 |
||||
| 2007 JASN IMPACT FACTOR 7.111 | HOME AUTHOR INFO EDITORIAL BOARD SUBSCRIBE FEEDBACK ALERTS HELP | |||
| CURRENT ISSUE | ARCHIVES | JASN Express | ONLINE SUBMISSION | |
|
||||
| 2007 JASN IMPACT FACTOR 7.111 | HOME AUTHOR INFO EDITORIAL BOARD SUBSCRIBE FEEDBACK ALERTS HELP | |||
| CURRENT ISSUE | ARCHIVES | JASN Express | ONLINE SUBMISSION | |
Institute for Cardiovascular Studies, College of Pharmacy, University of Houston, Houston, Texas.
Correspondence to Dr. Mustafa F. Lokhandwala, Department of Pharmacology, College of Pharmacy, University of Houston, Houston, TX 77204-5511. Phone: 713-743-1253; Fax: 713-743-5678; E-mail: MLokhandwala{at}uh.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
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 |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
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 x 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 x g for 5 min at
4°C. The supernatant was separated and further centrifuged at 37,000
x 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 x 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 x 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 |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
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).
|
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.
|
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.
|
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.
|
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).
|
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.
|
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 |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
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 |
|---|
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  Z. Li, C. Yu, Y. Han, H. Ren, W. Shi, C. Fu, D. He, L. Huang, C. Yang, X. Wang, et al. Inhibitory effect of D1-like and D3 dopamine receptors on norepinephrine-induced proliferation in vascular smooth muscle cells Am J Physiol Heart Circ Physiol, June 1, 2008; 294(6): H2761 - H2768. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Zeng, I. Armando, Y. Luo, G. M. Eisner, R. A. Felder, and P. A. Jose Dysregulation of dopamine-dependent mechanisms as a determinant of hypertension: studies in dopamine receptor knockout mice Am J Physiol Heart Circ Physiol, February 1, 2008; 294(2): H551 - H569. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Zeng, Z. Wang, H. Li, P. Yu, S. Zheng, L. Wu, L. D. Asico, U. Hopfer, G. M. Eisner, R. A. Felder, et al. D3 Dopamine Receptor Directly Interacts With D1 Dopamine Receptor in Immortalized Renal Proximal Tubule Cells Hypertension, March 1, 2006; 47(3): 573 - 579. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Zeng, Z. Wang, U. Hopfer, L. D. Asico, G. M. Eisner, R. A. Felder, and P. A. Jose Rat Strain Effects of AT1 Receptor Activation on D1 Dopamine Receptors in Immortalized Renal Proximal Tubule Cells Hypertension, October 1, 2005; 46(4): 799 - 805. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. A. Banday, A. H. Siddiqui, M. M. Menezes, and T. Hussain Insulin treatment enhances AT1 receptor function in OK cells Am J Physiol Renal Physiol, June 1, 2005; 288(6): F1213 - F1219. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Zeng, H. Sanada, H. Watanabe, G. M. Eisner, R. A. Felder, and P. A. Jose Functional genomics of the dopaminergic system in hypertension Physiol Genomics, November 17, 2004; 19(3): 233 - 246. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Tegeder and G. Geisslinger Opioids As Modulators of Cell Death and Survival--Unraveling Mechanisms and Revealing New Indications Pharmacol. Rev., September 1, 2004; 56(3): 351 - 369. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Zeng, D. Wang, L. D. Asico, W. J. Welch, C. S. Wilcox, U. Hopfer, G. M. Eisner, R. A. Felder, and P. A. Jose Aberrant D1 and D3 Dopamine Receptor Transregulation in Hypertension Hypertension, March 1, 2004; 43(3): 654 - 660. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. A. Banday, M. Asghar, T. Hussain, and M. F. Lokhandwala Dopamine-Mediated Inhibition of Renal Na,K-ATPase Is Reduced by Insulin Hypertension, June 1, 2003; 41(6): 1353 - 1358. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Narkar, T. Hussain, and M. Lokhandwala Role of tyrosine kinase and p44/42 MAPK in D2-like receptor-mediated stimulation of Na+, K+-ATPase in kidney Am J Physiol Renal Physiol, April 1, 2002; 282(4): F697 - F702. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
HOME
CURRENT ISSUE
ARCHIVES
JASN Express
ONLINE SUBMISSION
AUTHOR INFO
EDITORIAL BOARD SUBSCRIBE FEEDBACK ALERTS HELP |
---) 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;
---
), 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).
