not D3 or D4 receptors causes mitogenesis in OK cells; this mitogenic response is the result of Gi or Go protein activation and p44/42 MAPK phosphorylation. The phosphorylation of p44/42 MAPK requires D2 receptor-mediated decreases in cAMP accumulation, activation of tyrosine kinase, and transactivation of EGF receptors. Furthermore, these multiple pathways may converge on the activation of PKC, which subsequently leads to the phosphorylation of p44/42 MAPK.

Acknowledgments

This research was supported in part by National Institutes of Health Grant AG15031, from the National Institute on Aging, and DK53460.

References

  1. ↵
    Hussain T, Lokhandwala MF: Renal dopamine receptor function in hypertension. Hypertension 32: 187 -197, 1998
    OpenUrlAbstract/FREE Full Text
  2. ↵
    Jose PA, Eisner GM, Felder RA: Dopamine receptors in health and hypertension. Pharmacol Ther 80 : 149-182, 1998
    OpenUrlCrossRefPubMed
  3. ↵
    Hegde SS, Ricci A, Amenta F, Lokhandwala MF: Evidence from functional and autoradiographic studies for the presence of tubular dopamine-1 receptors and their involvement in the renal effects of fenoldopam. J Pharmacol Exp Ther 251: 1237 -1245, 1989
    OpenUrlAbstract/FREE Full Text
  4. ↵
    Sibley DR, Monsma FJ Jr: Molecular biology of dopamine receptors. Trends Pharmacol Sci 13: 61 -69, 1992
    OpenUrlCrossRefPubMed
  5. ↵
    Felder CC, McKelvey AM, Gitler MS, Eisner GM, Jose PA: Dopamine receptor subtypes in renal brush border and basolateral membrane. Kidney Int 36: 183 -193, 1989
    OpenUrlPubMed
  6. ↵
    Lokhandwala MF, Amenta F: Anatomical distribution and function of dopamine receptors in the kidney. FASEB J 5 : 3023-3030, 1991
    OpenUrlAbstract
  7. ↵
    Hussain T, Abdul-Wahab R, Lokhandwala MF: Bromocriptine stimulates Na+,K+-ATPase in renal proximal tubules via the cAMP pathway. Eur J Pharmacol 321: 259 -263, 1997
    OpenUrlCrossRefPubMed
  8. ↵
    Yamaguchi I, Walk SF, Jose PA, Felder RA: Dopamine D2L receptors stimulate Na+,K+-ATPase activity in murine LTK- cells. Mol Pharmacol 46 : 373-378, 1996
    OpenUrl
  9. ↵
    Valler L, Muca C, Magni M, Albert P, Bunzow J, Meldolesi J, Civelli O: Differential coupling of dopaminergic D2 receptors expressed in different cell types. J Biol Chem 265: 10320 -10326, 1990
    OpenUrlAbstract/FREE Full Text
  10. ↵
    Lledo P, Homburger V, Bockaert J, Vincent J: Differential G-protein mediated coupling of D2 dopamine receptors to K+ and Ca2+ currents in rat anterior pituitary cells. Neuron 8: 455 -463, 1992
    OpenUrlCrossRefPubMed
  11. ↵
    Ilasaca ML: Signaling from G-protein coupled receptors to mitogen activated protein (MAP) kinase cascades. Biochem Pharmacol 56: 269 -277, 1998
    OpenUrlCrossRefPubMed
  12. ↵
    Garrington TP, Johnson GL: Organization and regulation of mitogen-activated protein kinase signaling pathways. Curr Opin Cell Biol 11: 211-218, 1999
    OpenUrlCrossRefPubMed
  13. ↵
    Luo Y, Kokkonen GC, Wang X, Neve KA, Roth GS: D2 dopamine receptors stimulate mitogenesis through pertussis toxin-sensitive G proteins and Ras-involved ERK and SAP/JNK pathways in rat C6-D2L glioma cells. J Neurochem 71: 980 -990, 1998
    OpenUrlPubMed
  14. ↵
    Welsh GI, Hall DA, Warnes A, Strange PG, Proud CG: Activation of microtubule-associated protein kinase (ERK) and p70 S6 kinase by D2 dopamine receptors. J Neurochem 70: 2139 -2146, 1998
    OpenUrlPubMed
  15. ↵
    Zhen X, Wang H-Y, Uryu K, Cai G, Smith C, Friedman E: Activation of extracellular signal (erk)-regulated kinase by D4 dopamine receptors requires Src, Shc-Grb via Gi protein. Soc Neurosci Abstr 24: 340 , 1998
    OpenUrl
  16. ↵
    Cussac D, Tancredi A, Pasteau V, Millan MJ: Human dopamine D3 receptors mediate mitogen-activated protein kinase activation via a phosphatidylinositol 3-kinase and an atypical protein kinase C-dependent mechanism. Mol Pharmacol 56: 1025 -1030, 1999
    OpenUrlAbstract/FREE Full Text
  17. ↵
    Koyama H, Goodpasture C, Miller MM, Teplitz RL, Riggs AD: Establishment and characterization of a cell line from American opossum (Didelphys virginiana). In Vitro 14 : 239-246, 1978
    OpenUrlCrossRefPubMed
  18. ↵
    Wang TT, Lachance S, Delalandre A, Carriero S, Chan JSD: Dopaminergic receptors and angiotensinogen gene expression in opossum kidney cells. Am J Physiol 40: R519 -R527, 1996
    OpenUrl
  19. ↵
    Cheng L, Precht P, Frank D, Liang CT: Dopamine stimulation of cAMP production in cultured opossum kidney cells. Am J Physiol 258: F877 -F882, 1990
    OpenUrlAbstract/FREE Full Text
  20. ↵
    David C, Fishburn CS, Monsma FJ Jr, Sibley DR, Fuchs S: Synthesis and processing of D2 dopamine receptors. Biochemistry 32 : 8179-8183, 1993
    OpenUrlCrossRefPubMed
  21. ↵
    Lanau F, Brockhaus M, Pink JR, Franchet C, Wildt-Perinic D, Goepfert C, Probst A, Hartman DS: Development and characterization of antibodies against the N terminus of the human dopamine D4 receptor. J Neurochem 69: 2169 -2178, 1997
    OpenUrlPubMed
  22. ↵
    Dudley DT, Pang L, Decker SJ, Bridges AJ, Saltiel AR: A synthetic inhibitor of the mitogen-activated protein kinase cascade. Proc Natl Acad Sci USA 92: 7686 -7689, 1995
    OpenUrlAbstract/FREE Full Text
  23. ↵
    Hirota Y, Tuskazaki T, Yonekura A, Miyazaki Y, Osaki M, Shindo H, Yamashita S: Activation of specific MEK-ERK cascade is necessary for TGF-beta signaling and crosstalk with PKA and PKC pathways in cultured rat articular chondrocytes. Osteoarthritis Cartilage 8 : 241-247, 2000
    OpenUrlCrossRefPubMed
  24. ↵
    Mannon PJ, Mele JM: Peptide YY Y1 receptor activates mitogen activated protein kinase and proliferation in gut epithelial cells via the epidermal growth factor receptor. Biochem J 350 : 655-661, 2000
  25. ↵
    Wu J, Dent P, Jelinek T, Woltman A, Weber MJ, Sturgill W: Inhibition of the EGF-activated MAP kinase signaling pathway by adenosine 3′,5′-monophosphate. Science (Washington DC) 262: 1065 -1069, 1993
    OpenUrlAbstract/FREE Full Text
  26. ↵
    di Mari JF, Davis R, Safirstein RL: MAPK activation determines renal epithelial cell survival during oxidative injury. Am J Physiol 277: F195 -F203, 1999
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Dopamine D2 Receptor Activation Causes Mitogenesis via p44/42 Mitogen-Activated Protein Kinase in Opossum Kidney Cells
VIHANG A. NARKAR, TAHIR HUSSAIN, CARLOS PEDEMONTE, MUSTAFA F. LOKHANDWALA
JASN Sep 2001, 12 (9) 1844-1852;
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