Dopamine recruits D1A receptors to Na-K-ATPase-rich caveolar plasma membranes in rat renal proximal tubules

Meghna Trivedi, Vihang A. Narkar, Tahir Hussain, and Mustafa F. Lokhandwala

Heart and Kidney Institute, College of Pharmacy, University of Houston, Texas 77204-5041

Submitted 22 January 2004 ; accepted in final form 18 July 2004


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Activation of dopamine D1A receptors in renal proximal tubules causes inhibition of sodium transporters (Na-K-ATPase and Na/H exchanger), leading to a decrease in sodium reabsorption. In addition to being localized on the plasma membrane, D1A receptors are mainly present in intracellular compartments under basal conditions. We observed, using [3H]SCH-23390 binding and immunoblotting, that dopamine recruits D1A receptors to the plasma membrane in rat renal proximal tubules. Furthermore, radioligand binding and/or immunoblotting experiments using pharmacological modulators showed that dopamine-induced D1A receptor recruitment requires activation of cell surface D1-like receptors, activation of adenylyl cyclase, and intact endocytic vesicles with internal acidic pH. A key finding of this study was that these recruited D1A receptors were functional because they potentiated dopamine-induced [35S]GTP{gamma}S binding, cAMP accumulation, and Na-K-ATPase inhibition. Interestingly, dopamine increased immunoreactivity of D1A receptors specifically in caveolin-rich plasma membranes isolated by a sucrose density gradient. In support of this observation, coimmunoprecipitation studies showed that D1A receptors interacted with caveolin-2 in an agonist-dependent fashion. The caveolin-rich plasma membranes had a high content of the {alpha}1-subunit of Na-K-ATPase, which is a downstream target of D1A receptor signaling in proximal tubules. These results show that dopamine, via the D1-like receptor-adenylyl cyclase pathway, recruits D1A receptors to the plasma membrane. These newly recruited receptors couple to G proteins, increase cAMP, and participate in dopamine-mediated inhibition of Na-K-ATPase in proximal tubules. Moreover, dopamine-induced recruitment of D1A receptors to the caveolin-rich plasma membranes brings them in close proximity to targets such as Na-K-ATPase in proximal tubules of Sprague-Dawley rats.

G protein-coupled receptor; translocation


RENAL DOPAMINE PLAYS AN IMPORTANT role in the maintenance of sodium homeostasis during conditions of sodium overload. Under these conditions, dopamine is synthesized and released from proximal tubules of the kidney and contributes to maintenance of sodium balance (22, 42). Dopamine exerts its natriuretic and diuretic actions primarily via activation of D1A receptors in the proximal tubules (1, 23). In the proximal tubules, dopamine D1A receptors are coupled to activation of adenylyl cyclase (13) and phospholipase C (12). Activation of adenylyl cyclase causes stimulation of PKA, which in turn inhibits the Na/H exchanger on the apical brush-border membrane (13). On the other hand, activation of phospholipase C leads to PKC-mediated inhibition of the Na-K-ATPase located on the basolateral membrane (9). Inhibition of these sodium transporters decreases sodium reabsorption in the proximal tubules and in turn increases sodium and water excretion by the kidney (20). Therefore, in conditions of sodium overload, it is critical that the responses to dopamine be maintained for a longer period of time.

A prolonged response to endogenously produced or exogenously administered dopamine depends on the abundance of the D1A receptors on the cell surface. D1A receptors belong to the G protein-coupled receptor (GPCR) superfamily. Intracellular trafficking of GPCRs regulates their abundance at the cell surface. After synthesis, most GPCRs are transported to the plasma membrane, their primary site of action (36). The translocation of GPCRs to the plasma membrane after synthesis is thought to be a constitutive, agonist-independent process. This may be true for the receptors located predominantly on the plasma membrane under basal conditions. However, unlike most GPCRs, dopamine D1A receptors are primarily located in intracellular compartments under basal conditions in the kidney and heart (8, 31, 35). It has been shown that intracellular D1A receptors, on activation by an agonist, translocate to the plasma membrane in LLC-PK1 cells, a proximal tubule-like cell line (8). However, the ability of dopamine to recruit D1A receptors has not been demonstrated in the proximal tubules isolated from rodent kidneys. More importantly, the ability of these recruited receptors to couple to G proteins and to signal to downstream effector molecules (e.g., adenylyl cyclase, Na-K-ATPase) has not been evaluated. It is possible that dopamine-induced D1A receptor recruitment helps sustain the response to dopamine in proximal tubules.

The role of heterogeneously distributed cellular plasma membranes in the coordination of transcellular signaling events has been extensively studied during the past decade. Caveolae are invaginations in the plasma membranes and play a role in signal transduction and GPCR trafficking (28). Caveolae are thought to be important not only for internalization of GPCRs but also for creating compartmentation that facilitates/restricts interaction between receptors and downstream effector molecules (32, 34). In proximal tubules, D1A receptors are immunoprecipitated with caveolin-2, which is the only isoform of caveolin present in proximal tubules of rats (46). Moreover, effector molecules in D1-like receptor signaling pathways, like G proteins (Gs and Gq/11), PKA subunits, and protein phosphatase 2A, are also immunoprecipitated with caveolin-2 (46). Therefore, it is possible that recruitment of D1A receptors by dopamine to caveolar plasma membranes rich in effector molecules may accelerate transduction of D1A receptor signaling in proximal tubules.

In the first part of this study, we have characterized the effect of dopamine on D1A receptor recruitment to the plasma membrane in proximal tubules of Sprague-Dawley rats. We also have studied the signaling pathway by which dopamine recruits D1A receptors to the plasma membrane. In the second part, the functionality of the recruited D1A receptors has been tested by investigating the effect of dopamine-induced D1A receptor recruitment on the receptor-G protein-second messenger-effector axis. In the third part, we have evaluated the recruitment of D1A receptors to the specialized caveolar domains of the plasma membrane in proximal tubules of Sprague-Dawley rats. Moreover, we have assessed the distribution of effectors such as Na-K-ATPase and the Na/H exchanger in these domains to further identify the importance of compartmentalized recruitment of D1A receptors by dopamine.


    METHODS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials

The following chemicals and materials were purchased from the source indicated: [3H]SCH-23390, [35S]GTP{gamma}S (DuPont New England Nuclear, Boston, MA); cAMP radioimmunoassay kit (Amersham Bioscience, Piscataway, NJ); rabbit anti-rat D1A and D1B polyclonal antibodies, horseradish peroxidase-conjugated anti-rabbit antibodies, and chemiluminescent substrate (Alpha Diagnostics, San Antonio, TX); mouse anti-rabbit Na-K-ATPase {alpha}1-subunit monoclonal antibodies, rabbit anti-human Rab 5A and 5B polyclonal antibodies, and horseradish peroxidase-conjugated anti-mouse antibodies (Santa Cruz Biotechnology, Santa Cruz, CA); caveolin-2 antibodies (BD Biosciences, Lexington, KY); complete protease inhibitor cocktail (Boehringer Mannheim, Indianapolis, IN); Immobilon P membrane (Millipore, Bedford, MA); and X-ray film (Kodak, Rochester, NY). All the other chemicals were purchased from Sigma (St. Louis, MO) and were of highest grade available.

Animals

All experimental protocols were reviewed and approved by the University of Houston Institutional Animal Care and Use Committee. Male Sprague-Dawley rats (12–15 wk old, weighing 200–250 g) were used in all experiments (Harlan Sprague-Dawley, Indianapolis, IN). The rats were maintained in a temperature-controlled animal care facility, with a 12:12-h light-dark cycle. All the rats were provided standard rat chow containing 0.4% sodium (Purina Mills, St. Louis, MO) and tap water ad libitum.

Isolation of Proximal Tubules From Kidney

Proximal tubules were prepared using the Ficoll gradient method as previously described (9). The enriched proximal tubules were resuspended in 5 ml of modified Krebs-Henseleit buffer A (KHBA; in mM: 118 NaCl, 27.2 NaHCO3, 4 KCl, 1.25 CaCl2, 1.2 MgCl2, 1.0 KH2PO4, 5 glucose, and 10.0 HEPES at pH 7.4). For the Na-K-ATPase activity assay, the proximal tubules were resuspended in KHBC with no phosphate and 0.12 mM MgCl2. In an aliquot of the proximal tubular suspension, Trypan blue exclusion tests were performed to ensure >95% proximal tubular cell viability (14).

Drug Treatment of Proximal Tubules

The proximal tubular suspension (1 mg/ml protein) was treated with either vehicle (0.1% sodium metabisulfite for dopamine and DMSO for forskolin), dopamine (various concentrations as indicated), or forskolin (1 µM) at 37°C for 1, 5, or 15 min as indicated. For inhibitor studies, the proximal tubules were pretreated with SCH-23390 (1 µM) at 37°C for 15 min, bafilomycin A1 (20 nM) at room temperature for 10 min, or 2',5'-dideoxyadenosine 3'-triphosphate (DDA; 10 µM) at 37°C for 15 min before dopamine treatment. After the drug treatments, proximal tubules were rapidly frozen in dry ice/acetone. Before being frozen, protease inhibitor cocktail was added to proximal tubular suspension.

Preparation of Plasma Membranes

Plasma membranes (including both basolateral and brush border) were prepared from the proximal tubular suspension as described previously, with slight modifications (29). The freeze-thawed cell lysates were first centrifuged at 30,000 g at 4°C for 20 min to obtain pellets. The pellets were resuspended in 5 ml of homogenization buffer (50 mM Tris·HCl, 1 mM MgCl2, 0.2 mM PMSF, at pH 7.4) containing protease inhibitor cocktail and homogenized using a Wheaton homogenizer (20 strokes at setting 7). The homogenate was centrifuged at 2,500 g for 10 min to remove cellular debris and the nuclear fraction. The supernatant was centrifuged at 30,000 g at 4°C for 20 min to obtain membrane pellets. The pellet was resuspended in homogenization buffer and treated with 2.5 IU/ml catecholamine methyl transferase at 37°C for 15 min to degrade dopamine remaining in the membrane fraction. After incubation with catecholamine methyl transferase, the membrane fraction was washed three times with homogenization buffer by centrifugation at 30,000 g at 4°C for 10 min. Finally, the pellet was resuspended in binding buffer (250 µl; 50 mM Tris·HCl, 1 mM MgCl2, 0.2 mM sodium metabisulfite, pH 7.4) at 2 mg/ml protein concentration. The purity of plasma membranes was confirmed by enrichment of the Na-K-ATPase {alpha}1-subunit containing the nuclear fraction (by ~2-fold) and by decreasing levels of Rab 5A and 5B, an endosomal marker protein (by ~4-fold) compared with the total particulate fraction (data not shown). Plasma membranes, with the relatively constant degree of purity, were used for [3H]SCH-23390 binding, immunoblotting, and [35S]GTP{gamma}S binding experiments.

Radioligand ([3H]SCH-23390) Binding

To determine whether dopamine recruits D1-like receptors to the cell surface, [3H]SCH-23390 binding was performed in plasma membranes isolated from proximal tubules. The plasma membrane samples (50 µg) were incubated with 10 nM [3H]SCH-23390 (specific activity 86 Ci/mmol) in binding buffer (final volume 250 µl) at 25°C for 90 min. Nonspecific binding was defined using 10 µM unlabeled SCH-23390. Specific binding was calculated as the difference between the total and nonspecific binding. Here, we have used only 10 nM [3H]SCH-23390 because, in saturation binding experiments, 10 nM was the lowest concentration of [3H]SCH-23390 that represented quantitatively similar changes in maximum binding capacity (increased from 71.82 ± 4.87 to 108.75 ± 2.95 fmol/mg protein) by dopamine. Moreover, the affinity of [3H]SCH-23390 for D1-like receptors, as reflected by Kd, was not altered with dopamine treatment of proximal tubules (5.5 ± 0.6 vs. 5.9 ± 0.2 nM).

Immunoblotting of D1A Receptor Protein

Because SCH-23390 is a nonspecific D1-like receptor (D1A/D1 and D1B/D5) ligand, the recruitment of D1A vs. D1B was examined by using specific antibodies in immunoblotting. In these experiments, membrane samples (3 µg protein for D1A and 10 µg protein for D1B) were separated by 10% SDS-PAGE and electrophoretically blotted onto an Immobilon P membrane. The membrane blot was incubated with primary polyclonal antibodies (1:1,000 dilution): anti-D1A receptor or anti-D1B receptor antibodies, followed by horseradish peroxidase-conjugated secondary antibodies (1:1,000 dilution). The amount of protein loaded was in the linear range. The bands were detected with a chemiluminescent substrate on X-ray films and densitometrically quantified using Scion Image software provided by the National Institutes of Health (NIH).

Measurement of [35S]GTP{gamma}S Binding

The ability of newly recruited D1A receptors to couple to G proteins was studied by [35S]GTP{gamma}S binding in plasma membranes as described previously (19). Briefly, [35S]GTP{gamma}S binding was stimulated by various concentrations (1 nM-0.1 µM, 15 min, 37°C) of fenoldopam, a preferential D1-like receptor agonist. The assay was carried out in the presence of 0.6 nM (~100,000 cpm) of [35S]GTP{gamma}S (specific activity 1,250 Ci/mmol), 5 µg of membrane protein, and fenoldopam (1 nM-0.1 µM). Nonspecific [35S]GTP{gamma}S binding was determined in the presence of 100 µM unlabeled GTP{gamma}S. Binding in the control treatment group was normalized to 100% because there was no significant difference in basal GTP{gamma}S binding by treatment with either bafilomycin A1 or dopamine.

cAMP Accumulation in Proximal Tubules

The contribution of newly recruited D1A receptors to the dopamine-induced increase in cAMP accumulation was determined using bafilomycin A1 as an inhibitor of D1A receptor recruitment (8). Measurement of cAMP accumulation was performed as described previously (18). cAMP levels were measured with a cAMP radioimmunoassay kit. cAMP values were calculated and expressed as femtomoles per milligram protein using a cAMP acetylation standard curve (2–256 fmol) provided with the kit.

Na-K-ATPase Activity Assay in Proximal Tubules

Na-K-ATPase activity was measured as previously described (17) as the function of liberated inorganic phosphate and represented as percent inhibition (from basal).

Preparation of Caveolar Plasma Membranes

After the drug treatment (vehicle or 10 nM dopamine) for 1 min, rat proximal tubules were fractionated using a detergent-free method as described previously (33). After the drug treatment (vehicle or 10 nM dopamine) for 1 min, proximal tubules were washed with ice-cold KHBA and resuspended in 2 ml of 500 mM sodium carbonate, pH 11. Proximal tubules were homogenized using a Wheaton homogenizer (20 strokes at setting 7) and then a sonicator with three 20-s bursts, both on ice. The homogenate was diluted to 45% sucrose by addition of an equal volume of 90% sucrose in Mes buffer saline (MBS; 25 mM MES, 150 mM NaCl, pH 6.5) and loaded at the bottom of an ultracentrifuge tube. A 5–35% discontinuous sucrose gradient was formed by placing 4 ml of 35% sucrose prepared in MBS with 250 mM sodium carbonate then 4 ml of 5% sucrose (also in MBS/Na2CO3) on top of the sample. The gradient was centrifuged at 39,000 rpm in a SW40Ti rotor (Beckman Instruments) for 16–18 h at 4°C. Fraction 7 was collected as the caveolar plasma membrane, and fractions 1–4 (lower 45% sucrose layer) were collected as the noncaveolar plasma membranes. Preliminary immunoblotting experiments were performed to establish fraction 7 to be the caveolar plasma membrane fraction as it contained the maximum level of caveolin-2 protein. The topmost fraction (fraction 12) was considered to be the cytosolic fraction.

Isolation of Basolateral and Brush-Border Membranes

Basolateral and brush-border membranes were prepared from renal cortexes by using a previously described method (2, 39). Briefly, the cortexes were homogenized in 10 mM Tris·HCl buffer (pH 7.4) containing 250 mM sucrose and 0.1 mM PMSF (buffer A). The homogenate was centrifuged at 2,500 g for 15 min. The supernatant was recentrifuged at 24,000 for 20 min. The fluffy layer of the pellet was resuspended in buffer A. Percoll was added to the suspension and mixed thoroughly in a volume ratio of 1.0 ml percoll/11.5 ml suspension. The suspension was centrifuged at 30,000 g for 35 min. The lower dense fraction (for brush-border membrane) and the upper cloudy layer (for basolateral membrane) were separated and resuspended separately in buffer B (100 mM KCl, 100 mM mannitol, and 5 mM HEPES, pH 7.2) and centrifuged at 34,000 g for 30 min. The pellets were washed once more in buffer B. Finally, the pellets were suspended in buffer A with a protein concentration of ~2 mg/ml and stored frozen at –70°C until use. Protein was assayed using a Pierce BCA (bicinchoninic acid) reagent kit.

Immunoblotting of Na-K-ATPase, Na/H Exchanger, and Caveolin-2 Proteins

Basolateral and brush-border membranes were used to prepare loading samples containing SDS-Laemmli and bromophenol blue for immunoblotting. These loading samples [protein: 0.5 µg for Na-K-ATPase, 2 µg for Na/H exchanger (NHE3), and 4 µg for caveolin-2] were then resolved by 10% SDS-PAGE and electrophoretically blotted onto an Immobilon P membrane. The membrane blots were incubated first with primary antibodies, i.e., monoclonal anti-Na-K-ATPase {alpha}1-subunit antibodies (1:10,000), polyclonal anti-NHE3 antibodies (1:1,000), or monoclonal anti-caveolin-2 antibodies (1:250), followed by appropriate horseradish peroxidase-conjugated secondary antibodies, i.e., goat anti-rabbit (1:1,000) or goat-anti-mouse (1:4,000). The bands were detected with a chemiluminescent substrate on X-ray film and densitometrically quantified using Scion Image software provided by the NIH.

Coimmunoprecipitation of Caveolin-2 With D1A Receptors

Immunoprecipitation of D1A receptors. A previously described method (21) was used for immunoprecipitation of D1A receptors from proximal tubular cell lysates with slight modification. Briefly, proximal tubular cell lysates were suspended in coimmunoprecipitation buffer (50 mM Tris-base, 150 mM NaCl, 2 mM EDTA, 1 mM orthovanadate, and 1 mM PMSF, 1% Nonidet P-40, 0.5% sodium cholate, and protease inhibitor cocktail, pH 7.4). Next, these samples were incubated with D1A receptor antibody overnight to allow the formation of a D1A receptor-antibody complex. This complex was incubated for 2 h with protein A/G covalently bound to agarose beads (protein A/G-agarose). The D1A receptor-antibody-protein A/G complex attached to agarose beads was centrifuged, washed once with immunoprecipitation buffer, and finally with 50 mM Tris·HCl, pH 8.0. All the steps of immunoprecipitation were carried out on ice at 4°C. Finally, the D1A receptor-antibody-protein A/G complex was dissociated with 2x Laemmli buffer containing 125 mM Tris·HCl, 4% SDS, 5% {beta}-mercaptoethanol, and 20% glycerol at 37°C for 1 h. The samples were vortexed and centrifuged at room temperature, and the supernatant was used for electrophoresis.

Detection of caveolin-2 proteins interacting with the D1A receptor. The immunoprecipitated samples were resolved by 10% SDS-PAGE, and the proteins were electrotransferred on an Immobilon P membrane. The membrane was blocked with 5% nonfat milk in PBS with 0.1% Tween 20. Specific caveolin-2 antibodies (1:250) were used to detect interaction of D1A receptors with this protein. Horseradish peroxidase-conjugated secondary antibody (1:4,000) was used to probe primary antibodies, and the bands were visualized with an enhanced chemiluminiscence reagent kit. The same Immobilon P membranes were stripped off the antibody complex using stripping buffer and was used for immunoblotting of D1A receptors as described above. The band density of caveolin-2 proteins was normalized to the band density of D1A receptors.

Data Analysis

The data are represented as means ± SE of the number (n) of experiments. The data were analyzed using an unpaired Student's t-test and one-way ANOVA (with an appropriate post hoc test) as indicated. The difference was considered statistically significant when P < 0.05.


    RESULTS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Dopamine Recruits D1-Like Receptors to the Plasma Membrane

We found a >50% increase in specific [3H]SCH-23390 binding in plasma membranes prepared from proximal tubules treated with dopamine (1 and 10 nM) compared with vehicle (Fig. 1A). Moreover, this increase in [3H]SCH-23390 binding by dopamine was observed for up to 15 min of dopamine treatment (Fig. 1B).



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Fig. 1. Dopamine-induced recruitment of D1-like receptors. Proximal tubules were treated with either vehicle, or dopamine (DA; 1 or 10 nM) at 37°C for 1 min (A) or with dopamine (10 nM) at 37°C for 0, 1, 5, and 15 min (B). [3H]SCH-23390 binding experiments were performed in plasma membranes isolated from proximal tubules as described in METHODS. Basal [3H]SCH-23390 binding was 40.45 ± 5.69 (A) or 47.89 ± 6.79 fmol/mg protein (B). Values are means ± SE expressed as percent increase in specific [3H]SCH-23390 binding over vehicle or time 0 (n = 4–7). Significantly different from vehicle treatment (A) or from time 0 (B) by 1-way ANOVA, Dunnett's multiple comparison test (*P < 0.05).

 
Dopamine Recruits D1A, But Not D1B, Receptors to the Plasma Membrane

Dopamine increased abundance of D1A receptors, but not D1B receptors, on plasma membranes in proximal tubules. Dopamine (10 nM) caused an ~50% increase in density of D1A receptor protein in plasma membranes (Fig. 2A), whereas no significant change in the density of D1B receptor protein was seen in plasma membranes after dopamine treatment of proximal tubules (Fig. 2B).



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Fig. 2. Dopamine-induced recruitment of D1A, and not D1B, receptors. Proximal tubules were treated with either vehicle (V) or 10 nM dopamine (D) at 37°C for 1 min. Immunoblotting experiments using D1A (A and C) and D1B (B) antibodies were performed in plasma membranes (A and B) as well as in proximal tubular lysate (C; n = 4). Top: representative immunoblot. Bottom: graph indicating densitometric analysis (means ± SE) of the protein bands (n = 4). Significant difference between vehicle- and dopamine-treated groups by unpaired Student's t-test (*P < 0.05).

 
In addition, dopamine did not affect the immunoreactivity of D1A receptor protein in proximal tubular cell lysates (Fig. 2C), confirming that the effect of dopamine is due to recruitment of intracellular D1A receptors to the plasma membrane and not to an increase in affinity of the antibodies for receptors.

Dopamine Recruits D1A Receptors Via Activation of D1-Like Receptors

SCH-23390 completely blocked the dopamine-induced increase in density of D1A receptor protein on plasma membranes, while having no significant effect on D1A receptor density by itself (Fig. 3).



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Fig. 3. Inhibition of dopamine-induced D1A receptor recruitment by SCH-23390. Proximal tubules were pretreated with either distilled water or 1 µM SCH-23390 at 37°C for 15 min and then treated again with either 0.1% Na metabisulfite or 10 nM dopamine at 37°C for 1 min. C, distilled water+Na metabisulfite; S, SCH-23390+Na metabisulfite; D, distilled water+dopamine; D+S, SCH-23390+dopamine. Immunoblotting experiments using D1A receptor-specific antibodies were performed in plasma membranes as described in METHODS. Top: representative immunoblot. Bottom: graph indicating densitometric analysis (means ± SE) of the protein bands (n = 4). *Significant difference between vehicle (C) and D groups; #significant difference between D and D+S groups by 1-way ANOVA, Newman-Keuls multiple comparison test (both P < 0.05).

 
Bafilomycin A1 Inhibits Recruitment of D1A Receptors by Dopamine

Bafilomycin A1 is an inhibitor of H+-ATPases, which are located on endocytic vesicles with an internal acidic environment. Bafilomycin A1 significantly blocked the dopamine-induced increase in the abundance of D1A receptors on plasma membranes, as shown by [3H]SCH-23390 binding (Fig. 4A) and immunoblotting (Fig. 4B). There was no significant change in either [3H]SCH-23390 binding or D1A receptor protein density in plasma membranes by bafilomycin A1 alone. Moreover, increasing concentrations of bafilomycin A1 (1–200 nM) did not displace [3H]SCH-23390 binding (data not shown), establishing that bafilomycin A1 does not act as an antagonist for D1-like receptors.



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Fig. 4. Inhibition of dopamine-induced D1A receptor recruitment by bafilomycin A1. Proximal tubules were pretreated with either DMSO or 20 nM bafilomycin A1 at room temperature for 10 min and then treated with either vehicle or 10 nM dopamine at 37°C for 1 min. C, DMSO+Na metabisulfite; B, bafilomycin A1+Na metabisulfite; D, DMSO+dopamine; D+B, bafilomycin A1+dopamine. [3H]SCH-23390 binding experiments (A; n = 6) and immunoblotting (B; n = 5) were performed in plasma membranes isolated from proximal tubules. Basal [3H]SCH-23390 binding was 45.15 ± 8.16 fmol/mg protein. Values are means ± SE. *Significant difference between vehicle (C) and D groups; #significant difference between D and D+B groups by 1-way ANOVA, Newman-Keuls multiple comparison test (both P < 0.05).

 
Forskolin Recruits D1A Receptors to the Plasma Membrane

Forskolin caused a >100% increase in [3H]SCH-23390 binding (Fig. 5A) and a similar increase in the density of D1A receptor protein (Fig. 5B) in plasma membranes. Moreover, treatment of proximal tubules with dopamine in the presence of forskolin did not result in a further increase in [3H]SCH-23390 binding in plasma membranes (data not shown), suggesting a common pathway (adenylyl cyclase activation) for dopamine- and forskolin-induced recruitment of D1A receptors.



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Fig. 5. Forskolin-induced D1A receptor recruitment. Proximal tubules were treated with either vehicle (V) or 1 µM forskolin (F) at 37°C for 15 min. [3H]SCH-23390 binding experiments (A; n = 7) and immunoblotting (B; n = 4) were performed in plasma membranes as described in METHODS. Basal [3H]SCH-23390 binding was 44.17 ± 5.15 fmol/mg protein. Values are means ± SE. *Significant difference between V and F groups by unpaired Student's t-test (P < 0.05).

 
DDA Inhibits Recruitment of D1A Receptors by Dopamine

DDA, an inhibitor of adenylyl cyclase, significantly blocked the dopamine-induced increase in the immunoreactivity of D1A receptor protein on plasma membranes (Fig. 6). There was no significant change in D1A receptor protein density in plasma membranes using DDA alone (3).



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Fig. 6. Inhibition of dopamine-induced D1A receptor recruitment by 2',5'-dideoxyadenosine 3'-triphosphate (DDA). Proximal tubules were pretreated with either distilled water or 10 µM DDA at 37°C for 15 min and then treated again with either 0.1% Na metabisulfite or 10 nM dopamine at 37°C for 1 min. C, distilled water+Na metabisulfite; A, DDA+Na metabisulfite; D, distilled water+dopamine; D+A, DDA+dopamine. Immunoblotting experiments using D1A receptor-specific antibodies were performed in plasma membranes as described in METHODS. Top: representative immunoblot. Bottom: graph indicating densitometric analysis (means ± SE) of the protein bands (n = 5). *Significant difference between vehicle (C) and D groups; #significant difference between D and D+A groups by 1-way ANOVA, Newman-Keuls multiple comparison test (both P < 0.05).

 
Newly Recruited D1A Receptors Couple to G Proteins

The increase in [35S]GTP{gamma}S binding by fenoldopam was significantly higher at all concentrations (1 nM-0.1 µM) in plasma membranes isolated from dopamine-treated proximal tubules compared with vehicle-treated proximal tubules (Fig. 7). The maximal stimulation of ~12% in [35S]GTP{gamma}S binding was produced by 0.1 µM fenoldopam in plasma membranes of vehicle-treated proximal tubules compared with ~35% stimulation in plasma membranes of dopamine-treated proximal tubules. Furthermore, pretreatment of proximal tubules with bafilomycin A1 completely inhibited the dopamine-induced increase in [35S]GTP{gamma}S binding with fenoldopam. A concentration-dependent stimulation of [35S]GTP{gamma}S binding by fenoldopam was observed in all the groups. Basal GTP{gamma}S binding was not affected by either bafilomycin A1 or dopamine (Fig. 7).



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Fig. 7. G protein coupling of newly recruited D1A receptors. Proximal tubules were pretreated with either DMSO or 20 nM bafilomycin A1 at room temperature for 10 min and then treated with either 0.1% Na metabisulfite or dopamine (10 nM) at 37°C for 1 min. [35S]GTP{gamma}S binding experiments were performed in plasma membranes prepared from proximal tubules (n = 7). GTP{gamma}S binding was stimulated by various concentrations (1 nM-0.1 µM) of fenoldopam. Basal GTP{gamma}S binding was similar in all groups: 1.10 ± 0.12 pmol/mg protein in control, 1.11 ± 0.11 pmol/mg protein in bafilomycin A1-treated tubules, 1.21 ± 0.13 pmol/mg protein in dopamine-treated tubules, and 1.16 ± 0.13 pmol/mg protein in dopamine + Baf A1-treated tubules. Values are means ± SE. *Significant difference between vehicle- and dopamine-treated groups; #significant difference between dopamine- and dopamine+bafilomycin A1-treated groups by 1-way ANOVA, Newman-Keuls multiple comparison test (both P < 0.05).

 
Bafilomycin A1 Inhibits Dopamine-Induced Increase in cAMP Accumulation

The increase in cAMP observed in the presence of dopamine (10 nM, 5 min) was significantly inhibited by pretreatment of the proximal tubules with bafilomycin A1 (Table 1), suggesting the contribution of newly recruited receptors in the stimulation of adenylyl cyclase.


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Table 1. Effects of bafilomycin A1 on dopamine-induced accumulation of cAMP in proximal tubules

 
Dopamine-Induced Inhibition of Na-K-ATPase Activity Is Attenuated by Bafilomycin A1

Pretreatment of the proximal tubules with bafilomycin A1 partially blocked inhibition of Na-K-ATPase by dopamine at all concentrations (Table 2). Basal Na-K-ATPase activity in proximal tubules in the absence and presence of bafilomycin A1 was not significantly different (209.67 ± 17.00 and 199.67 ± 9.67 nmol Pi·mg protein–1·min–1, respectively). These results suggest that newly recruited D1A receptors are involved in the inhibition of Na-K-ATPase produced by dopamine.


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Table 2. Effects of bafilomycin A1 on dopamine-induced inhibition of Na-K-ATPase activity

 
Dopamine Recruits D1A Receptors to Caveolar Plasma Membranes

Caveolar plasma membranes, as determined by expression of caveolin-2 protein, were separated in fraction 7 and noncaveolar plasma membranes were separated in the lower band of 45% sucrose (fractions 1–4). Dopamine D1A receptor protein density was higher in noncaveolar plasma membranes compared with caveolar plasma membranes (Fig. 8, A and B). However, dopamine treatment of proximal tubules (10 nM) caused a two- to threefold increase in the density of D1A receptor protein (per microgram protein) in caveolar plasma membranes without causing a significant change in the density of D1A receptor protein on noncaveolar plasma membranes (Fig. 8, A and B). Moreover, D1A receptor density (per microgram protein) was reduced by ~70% in the cytoplasmic fraction with dopamine treatment (fraction 12, Fig. 8, A and B).



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Fig. 8. Recruitment of D1A receptor by dopamine to caveolin-rich plasma membranes. Proximal tubules were treated with either vehicle (V) or 10 nM dopamine (D) at 37°C for 1 min and fractionated by sucrose gradient as described in METHODS. Caveolin-2 and D1A receptor expression was determined by immunoblotting in fractions (indicated above blots). A: representative immunoblot of D1A receptors and caveolin-2 in various fractions. B: densitometric analysis (means ± SE) of D1A receptor protein bands, normalized to amount of protein loaded (n = 3–4). *D vs. V by unpaired Student's t-test (P < 0.05).

 
Expression of Na-K-ATPase and Na/H Exchanger in Caveolin-Rich Vs. Noncaveolar Plasma Membranes

The {alpha}1-subunit of Na-K-ATPase was more abundant in caveolin-rich plasma membranes than in noncaveolar plasma membranes (Fig. 9). On the other hand, NHE3 was excluded from caveolin-2-rich membranes (Fig. 9).



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Fig. 9. Distribution of Na-K-ATPase and Na/H exchanger in caveolar and noncaveolar plasma membranes. Proximal tubules were prepared from kidneys of Sprague-Dawley rats and were fractionated by sucrose gradient as described in METHODS. Expression of Na-K-ATPase {alpha}1-subunit and Na/H exchanger type 3 in various fractions was determined by immunoblotting using antibodies specific for these proteins. The blots are representative of 2 experiments with similar results.

 
Expression of Caveolin-2 in Basolateral Vs. Brush-Border Membranes

Because the Na-K-ATPase {alpha}1-subunit was more abundant in the caveolin-2-rich plasma membranes and NHE3 was not detected in this fraction, we wanted to examine expression of caveolin-2 in basolateral membranes (that are rich in Na-K-ATPase) and brush-border membranes (that are rich in the Na/H exchanger). Interestingly, caveolin-2 protein was detected in basolateral membranes, but not in brush-border membranes (Fig. 10). These results collectively suggest that dopamine recruits D1A receptors specifically to caveolin-rich plasma membranes that are also rich in Na-K-ATPase.



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Fig. 10. Caveolin-2 expression in basolateral vs. brush-border membrane. Proximal tubules were isolated from kidneys of Sprague-Dawley rats and were used to prepare basolateral and brush-border membranes as described in METHODS. Expression of cavelin-2, Na-K-ATPase {alpha}1-subunit, and Na/H exchanger type 3 in these fractions was determined by immunoblotting using specific antibodies for these proteins. The blots are representative of 2–4 experiments with similar results.

 
Effects of Dopamine on Coimmunoprecipitation of Caveolin-2 With D1A Receptors

Because the low-buoyancy caveolin-rich fraction may also contain noncaveolar lipid rafts, we investigated the interaction between D1A receptors and caveolin-2 by coimmunoprecipitation experiments in proximal tubular cell lysates. When proximal tubules were treated with dopamine, the amount of caveolin-2 immunoprecipitated with D1A receptors was increased significantly compared with that in vehicle-treated proximal tubules (Fig. 11). These results suggest dopamine increases interaction of D1A receptors with caveolin-2 and further confirms our results of the sucrose density gradient experiment.



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Fig. 11. Effects of dopamine on coimmunoprecipitation of caveolin-2 with D1A receptors in proximal tubules. Proximal tubules were prepared from kidneys of Sprague-Dawley rats and were treated with either vehicle (V) or 10 nM dopamine (D) at 37°C for 1 min. Proximal tubular cell lysates were used for immunoprecipitation of D1A receptors. Immunoprecipitated samples were then used for immunoblotting of caveolin-2 and D1A receptors as described in METHODS. Band density of caveolin-2{beta} proteins was normalized to immunoprecipitated D1A receptor band density. Values are means ± SE (n = 3). *D vs. V by unpaired Student's t-test (P < 0.05).

 

    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Our results show that dopamine, by activating D1-like receptors, recruits D1A receptors to the plasma membrane in proximal tubules of Sprague-Dawley rats. Moreover, D1A receptor recruitment by dopamine occurs through an increase in cAMP and requires the presence of intact endocytic vesicles. Newly recruited D1A receptors are functional because they couple to G proteins, increase cAMP, and participate in the inhibition of Na-K-ATPase. Moreover, dopamine recruits D1A receptors selectively to Na-K-ATPase-rich caveolar plasma membranes in proximal tubules.

In our study, recruitment of D1A receptors by dopamine was completely blocked by SCH-23390, a D1-like receptor antagonist. The proximal tubules of rat kidneys express both D1A and D1B receptors, which are indistinguishable in their ability to bind the D1-like receptor antagonist (30). Therefore, it is not clear at this point whether the activation of D1A receptors, D1B receptors, or both leads to recruitment of D1A receptors. However, in opossum kidney cells, which lack D1B receptors, dopamine is able to recruit D1A receptors to the plasma membrane (10). Therefore, it is unlikely that D1B receptors play a significant role in dopamine-induced recruitment of D1A receptors.

Bafilomycin A1 is an inhibitor of vacuolar H+-ATPases, which are mainly located in intracellular organelles of secretory pathways and play a role in membrane trafficking and protein sorting (5, 15, 26, 27, 41, 45). In our experiments, bafilomycin A1 blocked dopamine-induced D1A receptor recruitment. This suggests that intracellular D1A receptors are stored in and/or transported to the plasma membrane and then into the vesicles that possess vacuolar H+-ATPases, and translocation of D1A receptors from intracellular compartments to the plasma membrane requires internal acidification of intracellular vacuolar compartments. Alternatively, Yang et al. (44) have reported that bafilomycin A1 reduces insulin-induced translocation of GLUT4 in cardiomyocytes by making the intracellular pH acidic. It is possible that intracellular pH affects transport of D1A receptors. However, dopamine, unlike insulin, has not been shown to cause alkalinization of intracellular pH. Therefore, bafilomycin A1 blocks dopamine-induced D1A receptor recruitment most likely by disrupting the vesicles involved with their storage and/or transport. In our study with proximal tubules, we could not use other inhibitors of intracellular transport, such as microtubule-depolymerizing agents (nocodazole) or inhibitors of actin polymerization (cytochalasin D), because they require at least 1-h treatment, at which point the viability of proximal tubules was <70%.

Forskolin, a direct activator of adenylyl cyclase, recruited D1A receptors to the plasma membrane. In the presence of forskolin, dopamine did not cause a further increase in D1A receptor recruitment (data not shown), suggesting a common pathway (adenylyl cyclase activation) for dopamine- and forskolin-induced recruitment of D1A receptors. This agrees with the previous reports that D1A receptor recruitment in LLC-PK1 cells involves the cAMP-PKA pathway (7). More specifically, a {beta}2-adrenoceptor agonist and atrial natriuretic peptide recruit D1A receptors, by stimulating adenylyl cyclase or guanylyl cyclase, respectively (7, 16). Thus an increase in cAMP or cGMP is the proposed mechanism for homologous as well as heterologous sensitization of D1A receptors.

An important finding of our study is that the newly recruited D1A receptors are functional. We show in this paper that newly recruited D1A receptors couple to G proteins. Although [35S]GTP{gamma}S binding experiments do not differentiate between Gs and Gq proteins that couple to D1A receptors, newly recruited D1A receptors increase cAMP and participate in dopamine-induced inhibition of Na-K-ATPase activity. Therefore, we can suggest that newly recruited D1A receptors couple to both Gs and Gq proteins, because Gs coupling of D1A receptors leads to activation of adenylyl cyclase and Gq coupling causes inhibition of Na-K-ATPase in proximal tubules. Inhibition of Na-K-ATPase activity by dopamine occurs via the phospholipase C-PKC pathway (20). While we were preparing this manuscript, a study was published showing that newly recruited D1A receptors couple to the PKC signaling pathway (24). Our results further extend this finding by demonstrating that newly recruited D1A receptors also participate in the inhibition of Na-K-ATPase activity.

When effects of bafilomycin A1 on dopamine-induced cAMP accumulation were evaluated, the increase in cAMP by dopamine (in 5 min) was completely inhibited by pretreatment with bafilomycin A1. If bafilomycin A1 blocks the recruitment of D1A receptors, it should block the cAMP accumulation contributed only by the newly recruited receptors. However, when we measured cAMP accumulation with 1 min of dopamine treatment, we did not find a significant increase compared with control (data not shown), although the recruitment of D1A receptors occurred at 1 min of dopamine treatment. It is possible that the cAMP accumulation required for D1A receptor recruitment is a very modest amount, which may not be detected by the radioimmunoassay procedure employed in our study.

Recent studies have shown localization of GPCR-signaling molecules in caveolae. Breton et al. (6) have reported the absence of morphologically distinguishable caveolae in rat proximal tubules. This can be explained by the expression of only caveolin-2, which exists mainly as a monomer or homodimer, in proximal tubules (11, 25). Caveolin-2 requires caveolin-1 to form high-molecular-mass oligomers, which are necessary to regulate the formation of uniform caveolae-sized vesicles (25, 40). Although caveolae are not detected, caveolin-2 is involved in D1A receptor signaling in proximal tubules. In proximal tubules, D1A receptors, along with Gs, Gq/11, PKA, and phosphatase 2A, were shown to be localized in caveolin-2-rich plasma membranes by cell fractionation, immunofluorescence, and immunoprecipitation (46). Our observation that dopamine recruited D1A receptors to caveolar, but not to noncaveolar, plasma membranes supports the notion that caveolae act as spatial compartments in which specific ligand-induced signaling takes place. Agonist activation has been shown to translocate bradykinin B1 receptors from noncaveolar plasma membranes to caveolar plasma membranes (38). However, in our study, the increase in D1A receptor density in caveolar plasma membranes is due to recruitment of the receptors from cytoplasmic compartments and not due to translocation of D1A receptors from noncaveolar plasma membranes because 1) there is no significant change in D1A receptor density in noncaveolar plasma membranes and 2) there is a decrease in D1A receptor density in the cytoplasmic compartment. Moreover, the increase in D1A receptor density is two- to threefold in caveolar plasma membranes as opposed to only 50% in total plasma membranes. Our finding that intracellular D1A receptors are recruited selectively to caveolar plasma membranes and interact with caveolin-2 describes a novel mechanism for employing selective signaling in one region of the plasma membranes. Yu et al. (46) have shown the functionality of D1A receptors in caveolar plasma membranes by their ability to increase cAMP in proximal tubules. Moreover, we show that Na-K-ATPase protein is predominantly present in caveolar plasma membranes, where recruitment takes place. Therefore, recruitment of D1A receptor in caveolar plasma membrane potentiates a dopamine-induced inhibition of Na-K-ATPase in proximal tubules.

Dopamine-induced recruitment of D1A receptors to the plasma membrane is a cAMP-dependent process, as the adenylyl cyclase inhibitor DDA blocks this recruitment. However, the mechanism by which the increased levels of cAMP lead to vesicular transport of intracellular D1A receptors to the plasma membrane is a matter of speculation. It has been speculated that the cAMP-dependent kinase PKA plays a role in the recruitment of D1A receptors to the plasma membrane (7). There are two potential mechanisms by which PKA activation leads to recruitment of D1A receptors to the plasma membrane. The first is that PKA-dependent phosphorylation of (a) protein(s) potentiates the interaction of D1A receptor-carrying cargo vesicles with the microtubulin network and subsequently leads to recruitment of D1A receptors. This argument can be supported by the observation that disrupting the microtubulin network by nocodazole prevents L-dopa-induced translocation of D1A receptors to the plasma membrane (24). Possible candidates for proteins phosphorylated by PKA include 1) accessory protein complexes like the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and 2) adaptor proteins, like AP-1. SNARE protein complexes are involved in vesicular fusion events (43) and may promote fusion of D1A receptor cargo vesicles with the plasma membrane. Similarly, AP-1 is involved in trafficking of many proteins such as Na-K-ATPase (10). PKA phosphorylation of either of these proteins may be necessary for trafficking of D1A receptors to the plasma membrane. The second possibility is that PKA phosphorylation of a D1A receptor-interacting protein (DRiP), which retains D1A receptors in intracellular compartments, is necessary for D1A receptor recruitment. At the least, overexpression of one such DRiP, DRiP-78, resulted in the retention of D1A receptors in the endoplasmic reticulum (4). Because D1A receptors in intracellular compartments are thought to be located in vesicles, it is unlikely that DRiP-78 that is in the endoplasmic reticulum is responsible for intracellular localization of D1A receptors in proximal tubules. However, it is possible that PKA-dependent phosphorylation of other DRiP(s) results in release and consequent translocation of D1A receptors to the plasma membrane. Both of these possibilities are interesting, and additional detailed studies are required to identify the mechanism for the D1A recruitment process.

In summary, we have shown that dopamine recruits D1A receptors to Na-K-ATPase-rich caveolar plasma membrane via the D1-like receptor-cAMP pathway in proximal tubules of Sprague-Dawley rats. Furthermore, the recruited D1A receptors are functional in terms of G protein coupling, cAMP accumulation, and Na-K-ATPase inhibition. Using proximal tubules isolated from rodent kidneys as a system for studying the recruitment of D1A receptors will enable us to investigate the dopamine-induced recruitment of D1A receptors in various models, such as obesity and aging. It is likely that a defect in D1A receptor recruitment may be one of the causative factors for the reported diminished natriuretic and diuretic response to dopamine in these conditions.


    GRANTS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This study was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-58743.


    ACKNOWLEDGMENTS
 
Present address of V. A. Narkar: Integrative Biology and Pharmacology, Univ. of Texas Medical School-Houston, 6431 Fannin St., Houston, TX 77030.


    FOOTNOTES
 

Address for reprint requests and other correspondence: M. F. Lokhandwala, Heart and Kidney Institute, College of Pharmacy, Univ. of Houston, Houston, TX 77204-5041 (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.


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