AJP - Renal Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Renal Physiol 293: F306-F315, 2007. First published April 25, 2007; doi:10.1152/ajprenal.00108.2007
0363-6127/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/1/F306    most recent
00108.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Banday, A. A.
Right arrow Articles by Lokhandwala, M. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Banday, A. A.
Right arrow Articles by Lokhandwala, M. F.

Oxidative stress reduces renal dopamine D1 receptor-Gq/11{alpha} G protein-phospholipase C signaling involving G protein-coupled receptor kinase 2

Anees Ahmad Banday and Mustafa F. Lokhandwala

Heart and Kidney Institute, College of Pharmacy, University of Houston, Houston, Texas

Submitted 1 March 2007 ; accepted in final form 24 April 2007


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The dopamine D1 receptors (D1R), expressed in renal proximal tubules, participate in the regulation of sodium transport. A defect in the coupling of the D1R to its G protein/effector complex in renal tubules has been reported in various conditions associated with oxidative stress. Because G protein-coupled receptor kinases (GRKs) are known to play an important role in D1R desensitization, we tested the hypothesis that increased oxidative stress in obese Zucker rats may cause GRK2 upregulation and, subsequently, D1R dysfunction. Lean and obese rats were given normal diet or diet supplemented with antioxidant lipoic acid for 2 wk. Compared with lean rats, obese rats exhibited oxidative stress, D1R were uncoupled from Gq/11{alpha} at basal level, and SKF-38393 failed to elicit D1R-G protein coupling, stimulate phospholipase C (PLC), and inhibit Na-K-ATPase activity. These animals showed increased basal protein kinase C (PKC) activity and membranous translocation of GRK2 and increased GKR2-Gq/11{alpha} interaction and D1R serine phosphorylation. Enzymatic dephosphorylation of D1R restored SKF-38393-induced adenylyl cyclase stimulation but not PLC activation. Treatment of obese rats with lipoic acid restored D1R-G protein coupling and SKF-38393-induced PLC stimulation and Na-K-ATPase inhibition. Lipoic acid treatment also normalized PKC activity, GRK2 sequestration, and GKR2-Gq/11{alpha} interaction. In conclusion, these data show that oxidative stress increases PKC activity causing GRK2 membranous translocation. GRK2 interacts with Gq/11{alpha} and acts, at least in part, as a regulator of G protein signaling leading to the D1R-Gq/11{alpha} uncoupling, causing inability of SKF-38393 to stimulate PLC and inhibit Na/K-ATPase. Lipoic acid, while reducing oxidative stress, normalized PKC activity and restored D1R-Gq/11{alpha}-PLC signaling and the ability of SKF-38393 to inhibit Na-K-ATPase activity.

adenylyl cyclase; G proteins; sodium/potassium-adenosine 5'-triphosphatase; phospholipase C

DOPAMINE PRODUCED IN THE KIDNEY, mainly within the proximal tubules, serves as an important regulator of renal sodium transport (reviewed in Refs. 2, 18, 20). There are two subtypes of dopamine receptors belonging to the superfamily of G protein-coupled receptors (GPCRs) that mediate the actions of dopamine: the D1-like receptors (D1 and D5) stimulate and the D2-like receptors (D2, D3, and D4) inhibit adenylyl cyclase (AC) activity (2, 18, 20). Dopamine via activation of D1 receptors (D1R) produces an increase in sodium excretion by inhibiting the activities of sodium transporters in renal proximal tubules (2, 18, 20). D1R activation inhibits Na/H exchanger (NHE) activity in renal brush border membranes, probably via accumulation of cAMP and subsequent activation of cAMP-dependent protein kinase (2, 18, 20). On the other hand, numerous studies have shown that dopamine can inhibit Na-K-ATPase activity by activating G protein-linked pathways independently of cAMP-dependent protein kinase while involving protein kinase C (PKC) (2, 18, 20, 34).

Several studies have shown that the responsiveness of the D1R in the renal proximal tubules is decreased in animal models of genetic hypertension as well as in diabetes, obesity, and salt-sensitive hypertension (7, 18, 20, 30, 39, 42). Despite the impairment of D1R function in hypertension, there are no mutations in the coding region of receptor in patients with essential hypertension or in genetically hypertensive rats (33). In addition, the diminished responsiveness to agonist activation is not always caused by a decrease in renal D1R protein or expression. Rather, the D1R are uncoupled from the G protein/effector complex and fail to increase the second messenger levels, suggesting that a defect lies in DIR signaling (18, 20, 42).

Like many other GPCRs, the loss of responsiveness of D1R following agonist treatment reportedly involves their phosphorylation by a variety of protein kinases, including G protein-coupled receptor kinases (GRKs) (41). A recent study by Yu et al. (47) has shown that renal D1R are hyperphosphorylated and uncoupled from G proteins under basal state as well. In addition, Rankin et al. (31) have shown that GRKs can phosphorylate D1R in absence of agonist activation, resulting in constitutive desensitization. Our own studies involving animal models associated with oxidative stress such as obese Zucker rats, streptozotocin-treated rats, and Fisher 344 old rats as well as renal proximal tubular cultures exposed to insulin or H2O2 showed that D1R were constitutively phosphorylated and uncoupled from G proteins (3, 4, 7, 27). In addition, we found that treatment of these animals with antioxidants, while reducing oxidative stress, normalized GRK2 sequestration and receptor phosphorylation and restored D1R signaling (7, 12, 27). Although these reports provide substantial evidence related to the role of GRKs in D1R hyperphosphorylation and subsequent desensitization, most of these studies were focused on D1R coupling to Gs{alpha} and AC activation (6, 7, 12, 27). Numerous studies have demonstrated that renal D1R also mediates its functional effects through activation of the Gq/11{alpha} protein and its downstream effector, phospholipase C (PLC) (13, 14, 40, 48). Stimulation of the D1R-Gq/11{alpha}-PLC pathway results in the synthesis of inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DAG) and an increase in intracellular Ca2+ causing PKC activation, which in turn inhibits Na-K-ATPase activity (13, 14, 34, 40, 48). It is also well known that in most hypertensive models, dopamine fails to stimulate D1R signaling and reduce Na-K-ATPase activity (7, 18, 20, 30, 39). However, beyond a basic appreciation, little is known about the functioning of D1R-Gq/11{alpha}-PLC pathway under pathophysiological conditions. Therefore, the present study was designed to investigate whether uncoupling of D1R from Gq/11{alpha} and failure to stimulate PLC activity are contributing to receptor dysfunction in renal proximal tubules of obese rats. Since obese Zucker rats exhibit increased oxidative stress (1, 6, 7, 25), we wondered whether antioxidant supplementation could restore the D1R coupling to Gq/11{alpha} and, subsequently, receptor functional response. Both lean and obese Zucker rats were provided with lipoic acid, an antioxidant-supplemented diet, for 2 wk. Renal proximal tubules were studied for D1R coupling to G proteins and Na-K-ATPase inhibition as well as the signaling molecules PLC, GRK2, and PKC.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
R(+)-SKF-38393 hydrochloride [an active enantiomer of (±)-1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol hydrochloride], a D1R agonist, was purchased from Sigma-Aldrich (St. Louis, MO). Radiolabeled SCH-23390 hydrochloride [R(+)-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepin-7-ol hydrochloride], a D1R antagonist, guanosine 5'-({gamma}-thio)triphosphate ([35S]GTP{gamma}S), and phosphatidylinositol-4,5-bisphosphate ([3H]PIP2) were purchased from PerkinElmer (Shelton, CT). Antibodies for G proteins, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), GRKs, and D1R were obtained from Calbiochem-Novabiochem (La Jolla, CA), Research Diagnostics (Concord, MA), Santa Cruz Biotechnology (Santa Cruz, CA), and Chemicon International (Temecula, CA), respectively. PKC assay kit, glutathione colorimetric determination kit, superoxide dismutase (SOD) assay kit, and protease inhibitor (PI) cocktail tablets (Complete 11697498) were purchased from Promega (Madison, WI), OXIS International (Foster City, CA), Cayman Chemical (Ann Arbor, MI), and Roche Diagnostic (Indianapolis, IN), respectively. All other chemicals of the highest purity were purchased from Sigma-Aldrich.

Animals. Male lean and obese Zucker rats, 8–9 wk old, were purchased from Harlan (Indianapolis, IN), maintained at an ambient temperature with a 12:12-h light-dark cycle, and acclimatized to a local environment for at least 1 wk with ad libitum access to rat chow and tap water. The animal protocols in this research were approved by the University of Houston Institutional Animal Care and Use Committee. Both lean and obese animals were randomly divided into two groups; control groups, in which animals were continued on rat chow, and treated groups, in which animals were provided with 0.4% lipoic acid-supplemented rat chow for 2 wk. Food and water were changed every day to monitor daily food and water intake.

Preparation of renal proximal tubular suspension. Renal proximal tubular suspension was prepared as described earlier (10). Briefly, rats were anesthetized with pentobarbital sodium (50 mg/kg ip), and after a midline abdominal incision, the aorta was cannulated below the renal arteries to perfuse the kidneys with an enzyme solution of collagenase and hyaluronidase. Enrichment of proximal tubules was carried out using a 20% Ficoll gradient in Krebs buffer, and protein was determined using the bicinchoninic acid method (Pierce, Rockford-IL) with bovine serum albumin as a standard.

[35S]GTP{gamma}S binding to G proteins. Crude membrane preparations were obtained from renal proximal tubules from lean and obese Zucker rats. Proximal tubules were homogenized in 10 volumes of ice-cold homogenization buffer containing 25 mM HEPES, pH 7.4, 100 mM sucrose, 1 mM EGTA, 0.2% 2-mercaptoethanol, PI cocktail, and 0.04 mM phenylmethylsulfonyl fluoride (PMSF). The tissue homogenate was centrifuged for 5 min at 750 g (4°C). The resultant supernatant was then centrifuged for 10 min at 48,200 g (4°C). The pellet was resuspended in Krebs-Ringer buffer (KRB) containing 20 mm HEPES, pH 7.4, 154 mM NaCl, 4.8 mM KCl, 1.2 mM KHPO4, 1.2 mM MgCl2, PI cocktail, and 0.04 mM PMSF. Protein values were determined, and the assay mixture (250 µl) containing 200 µg of membrane protein and 2 nM [35S]GTP{gamma}S was incubated for 5 min at 25°C, followed by incubation in the absence or presence of D1R agonist (SKF-38393) for 5 min. In the experiments where D1R antagonist (SCH-23390) was used, membranes were incubated with antagonists for 10 min before exposure of [35S]GTP{gamma}S (15 min before exposure to the agonist). The reaction was terminated by the addition of 750 µl of ice-cold KRB containing 1 mM EDTA and 1 mM EGTA, followed by centrifugation at 16,000 g for 5 min at 4°C. The membrane pellets obtained were briefly sonicated in 500 µl of ice-cold 100 mM Tris·HCl immunoprecipitation buffer, pH 7.5, containing 200 mM NaCl, 2 mM MgCl2, 1 mM EDTA, 0.2% 2-mercaptoethanol, PI cocktail, and 0.04 mM PMSF, followed by addition of 1 volume of immunoprecipitation buffer. Normal rabbit serum was added (1:100 final dilution) to each reaction tube and incubated at 25°C with gentle shaking. After 30 min, 100 µl of protein A-bearing Staphylococcus aureus cells (Pansorbin cells) were added, and the incubation continued for another 30 min. The suspension was centrifuged (14,800 gfor 3 min), and the supernatant was combined with either specific Gq/11{alpha} or Go{alpha} antiserum at 1:1,000 dilution for 30 min at 25°C. This procedure was followed by the addition of 100 µl of Pansorbin cells, incubation for 30 min, and centrifugation. The pellet was washed, and the immune complex containing the antigen-antibody complex was resuspended in KRB, followed by brief sonication, and radioactivity was measured using liquid scintillation spectrometry. The radioactivity precipitated by the normal rabbit serum was considered as background and subtracted from all agonist-stimulated values (17).

Coprecipitation of [3H]SCH-23390 bound receptor with discrete G{alpha} proteins. To determine linkage between receptor and G proteins, 200 µg of membrane protein were solubilized in 1 ml of immunoprecipitation buffer with 0.2% cholate and 0.5% digitonin. Solubilized membranes were precleared by incubation with normal rabbit serum (1:100 dilution) at 4°C for 60 min, followed by an additional 30 min with 100 µl of Pansorbin cells. The suspension was centrifuged at 4°C, and the supernatant was combined with antisera (1:1,000 dilution) raised against specific peptides of Gq/11{alpha} or Go{alpha} proteins for 3 h at 4°C, followed by an additional 30-min incubation with 100 µl of Pansorbin cells. The mixture was centrifuged and washed, and the pellet was suspended and incubated for 30 min at 30°C in 500 µl of 50 mM Tris·HCl binding buffer, pH 7.5, which included 5 mM MgCl2 and 1 nM [3H]SCH-23390. Nonspecific binding was defined by the addition of 1 µM unlabeled SCH-23390. The reaction was terminated by the addition of 9 ml of ice-cold buffer and was immediately vacuum-filtered over Whatman GF/F filters. The amount of radioactivity on the filter was assessed using liquid scintillation counting, and specific [3H]SCH-23390 binding was determined (40).

Na-K-ATPase assay. Na-K-ATPase activity was determined as reported previously (10). Briefly, SKF-38393-induced Na-K-ATPase inhibition was determined in renal proximal tubular suspensions (1 mg protein/ml) incubated with or without 1 mM ouabain at 37°C for 15 min.

Assay of PLC activity using exogenously added [3H]PIP2 as a substrate. PLC activity was determined by using an exogenous source of [3H]PIP2 as the primary substrate for PLC and monitoring the release of [3H]IP3 (13, 14). Felder et al. (13, 14) have shown that under similar experimental conditions, incubation of [3H]PIP2 with renal membranes for 10–15 min did not result in the formation of [3H]PIP or [3H]PI (13, 14). [3H]PIP2 (0.005 µCi/assay tube) and phosphatidylserine (6 pg/mg protein) were dried to remove the organic solvents. The phospholipid residue was resuspended in sufficient PLC assay buffer (25 mM Tris-C1, 1.0 mM EGTA, 1.0 mM MgC12, 0.94 mM CaCl2, and 5 mM LiCl, pH 7.4) to provide 50 µl of emulsion per assay tube. The reaction mixture contained 150 µl of sonicated membranes (suspended in PLC assay buffer, 100–200 µg of protein) and 50 µl of distilled water, with or without SKF-38393. Following a 5-min preincubation of the membranes and SKF-38393 at 37°C, the reaction was started with the addition of 50 µl of phospholipid emulsion. After 10 min, the reaction was terminated by addition of lipid extraction medium [1 ml of methanol-chloroform, 2:1 (vol/vol)], followed by vigorous vortexing. Chloroform (0.5 ml) and distilled water (0.5 ml) were added to the samples and vortexed vigorously. Following a 20-min incubation at room temperature, the samples were centrifuged at 1,200 gfor 10 min. One milliliter of the upper aqueous layer was added to 5 ml of scintillation fluid, and the radioactive decay was counted in a liquid scintillation spectrophotometer.

PLC activity was also measured in myo-[3H]inositol-labeled membranes (data not shown). The renal proximal tubules were suspended in Dulbecco's modified Eagle's medium and incubated with myo-[3H]inositol under humidified 95% O2-5% CO2 atmosphere at 37°C for 90 min (13, 14). PLC activity was assayed by determining the release of inositol phosphates following the incubation of 200 µ1 of membrane suspension in PLC assay buffer with or without SKF-38393 (total volume of 250 µl). The reaction was terminated by the addition of 1 ml of methanol-chloroform [2:1 (vol/vol)], and the products were separated using the procedure of Felder et al. (13).

AC assay. The AC assay was performed using a modification of the method described by Salomon (32). The reaction mixture included 0.5 mM MgCl2, 0.5 mM 3-isobutyl-1-methylxanthine, 0.2 mM EGTA, 0.5 mM dithiothreitol, 1 µM GTP, 0.1 mM ATP, 2 mM phosphocreatine, 5 units of creatine phosphokinase, and 1 µCi of [{alpha}-32P]ATP (~2.2 x 106 cpm) in 10 mM imidazole buffer, pH 7.3, with or without SKF-38393. After preincubation at 30° for 5 min, the reaction was started by the addition of 50 µg of membrane protein. The reaction was terminated 10 min later by the addition of 300 µl of a solution containing 2% SDS, 25 mM ATP, and 1.3 mM cAMP. [32P]cAMP was separated using Dowex and alumina columns.

Immunoblotting. Proteins were solubilized in Laemmli buffer, separated by SDS-PAGE, and transferred to nitrocellulose membrane (38). The membranes were blocked and incubated with antisera directed against G proteins, GRK2, GAPDH, or D1R in 0.1% Tris-buffered saline, followed by incubation with horseradish peroxidase-conjugated secondary antibodies. Immunoprecipitation was performed as described previously (38).

Enzymatic dephosphorylation. Freshly prepared membranes were resuspended in Tris and EDTA buffer containing 2 mM MgCl2 and exposed to alkaline phosphatase (21, 47). The membranes (80 µg) were incubated with 20 units of alkaline phosphatase in a 100-µl reaction mixture at room temperature for 15–20 min. Okadaic acid (0.1 µM), a serine/threonine phosphatase inhibitor, was used to stop the reaction. The membranes were washed to remove the enzyme; protein was determined and assayed for AC and PLC activities.

PKC and SOD assay. Protein kinase C (PKC) was assayed as detailed in our previous publication (5). SOD activity was determined using a commercially available assay kit utilizing a tetrazolium salt for detection of superoxide radicals generated by xanthine oxidase and hypoxanthine.

Indexes of oxidative stress. Renal proximal tubular carboxymethyllysine was measured using enzyme-linked immunosorbent assay as described by Koo and Vaziri (24), and malondialdehyde was determined using the method of Mihara and Uchiyama (28). Glutathione was determined colorimetrically as described previously (6).

Statistical analysis. Differences between means were evaluated using the unpaired Student's t-test or analysis of variance with a Newman-Keuls multiple test, as appropriate. P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Obese animals were significantly heavier than lean rats (lean, 285.0 g; obese, 510 g). Lipoic acid had no effect on food or water intake and weight gain in lean or obese rats compared with respective controls (data not shown). Oxidative stress measured as content of malondialdehyde, a maker of lipid peroxidation, and carboxymethyllysine, a measure of advanced glycation end products, was significantly higher in obese compared with lean animals (Table 1). In addition, compared with lean rats, the levels of glutathione, a pivotal endogenous antioxidant, and SOD activity were markedly reduced in obese animals (Table 1). Supplementation of diet with lipoic acid reduced the malondialdehyde and carboxymethyllysine levels and restored glutathione levels and SOD activity in obese animals (Table 1).


View this table:
[in this window]
[in a new window]

  		Table 1. Effect of lipoic acid on oxidative markers in renal proximal tubules from lean and obese Zucker rats

 
Lipoic acid restored SKF-38393-induced Na-K-ATPase inhibition in obese rats. Incubation of proximal tubules with SKF-38393, a D1R agonist, caused concentration-dependent inhibition of Na-K-ATPase activity in lean rats. In obese rats, SKF-38393 failed to cause significant inhibition of Na-K-ATPase activity (Fig. 1A). Lipoic acid treatment restored SKF-38393-induced inhibition of Na-K-ATPase activity in obese animals but had no effect in lean rats. In addition, the D1R-mediated inhibition of Na-K-ATPase activity was PKC sensitive, given that SKF-38393 failed to inhibit renal proximal tubular sodium pump activity from lean rats in the presence of the PKC inhibitor chelerythrine chloride. However, the PKA inhibitor H-89 failed to show any effect (Fig. 1B). The basal PKC activity was higher in renal proximal tubular homogenates from obese compared with lean rats (Table 1). Treatment with lipoic acid normalized the PKC activity in obese rats but had no effect in lean rats (Table 1). The Na-K-ATPase {alpha}1-subunit protein content (data not shown) as well as basal activity (nmol·mg protein–1·min–1) were similar in all four experimental groups (lean control, 257.3 ± 30.2; obese control, 288.2 ± 32.3; lean lipoic acid, 267.3 ± 25.6; and obese lipoic acid, 248.6 ± 35.6).


Figure 1
View larger version (24K):
[in this window]
[in a new window]

  		Fig. 1. Effect of lipoic acid on SKF-38393, a dopamine D1 receptor (D1R) agonist, on Na-K-ATPase inhibition. A: renal proximal tubules from lean control (LC), obese control (OC), lean treated (LT), and obese treated (OT) animals were challenged with the indicated doses of SKF-38393 for 20 min. The data are expressed as a percentage of inhibition produced by the indicated concentration of SKF-38393. A single experiment is shown, representative of 6 individual experiments performed in triplicate. B: renal proximal tubules from lean rats were incubated with 1 µM chelerythrine chloride (CCl) and 1 µM H-89, pharmacological inhibitors of PKC and PKA, respectively, followed by challenge with 1 µM SKF-38393 for 20 min. Bars represent means ± SE of 6 different experiments performed in triplicate. *P < 0.05 compared with respective basal state using Student's t-test.

 
Lipoic acid restored D1R-G protein coupling in obese animals. As shown in Fig. 2A, varying concentrations of SKF-38393 elicited increased [35S]GTP{gamma}S membrane binding in renal proximal tubules from lean rats but not in obese rats. Next, we tried to investigate more specific coupling of D1R to G protein subtype Gq/11{alpha}. The proximal tubular membranes were incubated with SKF-38393 and [35S]GTP{gamma}S and immunoprecipitated with Gq/11{alpha} antibodies. As shown in Fig. 2B, the coupling of D1R to G proteins was reduced in obese rats compared with lean rats. The coupling was specific to D1R and Gq/11{alpha} in that the D1R antagonist was able to block it and the Go{alpha} subunit failed to precipitate significant [35S]GTP{gamma}S activity. Similar to agonist-mediated coupling, the basal D1R coupling to Gq/11{alpha} was also reduced in obese compared with lean rats (Fig. 2C). The D1R binding sites that coimmunoprecipitated with Gq/11{alpha} were significantly reduced in obese compared with lean rats. The reduced D1R-Gq/11{alpha} coimmunoprecipitation cannot be attributed to a decrease in protein content of D1R or Gq/11{alpha}, since both the proteins are expressed equally in lean and obese rats (data not shown).


Figure 2
View larger version (15K):
[in this window]
[in a new window]

  		Fig. 2. Effect of lipoic acid on SKF-38393, a D1R agonist, on guanosine 5'-({gamma}-thio)triphosphate (GTP{gamma}S) membrane binding. A: proximal tubular membranes from LC, OC, LT, and OT rats were incubated with varying concentrations of SKF-38393 and ~100,000 cpm of [35S]GTP{gamma}S. The data are expressed as a percentage of the increase in [35S]GTP{gamma}S membrane binding over basal by the indicated dose of SKF-38393. A single experiment is shown, representative of 6 individual experiments performed in triplicate. B: membranes were incubated with 2 nM [35S]GTP{gamma}S in the presence or absence of 10 µM SKF-38393, and G proteins were immunoprecipitated with Gq/11{alpha} or Go{alpha} antibodies. In experiments in which the D1R antagonist SCH-23390 was used, the cells were preincubated with antagonist before SKF-38393 challenge. Bars represent (means ± SE) immunoprecipitated [35S]GTP{gamma}S bound to G proteins from 5–6 individual experiments performed in triplicate. C: to determine basal D1R-G protein coupling, membrane G proteins were immunoprecipitated (IP) with Gq/11{alpha} or Go{alpha} antibodies and incubated with D1R ligand in 1 nM [3H]SCH-23390. Bars represent (means ± SE) [3H]SCH-23390 binding to D1R that were coimmunoprecipitated with G proteins from 5–6 different experiments performed in triplicate. *P < 0.05 compared with LC, LT, and OT using 1-way ANOVA followed by Newman-Keuls multiple test.

 
Lipoic acid restored SKF-38393-mediated PLC stimulation in obese rats. SKF-38393 caused a concentration dependent increase in PLC activity, as evidenced by hydrolysis of exogenously added [3H]PIP2, in renal proximal tubular membranes from lean rats (Fig. 3A). A decreased concentration-response relationship was found in renal membranes from obese rats. Treatment of obese rats with lipoic acid restored D1R-mediated PLC stimulation but had no effect in lean rats (Fig. 3A). Also, lipoic acid treatment had no effect on basal PLC activity in either lean or obese rats (data not shown). Basal as well deoxycholate-stimulated PLC activity was similar in lean and obese rats (Fig. 3B). In addition, preincubation of proximal tubular membranes with D1R antagonist SCH-23390 reduced the SKF-38393-induced PLC stimulation in lean rats (Fig. 3C).


Figure 3
View larger version (24K):
[in this window]
[in a new window]

  		Fig. 3. Effect of lipoic acid on SKF-38393, a D1R agonist, on phospholipase C (PLC) stimulation. A: renal proximal tubular membranes from LC, OC, LT, and OT rats were incubated with [3H]phosphatidylinositol-4,5-bisphosphate ([3H]PIP2) and challenged with indicated doses of SKF-38393 for 15 min. The data are expressed as a percentage of stimulation produced by the indicated concentration of SKF-38393. A single experiment is shown, representative of 6 individual experiments performed in triplicate. B: membranes were incubated with 3 mM deoxycholate (DOC). C: membranes from lean rats were incubated with 10 µM SKF-38393 in the presence and absence of 100 µM SCH-23390. Bars represent means ± SE of 6 different experiments performed in triplicate. *P < 0.05 compared with basal using Student's t-test (A) and 1-way ANOVA followed by post hoc Newman-Keuls multiple test (B).

 
Lipoic acid normalized GRK2 sequestration in obese rats. Proximal tubular membranes from obese rats showed a 50% increase in GRK2 protein levels compared with lean rats (Fig. 4A), whereas the cytosolic fraction from obese rats had a comparable decrease in GRK2 protein content (data not shown), indicating translocation of GRK2 to the membranes. The whole cell lysate from lean and obese rats showed similar GRK2 protein expression (Fig. 4B). Supplementation of lipoic acid to obese rats abolished the GRK2 membrane translocation (Fig. 4A). Lipoic acid treatment did not change the GRK2 protein levels in cell homogenates (Fig. 4B).


Figure 4
View larger version (38K):
[in this window]
[in a new window]

  		Fig. 4. Effect of lipoic acid on G protein-coupled receptor kinase 2 (GRK2) expression and membranous translocation. A: renal proximal tubular membranes from LC, OC, LT, and OT rats were immunoblotted for GRK2 protein. B: proximal tubular homogenate was immunoblotted for GRK2 and normalized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein. Blots shown are a single experiment, representative of 6 individual experiments, whereas bars represent (means ± SE) density arbitrary units from 6 different experiments. *P < 0.05 compared with LC, LT, and OT using 1-way ANOVA followed by post hoc Newman-Keuls multiple test.

 
Lipoic acid reduced GRK2-Gq/11{alpha} coimmunoprecipitation in obese rats. As shown in Fig. 5A, the immunological experiments using Gq/11{alpha} antibodies for immunoprecipitation revealed increased coimmunoprecipitation of GRK2 with Gq/11{alpha} in proximal tubular membranes from obese compared with lean rats. Conversely, the immunoprecipitation with GRK2 antibodies also showed increased interaction between GRK2 and Gq/11{alpha} in obese rats (Fig. 5B). Treatment with lipoic acid decreased the coimmunoprecipitation of GRK2 and Gq/11{alpha} in proximal tubular membranes from obese rats (Fig. 5, Aand B).


Figure 5
View larger version (35K):
[in this window]
[in a new window]

  		Fig. 5. Effect of lipoic acid on GRK2 and Gq/11{alpha} interaction. A: renal proximal tubular membranes from LC, OC, LT, and OT rats were immunoprecipitated (IP) with Gq/11{alpha} antibodies and immunoblotted (IB) for GRK2 and Gq/11{alpha} protein. B: renal proximal tubular membranes were immunoprecipitated with GRK2 antibodies and immunoblotted for Gq/11{alpha} and GRK2 protein. Blots shown are a single experiment, representative of 6 individual experiments, whereas bars represent (means ± SE) density arbitrary units from 6 different experiments. *P < 0.05 compared with LC, LT, and OT using 1-way ANOVA followed by post hoc Newman-Keuls multiple test.

 
Alkaline phosphatase treatment restored D1R-mediated AC stimulation but not PLC stimulation in obese rats. A 20-min alkaline phosphatase treatment of renal proximal tubules from lean and obese rats caused a significant decrease in membrane D1R phosphorylation (Fig. 6A). Incubation of proximal tubular membranes with 100 µM SKF-38393 caused significant AC stimulation in lean rats but not in obese rats (Fig. 6B). Pretreatment of membranes from obese rats with alkaline phosphatase restored the D1R-mediated AC stimulation in obese rats but had no effect in lean rats (Fig. 6B). The basal as well forskolin (10 µM)-induced AC activity (pmol·mg protein–1·min–1) was similar in lean and obese rats (lean basal, 60.2 ± 12; lean forskolin, 203.3 ± 42.3; obese basal, 55.3 ± 17.6; obese forskolin, 193.3 ± 37.8). SKF-38393 also failed to show significant PLC stimulation in obese compared with leans rats. In addition, pretreatment of membranes with alkaline phosphatase failed to restore the SKF-38393-induced PLC stimulation in renal proximal tubular membranes from obese rats (Fig. 6C). As aforementioned, the basal as well deoxycholate-stimulated PLC activities were similar in lean and obese rats (Fig. 3B).


Figure 6
View larger version (25K):
[in this window]
[in a new window]

  		Fig. 6. Effect of enzymatic dephosphorylation on SKF-38393, a D1R agonist, on adenylyl cyclase (AC) and PLC stimulation. A: renal proximal tubular membranes were treated with alkaline phosphatase (AP) and then D1R were immunoprecipitated, followed by immunoblotting for D1R protein and serine phosphorylation. Blots shown are a single experiment, representative of 6 individual experiments. B: membranes were treated with AP and challenged with 10 µM SKF-38393. The data are expressed as a percentage of SKF-38393-induced AC stimulation over basal, and bars represent means ± SE of 6 different experiments performed in triplicate. C: membranes were treated with AP and challenged with 100 µM SKF-38393. The data are expressed as a percentage of SKF-38393-induced PLC activation over basal, and bars represent means ± SE of 6 different experiments performed in triplicate. AP treatment did not change the basal AC or PLC stimulation. *P < 0.05 compared with lean using Student's t-test.

 

   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study we have provided evidence that in renal proximal tubules from obese rats, oxidative stress increases PKC activity and causes membranous translocation of GRK2. Upon translocation, GRK2 interacts with Gq/11{alpha} and acts, at least in part, as a regulator of G protein signaling (RGS) leading to the D1R-Gq/11{alpha} uncoupling. In agreement with our previous studies, we found that SKF-38393, a D1R agonist, failed to elicit D1R-G protein coupling and inhibit Na-K-ATPase activity in obese rats. SKF-38393 caused a concentration-dependent increase in PLC activity in lean rats but failed to cause similar stimulation in obese rats. In addition, we found increased D1R phosphorylation in obese rats and observed that alkaline phosphatase treatment dephosphorylated the membranes and rescued D1R-AC signaling but failed to restore D1R-PLC pathway. These studies suggest that in addition to phosphorylation, GRK2-Gq/11{alpha} interaction also contributes to D1R dysfunction. Therefore, the inability of SKF-38393 to inhibit Na-K-ATPase activity is due to uncoupling of D1R from Gq/11{alpha} and failure to activate PLC. Finally, the treatment of obese animals with antioxidant lipoic acid ameliorated oxidative stress, normalized PKC activity and GRK2 sequestration, and restored SKF-38393-mediated D1R-G protein coupling, PLC activation, and Na-K-ATPase inhibition, suggesting the role of oxidative stress in D1R dysfunction (Fig. 7).


Figure 7
View larger version (20K):
[in this window]
[in a new window]

  		Fig. 7. Proposed mechanism of oxidative stress-mediated renal D1R dysfunction in obese animals. Increased basal PKC activity contributes to GRK2 membrane translocation. GRK2 interacts with Gq/11{alpha}, causing D1R-G protein uncoupling and, subsequently, failure to activate PLC and inhibit Na-K-ATPase (NKA) activity. CML, carboxymethyllysine; DAG, diacylglycerol; DA, dopamine; GSH, glutathione; LA, lipoic acid; IP3, inositol 1,4,5-trisphosphate; MDA, malondialdehyde; SOD, superoxide dismutase. Symbols: +, positive modulation; –, negative modulation; ?, isoform not known; {downarrow}, inhibition/reduction.

 
There are several studies demonstrating that in diabetes there exists not only an increased level of circulating markers of free radical-induced damage but also reduced antioxidant defenses (reviewed in Refs. 8, 19). In agreement with our previous studies, we found that obese animals exhibit oxidative stress as evidenced by increased lipid peroxidation and advanced glycation end products along with decreased levels of glutathione and SOD activity, indicating a compromised antioxidant mechanism (6, 7). Diabetes can induce oxidative stress via several mechanisms (8, 19). These include glucose autoxidation, formation of advanced glycation end products, and activation of polyol pathway (8, 19). Other circulating factors, such as free fatty acid and leptin, also contribute to increased reactive oxygen species generation (8, 19). In agreement with these findings, our group has previously shown that obese Zucker rats are hyperinsulinemic, hyperlipidemic, and hyperglycemic, a hallmark of type 2 diabetes-associated obesity (6, 7). Because the pathogenesis of diabetes as well as its complications involves oxidative stress, the use of antioxidant is an appealing concept (8, 19). Our data show that lipoic acid not only reduces the oxidative stress but restores the normal redox status by normalizing the antioxidant defense mechanism, such as glutathione and SOD.

There is a remarkable parallel in the abnormal dopamine signaling, via D1R, and oxidative stress in hypertension and diabetes (6, 7, 26, 27). White and Sidhu (42) have shown that in spontaneously hypertensive rats (SHR), the renal D1R-G protein coupling defect is contributed by increased oxidative stress. Our own studies in normotensive Fisher 344 rats and streptozotocin-induced type 1 diabetic rats as well as in hypertensive animals provide a convincing correlation between oxidative stress and defective renal D1R function (6, 7, 12, 26, 27). In agreement with our previous findings, incubation of proximal tubules from lean rats with SKF-38393, a D1R agonist, caused a dose-dependent decrease in Na-K-ATPase activity (7). However, no such effect was found in obese animals, whereas treatment of these animals with lipoic acid restored the D1R function, thus providing evidence for the role of oxidative stress in defective D1R signaling. Similar defects have been observed in SHR and Dahl salt-sensitive rats, in which D1R fail to inhibit the NHE3 and Na-K-ATPase activities (18, 20, 30, 35, 42). Although, the exact mechanism for the failure of D1R to inhibit sodium transporters is not known, it is widely perceived that defect in coupling of D1R to G proteins and subsequent impaired production of cytoplasmic messengers could be a contributing factor (18, 20, 35, 42).

Evidence to date indicates that stimulated D1R couple to both Gs{alpha} and Gq/11{alpha} and activate AC and PLC, causing inhibition of the sodium transporters NHE3 and Na-K-ATPase (2, 13, 14, 18, 20, 29, 40, 49). The stimulation of AC causes cAMP accumulation and activates PKA, which leads to the inhibition of NHE3 (2, 18, 20). On the other hand, the mechanisms responsible for DIR-mediated Na-K-ATPase inhibition are elusive. In the present study we also found that in lean rats, SKF-38393-induced inhibition of Na-K-ATPase activity was blocked by the PKC-specific inhibitor chelerythrine chloride, whereas the PKA inhibitor H-89 had no effect. Previous studies have shown that in proximal tubules D1R inhibits Na-K-ATPase, involving a PLC-mediated increase in IP3 and DAG levels and subsequent PKC activation, whereas forskolin or dibutyryl-cAMP failed to decrease pump activity (2, 13, 14, 18, 20, 34). Although the role of cAMP-dependent kinase in renal D1R-mediated Na-K-ATPase inhibition seems to be controversial, surprisingly, most of studies related to D1R desensitization have been confined to AC stimulation (31, 41, 47). Therefore, in the present study we investigated the role of oxidative stress-mediated defect in D1R-Gq/11{alpha}-PLC signaling. We found that incubation of proximal tubular membranes from obese rats with SKF-38393 failed to increase the hydrolysis of PIP2, suggesting a defect in D1R-mediated PLC stimulation. Treatment of obese animals with lipoic acid restored PLC stimulation in response to SKF-38393. The failure of D1R to activate PLC and increase IP3 and DAG levels may explain the inability of SKF-38393 to inhibit Na-K-ATPase. The defect cannot be attributed to changes in activities of PLC or Na-K-ATPase, because basal activities of these molecules were similar in lean and obese rats. Also, deoxycholate, a direct PLC activator, stimulated PLC to a similar extent in lean and obese rats.

The observation that D1R failed to stimulate PLC and inhibit Na-K-ATPase in obese rats despite similar basal activities indicates that the defect lies proximal to these molecules. Of the many mechanisms, D1R-G protein uncoupling has been shown to contribute to D1R desensitization. Previously, our group has shown in obese Zucker rats that D1R are uncoupled from Gs{alpha} and that tempol supplementation restored the coupling, indicating the role of oxidative stress (6, 7). In agreement with these studies we also found that SKF-38393 elicited a concentration-dependent increase in [35S]GTP{gamma}S binding in lean rats but not in obese rats. In addition, our results show that SKF-38393 failed to stimulate Gq/11{alpha} in obese rats. Furthermore, immunological experiments showed decreased coimmunoprecipitation of D1R with Gq/11{alpha} in obese compared with lean rats. These results suggest that D1R are uncoupled from Gq/11{alpha} at the basal state and also fail to interact with G proteins after stimulation. Therefore, the inability of stimulated D1R to couple with G proteins may explain the failure of SKF-38393 to activate PLC and inhibit Na-K-ATPase activity in obese animals. Treatment of obese rats with lipoic acid restored the basal as well as agonist-mediated coupling, thus confirming the role of oxidative stress in D1R-G protein uncoupling.

Desensitization of many GPCRs, including D1R, is thought to largely involve their phosphorylation by either second messenger-activated kinases (e.g., PKA or PKA) or GRKs (16, 20, 31, 33, 47). In renal tissues, the serine phosphorylation of the D1R is mainly carried out by GRKs (15, 31, 33, 41). We have previously reported that in obese rats, D1R hyperphosphorylation is mostly contributed by GRK4 and GRK2 (38). Treatment of these rats with the insulin sensitizer rosiglitazone normalized GRKs, reduced D1R phosphorylation, and restored coupling to G proteins (38). In the present study, we also observed an increased translocation of GRK2 to the membranes as well as hyper D1R phosphorylation in obese rats. Treatment of these rats with lipoic acid normalized GRK2 sequestration and restored D1R function, indicating the role of GRK2 in D1R dysfunction. The observation that in obese animals the increased PKC activity was associated with GRK2 translocation and lipoic acid whereas reducing PKC activity also normalized GRK2 sequestration indicates a signal transduction cross talk. In this regard, there are reports suggesting that GRK2 can be phosphorylated by PKC, which leads to its membrane translocation and increased activity (11, 46). Interestingly, numerous reports have suggested that PKC is stimulated by oxidative stress as well as hyperglycemia (reviewed in Ref. 8), both of which are present in obese rats. Oxidative stress can stimulate PKC by direct tyrosine phosphorylation or via activation of phospholipase D, which leads to DAG synthesis (22, 23, 37). On the other hand, hyperglycemia can increase PKC activity via de novo DAG synthesis from glycolytic intermediates (8). In a previous study (3), our group found that exposure of renal proximal tubular cells to H2O2, an oxidant, caused PKC-dependent GRK2 membranous translocation.

In vitro dephosphorylation of membranes from obese rats with alkaline phosphatase restored only D1R-mediated AC stimulation, not PLC activation, suggesting that the defect in D1R-Gq/11{alpha}-PLC pathway is either independent of receptor phosphorylation or involves additional mechanisms. Recently, it has been shown that at least two members of the GRK family, GRK2 and GRK3, are able to suppress Gq/11{alpha}-coupled receptor/PLC signaling even in the absence of kinase activity (9, 43). GRK2-mediated, phosphorylation-independent receptor regulation has now been demonstrated not only in recombinant systems but also for endogenous receptors in cell lines and in primary cultured hippocampal neurons (44, 45). Phosphorylation-independent receptor regulation by GRK2 is mediated through a specific interaction of GTP-loaded Gq/11{alpha} with the RGS homology (RH) domain situated at the NH2 terminus of GRK2 (9, 36). Using immunological experiments, we also found that membranous GRK2 from obese rats showed increased coimmunoprecipitation with Gq/11{alpha}. Also, Gq/11{alpha} antisera showed similar increased coprecipitation of these proteins, suggesting that membranous translocation of GRK2 results in its interaction with Gq/11{alpha} and therefore may act as an RGS. Interestingly, treatment of obese rats with lipoic acid reversed the GRK2-Gq/11{alpha} interaction to levels similar to those observed in lean rats. Although these data highlight a novel mechanism by which GRK2 can regulate D1R signaling in renal proximal tubules, they do not exclude the possibility that GRK2-mediated receptor phosphorylation is also an important mechanism in D1R desensitization in obese animals. Interestingly, various reports have shown that human renal D1R phosphorylation is mainly contributed by GRK4 with minor contribution from GRK2 (15, 33, 41). Therefore, it is possible that GRK4-mediated phosphorylation may be responsible for modulating D1R-Gs{alpha}-AC pathway, whereas GRK2, functioning as an RGS, regulates D1R-Gq/11{alpha}-PLC signaling.

In conclusion, our data indicate that endogenous GRK2 is a key regulator of renal D1R signaling, utilizing both phosphorylation-dependent and -independent mechanisms to regulate receptor activity. In obese rats the increased oxidative stress causes PKC activation and translocation of GRK 2 to membranes. GRK 2, at least in part, interacts with Gq/11{alpha}, acts as an RGS, and decreases D1R coupling to Gq/11{alpha}. The D1R-G protein uncoupling diminishes the ability of SKF-38393 to stimulate PLC activity and inhibit Na-K-ATPase activity. Antioxidant lipoic acid, while reducing oxidative stress, restores D1R function, providing a substantial evidence for the role of reactive oxygen species in defective D1R signaling in conditions associated with oxidative stress.


   GRANTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported in part by National Institute on Aging Grant AG-25056.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. F. Lokhandwala, Univ. of Houston, 4800 Calhoun Rd., S & R-2 Bldg., Houston, TX 77204 (e-mail: mlokhandwala{at}uh.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Alderson NL, Chachich ME, Youssef NN, Beattie RJ, Nachtigal M, Thorpe SR, Baynes JW. The AGE inhibitor pyridoxamine inhibits lipemia and development of renal and vascular disease in Zucker obese rats. Kidney Int 63: 2123–2133, 2003.[CrossRef][ISI][Medline]
  2. Aperia AC. Intrarenal dopamine: a key signal in the interactive regulation of sodium metabolism. Annu Rev Physiol 62: 621–647, 2000.[CrossRef][ISI][Medline]
  3. Asghar M, Banday AA, Fardoun RZ, Lokhandwala MF. Hydrogen peroxide causes uncoupling of dopamine D1-like receptors from G proteins via a mechanism involving protein kinase C and G-protein-coupled receptor kinase 2. Free Radic Biol Med 40: 13–20, 2006.[CrossRef][ISI][Medline]
  4. Asghar M, Hussain T, Lokhandwala MF. Higher basal serine phosphorylation of D1A receptors in proximal tubules of old Fischer 344 rats. Am J Physiol Renal Physiol 283: F350–F355, 2002.[Abstract/Free Full Text]
  5. Asghar M, Hussain T, Lokhandwala MF. Overexpression of PKC-betaI and -{delta} contributes to higher PKC activity in the proximal tubules of old Fischer 344 rats. Am J Physiol Renal Physiol 285: F1100–F1107, 2003.[Abstract/Free Full Text]
  6. Banday AA, Fazili FR, Marwaha A, Lokhandwala MF. Mitogen-activated protein kinase upregulation reduces renal D1 receptor affinity and G-protein coupling in obese rats. Kidney Int 71: 397–406, 2007.[CrossRef][ISI][Medline]
  7. Banday AA, Marwaha A, Tallam LS, Lokhandwala MF. Tempol reduces oxidative stress, improves insulin sensitivity, decreases renal dopamine D1 receptor hyperphosphorylation, and restores D1 receptor-G-protein coupling and function in obese Zucker rats. Diabetes 54: 2219–2226, 2005.[Abstract/Free Full Text]
  8. Brownlee M. Biochemistry and molecular cell biology of diabetic complications. Nature 414: 813–820, 2001.[CrossRef][Medline]
  9. Carman CV, Parent JL, Day PW, Pronin AN, Sternweis PM, Wedegaertner PB, Gilman AG, Benovic JL, Kozasa T. Selective regulation of G{alpha}q/11 by an RGS domain in the G protein-coupled receptor kinase, GRK2. J Biol Chem 274: 34483–34492, 1999.[Abstract/Free Full Text]
  10. Chen C, Beach RE, Lokhandwala MF. Dopamine fails to inhibit renal tubular sodium pump in hypertensive rats. Hypertension 21: 364–372, 1993.[Abstract/Free Full Text]
  11. Chuang TT, LeVine H 3rd, De Blasi A. Phosphorylation and activation of beta-adrenergic receptor kinase by protein kinase C. J Biol Chem 270: 18660–18665, 1995.[Abstract/Free Full Text]
  12. Fardoun RZ, Asghar M, Lokhandwala M. Role of oxidative stress in defective renal dopamine D1 receptor-G protein coupling and function in old Fischer 344 rats. Am J Physiol Renal Physiol 291: F945–F951, 2006.[Abstract/Free Full Text]
  13. Felder CC, Blecher M, Jose PA. Dopamine-1-mediated stimulation of phospholipase C activity in rat renal cortical membranes. J Biol Chem 264: 8739–8745, 1989.[Abstract/Free Full Text]
  14. Felder CC, Jose PA, Axelrod J. The dopamine-1 agonist, SKF 82526, stimulates phospholipase-C activity independent of adenylate cyclase. J Pharmacol Exp Ther 248: 171–175, 1989.[Abstract/Free Full Text]
  15. Felder RA, Sanada H, Xu J, Yu PY, Wang Z, Watanabe H, Asico LD, Wang W, Zheng S, Yamaguchi I, Williams SM, Gainer J, Brown NJ, Hazen-Martin D, Wong LJ, Robillard JE, Carey RM, Eisner GM, Jose PA. G protein-coupled receptor kinase 4 gene variants in human essential hypertension. Proc Natl Acad Sci USA 99: 3872–3877, 2002.[Abstract/Free Full Text]
  16. Ferguson SS. Evolving concepts in G protein-coupled receptor endocytosis: the role in receptor desensitization and signaling. Pharmacol Rev 53: 1–24, 2001.[Abstract/Free Full Text]
  17. Friedman E, Butkerait P, Wang HY. Analysis of receptor-stimulated and basal guanine nucleotide binding to membrane G proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Anal Biochem 214: 171–178, 1993.[CrossRef][ISI][Medline]
  18. Hussain T, Lokhandwala MF. Renal dopamine receptor function in hypertension. Hypertension 32: 187–197, 1998.[Abstract/Free Full Text]
  19. Jay D, Hitomi H, Griendling KK. Oxidative stress and diabetic cardiovascular complications. Free Radic Biol Med 40: 183–192, 2006.[CrossRef][ISI][Medline]
  20. Jose PA, Eisner GM, Felder RA. Renal dopamine receptors in health and hypertension. Pharmacol Ther 80: 149–182, 1998.[CrossRef][ISI][Medline]
  21. Kim SO, Houtman JC, Jiang J, Ruppert JM, Bertics PJ, Frank SJ. Growth hormone-induced alteration in ErbB-2 phosphorylation status in 3T3-F442A fibroblasts. J Biol Chem 274: 36015–36024, 1999.[Abstract/Free Full Text]
  22. Konishi H, Tanaka M, Takemura Y, Matsuzaki H, Ono Y, Kikkawa U, Nishizuka Y. Activation of protein kinase C by tyrosine phosphorylation in response to H2O2. Proc Natl Acad Sci USA 94: 11233–11237, 1997.[Abstract/Free Full Text]
  23. Konishi H, Yamauchi E, Taniguchi H, Yamamoto T, Matsuzaki H, Takemura Y, Ohmae K, Kikkawa U, Nishizuka Y. Phosphorylation sites of protein kinase C{delta} in H2O2-treated cells and its activation by tyrosine kinase in vitro. Proc Natl Acad Sci USA 98: 6587–6592, 2001.[Abstract/Free Full Text]
  24. Koo JR, Vaziri ND. Effects of diabetes, insulin and antioxidants on NO synthase abundance and NO interaction with reactive oxygen species. Kidney Int 63: 195–201, 2003.[CrossRef][ISI][Medline]
  25. Laight DW, Desai KM, Gopaul NK, Anggard EE, Carrier MJ. F2-isoprostane evidence of oxidant stress in the insulin resistant, obese Zucker rat: effects of vitamin E. Eur J Pharmacol 377: 89–92, 1999.[CrossRef][ISI][Medline]
  26. Marwaha A, Banday AA, Lokhandwala MF. Reduced renal dopamine D1 receptor function in streptozotocin-induced diabetic rats. Am J Physiol Renal Physiol 286: F451–F457, 2004.[Abstract/Free Full Text]
  27. Marwaha A, Lokhandwala MF. Tempol reduces oxidative stress and restores renal dopamine D1-like receptor-G protein coupling and function in hyperglycemic rats. Am J Physiol Renal Physiol 291: F58–F66, 2006.[Abstract/Free Full Text]
  28. Mihara M, Uchiyama M. Determination of malonaldehyde precursor in tissues by thiobarbituric acid test. Anal Biochem 86: 271–278, 1978.[CrossRef][ISI][Medline]
  29. Missale C, Nash SR, Robinson SW, Jaber M, Caron MG. Dopamine receptors: from structure to function. Physiol Rev 78: 189–225, 1998.[Abstract/Free Full Text]
  30. Nishi A, Eklof AC, Bertorello AM, Aperia A. Dopamine regulation of renal Na+,K+-ATPase activity is lacking in Dahl salt-sensitive rats. Hypertension 21: 767–771, 1993.[Abstract/Free Full Text]
  31. Rankin ML, Marinec PS, Cabrera DM, Wang Z, Jose PA, Sibley DR. The D1 dopamine receptor is constitutively phosphorylated by G protein-coupled receptor kinase 4. Mol Pharmacol 69: 759–769, 2006.[Abstract/Free Full Text]
  32. Salomon Y, Londos C, Rodbell M. A highly sensitive adenylate cyclase assay. Anal Biochem 58: 541–548, 1974.[CrossRef][ISI][Medline]
  33. Sanada H, Jose PA, Hazen-Martin D, Yu PY, Xu J, Bruns DE, Phipps J, Carey RM, Felder RA. Dopamine-1 receptor coupling defect in renal proximal tubule cells in hypertension. Hypertension 33: 1036–1042, 1999.[Abstract/Free Full Text]
  34. Satoh T, Cohen HT, Katz AI. Different mechanisms of renal Na-K-ATPase regulation by protein kinases in proximal and distal nephron. Am J Physiol Renal Fluid Electrolyte Physiol 265: F399–F405, 1993.[Abstract/Free Full Text]
  35. Sela S, White BH, Uh M, Kimura K, Patel S, Sidhu A. Dysfunctional D1A receptor-G-protein coupling in proximal tubules of spontaneously hypertensive rats is not due to abnormal G-proteins. J Hypertens 15: 259–267, 1997.[CrossRef][ISI][Medline]
  36. Sterne-Marr R, Tesmer JJ, Day PW, Stracquatanio RP, Cilente JA, O'Connor KE, Pronin AN, Benovic JL, Wedegaertner PB. G protein-coupled receptor kinase 2/G{alpha}q/11 interaction. A novel surface on a regulator of G protein signaling homology domain for binding G{alpha} subunits. J Biol Chem 278: 6050–6058, 2003.[Abstract/Free Full Text]
  37. Taher MM, Garcia JG, Natarajan V. Hydroperoxide-induced diacylglycerol formation and protein kinase C activation in vascular endothelial cells. Arch Biochem Biophys 303: 260–266, 1993.[CrossRef][ISI][Medline]
  38. Trivedi M, Lokhandwala MF. Rosiglitazone restores renal D1A receptor-Gs protein coupling by reducing receptor hyperphosphorylation in obese rats. Am J Physiol Renal Physiol 289: F298–F304, 2005.[Abstract/Free Full Text]
  39. Tsuchida H, Imai G, Shima Y, Satoh T, Owada S. Mechanism of sodium load-induced hypertension in non-insulin dependent diabetes mellitus model rats: defective dopaminergic system to inhibit Na-K-ATPase activity in renal epithelial cells. Hypertens Res 24: 127–135, 2001.[CrossRef][ISI][Medline]
  40. Wang HY, Undie AS, Friedman E. Evidence for the coupling of Gq protein to D1-like dopamine sites in rat striatum: possible role in dopamine-mediated inositol phosphate formation. Mol Pharmacol 48: 988–994, 1995.[Abstract]
  41. Watanabe H, Xu J, Bengra C, Jose PA, Felder RA. Desensitization of human renal D1 dopamine receptors by G protein-coupled receptor kinase 4. Kidney Int 62: 790–798, 2002.[CrossRef][ISI][Medline]
  42. White BH, Sidhu A. Increased oxidative stress in renal proximal tubules of the spontaneously hypertensive rat: a mechanism for defective dopamine D1A receptor/G-protein coupling. J Hypertens 16: 1659–1665, 1998.[CrossRef][ISI][Medline]
  43. Willets JM, Challiss RA, Kelly E, Nahorski SR. G protein-coupled receptor kinases 3 and 6 use different pathways to desensitize the endogenous M3 muscarinic acetylcholine receptor in human SH-SY5Y cells. Mol Pharmacol 60: 321–330, 2001.[Abstract/Free Full Text]
  44. Willets JM, Mistry R, Nahorski SR, Challiss RA. Specificity of g protein-coupled receptor kinase 6-mediated phosphorylation and regulation of single-cell m3 muscarinic acetylcholine receptor signaling. Mol Pharmacol 64: 1059–1068, 2003.[Abstract/Free Full Text]
  45. Willets JM, Nash MS, Challiss RA, Nahorski SR. Imaging of muscarinic acetylcholine receptor signaling in hippocampal neurons: evidence for phosphorylation-dependent and -independent regulation by G-protein-coupled receptor kinases. J Neurosci 24: 4157–4162, 2004.[Abstract/Free Full Text]
  46. Winstel R, Freund S, Krasel C, Hoppe E, Lohse MJ. Protein kinase cross-talk: membrane targeting of the beta-adrenergic receptor kinase by protein kinase C. Proc Natl Acad Sci USA 93: 2105–2109, 1996.[Abstract/Free Full Text]
  47. Yu P, Asico LD, Luo Y, Andrews P, Eisner GM, Hopfer U, Felder RA, Jose PA. D1 dopamine receptor hyperphosphorylation in renal proximal tubules in hypertension. Kidney Int 70: 1072–1079, 2006.[CrossRef][ISI][Medline]
  48. Yu PY, Asico LD, Eisner GM, Jose PA. Differential regulation of renal phospholipase C isoforms by catecholamines. J Clin Invest 95: 304–308, 1995.[ISI][Medline]
  49. Zeng C, Eisner GM, Felder RA, Jose PA. Dopamine receptor and hypertension. Curr Med Chem Cardiovasc Hematol Agents 3: 69–77, 2005.[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Renal Physiol.Home page
A. A. Banday and M. F. Lokhandwala
Oxidative stress-induced renal angiotensin AT1 receptor upregulation causes increased stimulation of sodium transporters and hypertension
Am J Physiol Renal Physiol, September 1, 2008; 295(3): F698 - F706.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
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]

Home page
 HypertensionHome page
C. Zeng, V. A. M. Villar, G. M. Eisner, S. M. Williams, R. A. Felder, and P. A. Jose
G Protein-Coupled Receptor Kinase 4: Role in Blood Pressure Regulation
Hypertension, June 1, 2008; 51(6): 1449 - 1455.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/1/F306    most recent
00108.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted