Dopamine recruits D 1A receptors to Na-K-ATPase-rich caveolar plasma membranes i - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-22 08:09 |只看该作者 |倒序浏览 |打印

作者：Meghna Trivedi, Vihang A. Narkar, Tahir Hussain, and Mustafa F. Lokhandwala作者单位：Heart and Kidney Institute, College of Pharmacy, University of Houston, Texas 77204-5041
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      【摘要】
   Activation of dopamine D 1A 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, D 1A receptors are mainly present in intracellular compartments under basal conditions. We observed, using [ 3 H]SCH-23390 binding and immunoblotting, that dopamine recruits D 1A receptors to the plasma membrane in rat renal proximal tubules. Furthermore, radioligand binding and/or immunoblotting experiments using pharmacological modulators showed that dopamine-induced D 1A receptor recruitment requires activation of cell surface D 1 -like receptors, activation of adenylyl cyclase, and intact endocytic vesicles with internal acidic pH. A key finding of this study was that these recruited D 1A receptors were functional because they potentiated dopamine-induced [ 35 S]GTP S binding, cAMP accumulation, and Na-K-ATPase inhibition. Interestingly, dopamine increased immunoreactivity of D 1A receptors specifically in caveolin-rich plasma membranes isolated by a sucrose density gradient. In support of this observation, coimmunoprecipitation studies showed that D 1A receptors interacted with caveolin-2 in an agonist-dependent fashion. The caveolin-rich plasma membranes had a high content of the 1 -subunit of Na-K-ATPase, which is a downstream target of D 1A receptor signaling in proximal tubules. These results show that dopamine, via the D 1 -like receptor-adenylyl cyclase pathway, recruits D 1A 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 D 1A 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. & P! M; c& V4 P* ^
      【关键词】 G proteincoupled 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 D 1A receptors in the proximal tubules ( 1, 23 ). In the proximal tubules, dopamine D 1A 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.
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A prolonged response to endogenously produced or exogenously administered dopamine depends on the abundance of the D 1A receptors on the cell surface. D 1A 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 D 1A receptors are primarily located in intracellular compartments under basal conditions in the kidney and heart ( 8, 31, 35 ). It has been shown that intracellular D 1A receptors, on activation by an agonist, translocate to the plasma membrane in LLC-PK 1 cells, a proximal tubule-like cell line ( 8 ). However, the ability of dopamine to recruit D 1A 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 D 1A receptor recruitment helps sustain the response to dopamine in proximal tubules.
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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, D 1A receptors are immunoprecipitated with caveolin-2, which is the only isoform of caveolin present in proximal tubules of rats ( 46 ). Moreover, effector molecules in D 1 -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 D 1A receptors by dopamine to caveolar plasma membranes rich in effector molecules may accelerate transduction of D 1A receptor signaling in proximal tubules.

In the first part of this study, we have characterized the effect of dopamine on D 1A receptor recruitment to the plasma membrane in proximal tubules of Sprague-Dawley rats. We also have studied the signaling pathway by which dopamine recruits D 1A receptors to the plasma membrane. In the second part, the functionality of the recruited D 1A receptors has been tested by investigating the effect of dopamine-induced D 1A receptor recruitment on the receptor-G protein-second messenger-effector axis. In the third part, we have evaluated the recruitment of D 1A 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 D 1A receptors by dopamine.3 R+ a. ~" A" h1 X/ B/ a9 i
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METHODS' b/ \- h5 k; S/ [! z6 H

Materials/ b1 l$ k, v& N+ I# n$ s
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The following chemicals and materials were purchased from the source indicated: [ 3 H]SCH-23390, [ 35 S]GTP S (DuPont New England Nuclear, Boston, MA); cAMP radioimmunoassay kit (Amersham Bioscience, Piscataway, NJ); rabbit anti-rat D 1A and D 1B polyclonal antibodies, horseradish peroxidase-conjugated anti-rabbit antibodies, and chemiluminescent substrate (Alpha Diagnostics, San Antonio, TX); mouse anti-rabbit Na-K-ATPase 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.
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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.
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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 NaHCO 3, 4 KCl, 1.25 CaCl 2, 1.2 MgCl 2, 1.0 KH 2 PO 4, 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 MgCl 2. In an aliquot of the proximal tubular suspension, Trypan blue exclusion tests were performed 95% proximal tubular cell viability ( 14 )., a' e& l5 |- g8 `1 Q3 r
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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 A 1 (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.$ `1 u& s- z7 {) ?5 m

Preparation of Plasma Membranes
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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 MgCl 2, 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 MgCl 2, 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 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 [ 3 H]SCH-23390 binding, immunoblotting, and [ 35 S]GTP S binding experiments.' L0 h/ Y8 }3 w5 D' z; t

Radioligand ([ 3 H]SCH-23390) Binding

To determine whether dopamine recruits D 1 -like receptors to the cell surface, [ 3 H]SCH-23390 binding was performed in plasma membranes isolated from proximal tubules. The plasma membrane samples (50 µg) were incubated with 10 nM [ 3 H]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 [ 3 H]SCH-23390 because, in saturation binding experiments, 10 nM was the lowest concentration of [ 3 H]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 [ 3 H]SCH-23390 for D 1 -like receptors, as reflected by K d, was not altered with dopamine treatment of proximal tubules (5.5 ± 0.6 vs. 5.9 ± 0.2 nM).
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Immunoblotting of D 1A Receptor Protein

Because SCH-23390 is a nonspecific D 1 -like receptor (D 1A /D 1 and D 1B /D 5 ) ligand, the recruitment of D 1A vs. D 1B was examined by using specific antibodies in immunoblotting. In these experiments, membrane samples (3 µg protein for D 1A and 10 µg protein for D 1B ) 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-D 1A receptor or anti-D 1B 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).
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Measurement of [ 35 S]GTP S Binding
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The ability of newly recruited D 1A receptors to couple to G proteins was studied by [ 35 S]GTP S binding in plasma membranes as described previously ( 19 ). Briefly, [ 35 S]GTP S binding was stimulated by various concentrations (1 nM-0.1 µM, 15 min, 37°C) of fenoldopam, a preferential D 1 -like receptor agonist. The assay was carried out in the presence of 0.6 nM ( 100,000 cpm) of [ 35 S]GTP S (specific activity 1,250 Ci/mmol), 5 µg of membrane protein, and fenoldopam (1 nM-0.1 µM). Nonspecific [ 35 S]GTP S binding was determined in the presence of 100 µM unlabeled GTP S. Binding in the control treatment group was normalized to 100% because there was no significant difference in basal GTP S binding by treatment with either bafilomycin A 1 or dopamine.

cAMP Accumulation in Proximal Tubules% J* K6 k! h  l, M6 V+ {
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The contribution of newly recruited D 1A receptors to the dopamine-induced increase in cAMP accumulation was determined using bafilomycin A 1 as an inhibitor of D 1A 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.+ }1 U9 b. }; p# k; h. q! [6 P: S

Na-K-ATPase Activity Assay in Proximal Tubules. p  B  |7 N2 d# b/ I4 j4 c- |

Na-K-ATPase activity was measured as previously described ( 17 ) as the function of liberated inorganic phosphate and represented as percent inhibition (from basal).4 |: T  V" V; K+ ~. C  H9 G

Preparation of Caveolar Plasma Membranes' `6 g8 \/ K+ E- V! k

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/Na 2 CO 3 ) 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.5 P! p" J: v7 X# i" Q

Immunoblotting of Na-K-ATPase, Na/H Exchanger, and Caveolin-2 Proteins+ Z  j8 |) [0 w% h: P
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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 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.
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Coimmunoprecipitation of Caveolin-2 With D 1A Receptors

Immunoprecipitation of D 1A receptors. A previously described method ( 21 ) was used for immunoprecipitation of D 1A 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 D 1A receptor antibody overnight to allow the formation of a D 1A receptor-antibody complex. This complex was incubated for 2 h with protein A/G covalently bound to agarose beads (protein A/G-agarose). The D 1A 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 D 1A receptor-antibody-protein A/G complex was dissociated with 2 x Laemmli buffer containing 125 mM Tris·HCl, 4% SDS, 5% -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.9 e9 z9 h! G, n

Detection of caveolin-2 proteins interacting with the D 1A 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 D 1A 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 D 1A receptors as described above. The band density of caveolin-2 proteins was normalized to the band density of D 1A receptors.3 F; U- f  c+ e* c4 g% G. d' n

Data Analysis
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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 6 o5 G" Q! Z* Y3 D$ d; m

RESULTS

Dopamine Recruits D 1 -Like Receptors to the Plasma Membrane 50% increase in specific [ 3 H]SCH-23390 binding in plasma membranes prepared from proximal tubules treated with dopamine (1 and 10 nM) compared with vehicle ( Fig. 1 A ). Moreover, this increase in [ 3 H]SCH-23390 binding by dopamine was observed for up to 15 min of dopamine treatment ( Fig. 1 B ).' C0 \. [4 r. J* t* m1 {6 j

Fig. 1. Dopamine-induced recruitment of D 1 -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 ). [ 3 H]SCH-23390 binding experiments were performed in plasma membranes isolated from proximal tubules as described in METHODS. Basal [ 3 H]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 [ 3 H]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
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Dopamine Recruits D 1A, But Not D 1B, Receptors to the Plasma Membrane4 k5 A6 x+ y# x* J$ |

Dopamine increased abundance of D 1A receptors, but not D 1B receptors, on plasma membranes in proximal tubules. Dopamine (10 nM) caused an 50% increase in density of D 1A receptor protein in plasma membranes ( Fig. 2 A ), whereas no significant change in the density of D 1B receptor protein was seen in plasma membranes after dopamine treatment of proximal tubules ( Fig. 2 B ).
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Fig. 2. Dopamine-induced recruitment of D 1A, and not D 1B, receptors. Proximal tubules were treated with either vehicle (V) or 10 nM dopamine (D) at 37°C for 1 min. Immunoblotting experiments using D 1A ( A and C ) and D 1B ( 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 7 X; j& z& p7 h6 g0 y

In addition, dopamine did not affect the immunoreactivity of D 1A receptor protein in proximal tubular cell lysates ( Fig. 2 C ), confirming that the effect of dopamine is due to recruitment of intracellular D 1A receptors to the plasma membrane and not to an increase in affinity of the antibodies for receptors.
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Dopamine Recruits D 1A Receptors Via Activation of D 1 -Like Receptors

SCH-23390 completely blocked the dopamine-induced increase in density of D 1A receptor protein on plasma membranes, while having no significant effect on D 1A receptor density by itself ( Fig. 3 ).

Fig. 3. Inhibition of dopamine-induced D 1A 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 D 1A 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 . A% E$ f+ V9 {9 [4 i* P( d

Bafilomycin A 1 Inhibits Recruitment of D 1A Receptors by Dopamine

Bafilomycin A 1 is an inhibitor of H -ATPases, which are located on endocytic vesicles with an internal acidic environment. Bafilomycin A 1 significantly blocked the dopamine-induced increase in the abundance of D 1A receptors on plasma membranes, as shown by [ 3 H]SCH-23390 binding ( Fig. 4 A ) and immunoblotting ( Fig. 4 B ). There was no significant change in either [ 3 H]SCH-23390 binding or D 1A receptor protein density in plasma membranes by bafilomycin A 1 alone. Moreover, increasing concentrations of bafilomycin A 1 (1-200 nM) did not displace [ 3 H]SCH-23390 binding (data not shown), establishing that bafilomycin A 1 does not act as an antagonist for D 1 -like receptors.
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Fig. 4. Inhibition of dopamine-induced D 1A receptor recruitment by bafilomycin A 1. Proximal tubules were pretreated with either DMSO or 20 nM bafilomycin A 1 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 A 1  Na metabisulfite; D, DMSO dopamine; D B, bafilomycin A 1  dopamine. [ 3 H]SCH-23390 binding experiments ( A; n = 6) and immunoblotting ( B; n = 5) were performed in plasma membranes isolated from proximal tubules. Basal [ 3 H]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 $ l! m* w- l( _
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Forskolin Recruits D 1A Receptors to the Plasma Membrane 100% increase in [ 3 H]SCH-23390 binding ( Fig. 5 A ) and a similar increase in the density of D 1A receptor protein ( Fig. 5 B ) in plasma membranes. Moreover, treatment of proximal tubules with dopamine in the presence of forskolin did not result in a further increase in [ 3 H]SCH-23390 binding in plasma membranes (data not shown), suggesting a common pathway (adenylyl cyclase activation) for dopamine- and forskolin-induced recruitment of D 1A receptors.- G+ T9 m; H& W/ ?- w
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Fig. 5. Forskolin-induced D 1A receptor recruitment. Proximal tubules were treated with either vehicle (V) or 1 µM forskolin (F) at 37°C for 15 min. [ 3 H]SCH-23390 binding experiments ( A; n = 7) and immunoblotting ( B; n = 4) were performed in plasma membranes as described in METHODS. Basal [ 3 H]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
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DDA Inhibits Recruitment of D 1A Receptors by Dopamine! A$ P7 `. w" U$ K

DDA, an inhibitor of adenylyl cyclase, significantly blocked the dopamine-induced increase in the immunoreactivity of D 1A receptor protein on plasma membranes ( Fig. 6 ). There was no significant change in D 1A receptor protein density in plasma membranes using DDA alone ( 3 ).

Fig. 6. Inhibition of dopamine-induced D 1A 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 D 1A 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
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Newly Recruited D 1A Receptors Couple to G Proteins

The increase in [ 35 S]GTP 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 [ 35 S]GTP 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 A 1 completely inhibited the dopamine-induced increase in [ 35 S]GTP S binding with fenoldopam. A concentration-dependent stimulation of [ 35 S]GTP S binding by fenoldopam was observed in all the groups. Basal GTP S binding was not affected by either bafilomycin A 1 or dopamine ( Fig. 7 ).# }+ A$ b& [- z' z
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Fig. 7. G protein coupling of newly recruited D 1A receptors. Proximal tubules were pretreated with either DMSO or 20 nM bafilomycin A 1 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. [ 35 S]GTP S binding experiments were performed in plasma membranes prepared from proximal tubules ( n = 7). GTP S binding was stimulated by various concentrations (1 nM-0.1 µM) of fenoldopam. Basal GTP 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 A 1 -treated tubules, 1.21 ± 0.13 pmol/mg protein in dopamine-treated tubules, and 1.16 ± 0.13 pmol/mg protein in dopamine Baf A 1 -treated tubules. Values are means ± SE. *Significant difference between vehicle- and dopamine-treated groups; # significant difference between dopamine- and dopamine bafilomycin A 1 -treated groups by 1-way ANOVA, Newman-Keuls multiple comparison test (both P
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Bafilomycin A 1 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 A 1 ( Table 1 ), suggesting the contribution of newly recruited receptors in the stimulation of adenylyl cyclase.5 M, h( S* g  z1 c2 J

Table 1. Effects of bafilomycin A 1 on dopamine-induced accumulation of cAMP in proximal tubules) S5 U+ u! Q3 Y. A" b0 B
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Dopamine-Induced Inhibition of Na-K-ATPase Activity Is Attenuated by Bafilomycin A 1) ]$ k1 @7 ~/ V! ]5 y8 _

Pretreatment of the proximal tubules with bafilomycin A 1 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 A 1 was not significantly different (209.67 ± 17.00 and 199.67 ± 9.67 nmol P i ·mg protein -1 ·min -1, respectively). These results suggest that newly recruited D 1A receptors are involved in the inhibition of Na-K-ATPase produced by dopamine.

Table 2. Effects of bafilomycin A 1 on dopamine-induced inhibition of Na-K-ATPase activity
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Dopamine Recruits D 1A Receptors to Caveolar Plasma Membranes
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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 D 1A 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 D 1A receptor protein (per microgram protein) in caveolar plasma membranes without causing a significant change in the density of D 1A receptor protein on noncaveolar plasma membranes ( Fig. 8, A and B ). Moreover, D 1A receptor density (per microgram protein) was reduced by 70% in the cytoplasmic fraction with dopamine treatment ( fraction 12, Fig. 8, A and B ).9 g/ g& c( p4 k( q3 c/ \3 e

Fig. 8. Recruitment of D 1A 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 D 1A receptor expression was determined by immunoblotting in fractions (indicated above blots). A : representative immunoblot of D 1A receptors and caveolin-2 in various fractions. B : densitometric analysis (means ± SE) of D 1A receptor protein bands, normalized to amount of protein loaded ( n = 3-4). *D vs. V by unpaired Student's t -test ( P & L6 @/ O$ L/ y
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Expression of Na-K-ATPase and Na/H Exchanger in Caveolin-Rich Vs. Noncaveolar Plasma Membranes" q8 }; L- ^2 M) C/ e; L+ `

The 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 ).* p. E. `% F9 W7 A7 B, f- m
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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 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
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Because the Na-K-ATPase 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 D 1A 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 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.
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Effects of Dopamine on Coimmunoprecipitation of Caveolin-2 With D 1A Receptors% Z) V4 o9 X: T4 F
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Because the low-buoyancy caveolin-rich fraction may also contain noncaveolar lipid rafts, we investigated the interaction between D 1A 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 D 1A receptors was increased significantly compared with that in vehicle-treated proximal tubules ( Fig. 11 ). These results suggest dopamine increases interaction of D 1A receptors with caveolin-2 and further confirms our results of the sucrose density gradient experiment.

Fig. 11. Effects of dopamine on coimmunoprecipitation of caveolin-2 with D 1A 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 D 1A receptors. Immunoprecipitated samples were then used for immunoblotting of caveolin-2 and D 1A receptors as described in METHODS. Band density of caveolin-2 proteins was normalized to immunoprecipitated D 1A receptor band density. Values are means ± SE ( n = 3). *D vs. V by unpaired Student's t -test ( P # d+ w& S8 x9 k6 a' H
* P: f- G( [# o4 F  X2 X8 G
DISCUSSION" e% |9 {4 F; A% i3 H
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Our results show that dopamine, by activating D 1 -like receptors, recruits D 1A receptors to the plasma membrane in proximal tubules of Sprague-Dawley rats. Moreover, D 1A receptor recruitment by dopamine occurs through an increase in cAMP and requires the presence of intact endocytic vesicles. Newly recruited D 1A receptors are functional because they couple to G proteins, increase cAMP, and participate in the inhibition of Na-K-ATPase. Moreover, dopamine recruits D 1A receptors selectively to Na-K-ATPase-rich caveolar plasma membranes in proximal tubules.

In our study, recruitment of D 1A receptors by dopamine was completely blocked by SCH-23390, a D 1 -like receptor antagonist. The proximal tubules of rat kidneys express both D 1A and D 1B receptors, which are indistinguishable in their ability to bind the D 1 -like receptor antagonist ( 30 ). Therefore, it is not clear at this point whether the activation of D 1A receptors, D 1B receptors, or both leads to recruitment of D 1A receptors. However, in opossum kidney cells, which lack D 1B receptors, dopamine is able to recruit D 1A receptors to the plasma membrane ( 10 ). Therefore, it is unlikely that D 1B receptors play a significant role in dopamine-induced recruitment of D 1A receptors.7 U( s+ `6 r0 Z) k" b' M0 X

Bafilomycin A 1 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 A 1 blocked dopamine-induced D 1A receptor recruitment. This suggests that intracellular D 1A receptors are stored in and/or transported to the plasma membrane and then into the vesicles that possess vacuolar H -ATPases, and translocation of D 1A receptors from intracellular compartments to the plasma membrane requires internal acidification of intracellular vacuolar compartments. Alternatively, Yang et al. ( 44 ) have reported that bafilomycin A 1 reduces insulin-induced translocation of GLUT4 in cardiomyocytes by making the intracellular pH acidic. It is possible that intracellular pH affects transport of D 1A receptors. However, dopamine, unlike insulin, has not been shown to cause alkalinization of intracellular pH. Therefore, bafilomycin A 1 blocks dopamine-induced D 1A 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 0 V2 Y# ~% S. q7 s
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Forskolin, a direct activator of adenylyl cyclase, recruited D 1A receptors to the plasma membrane. In the presence of forskolin, dopamine did not cause a further increase in D 1A receptor recruitment (data not shown), suggesting a common pathway (adenylyl cyclase activation) for dopamine- and forskolin-induced recruitment of D 1A receptors. This agrees with the previous reports that D 1A receptor recruitment in LLC-PK 1 cells involves the cAMP-PKA pathway ( 7 ). More specifically, a 2 -adrenoceptor agonist and atrial natriuretic peptide recruit D 1A 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 D 1A receptors.! Q4 G! n$ m5 E' k6 U

An important finding of our study is that the newly recruited D 1A receptors are functional. We show in this paper that newly recruited D 1A receptors couple to G proteins. Although [ 35 S]GTP S binding experiments do not differentiate between Gs and Gq proteins that couple to D 1A receptors, newly recruited D 1A receptors increase cAMP and participate in dopamine-induced inhibition of Na-K-ATPase activity. Therefore, we can suggest that newly recruited D 1A receptors couple to both Gs and Gq proteins, because Gs coupling of D 1A 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 D 1A receptors couple to the PKC signaling pathway ( 24 ). Our results further extend this finding by demonstrating that newly recruited D 1A receptors also participate in the inhibition of Na-K-ATPase activity.

When effects of bafilomycin A 1 on dopamine-induced cAMP accumulation were evaluated, the increase in cAMP by dopamine (in 5 min) was completely inhibited by pretreatment with bafilomycin A 1. If bafilomycin A 1 blocks the recruitment of D 1A 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 D 1A receptors occurred at 1 min of dopamine treatment. It is possible that the cAMP accumulation required for D 1A receptor recruitment is a very modest amount, which may not be detected by the radioimmunoassay procedure employed in our study., W2 X7 O  }8 m1 K6 e3 e
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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 D 1A receptor signaling in proximal tubules. In proximal tubules, D 1A 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 D 1A 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 B 1 receptors from noncaveolar plasma membranes to caveolar plasma membranes ( 38 ). However, in our study, the increase in D 1A receptor density in caveolar plasma membranes is due to recruitment of the receptors from cytoplasmic compartments and not due to translocation of D 1A receptors from noncaveolar plasma membranes because 1 ) there is no significant change in D 1A receptor density in noncaveolar plasma membranes and 2 ) there is a decrease in D 1A receptor density in the cytoplasmic compartment. Moreover, the increase in D 1A receptor density is two- to threefold in caveolar plasma membranes as opposed to only 50% in total plasma membranes. Our finding that intracellular D 1A 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 D 1A 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 D 1A receptor in caveolar plasma membrane potentiates a dopamine-induced inhibition of Na-K-ATPase in proximal tubules.

Dopamine-induced recruitment of D 1A 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 D 1A 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 D 1A receptors to the plasma membrane ( 7 ). There are two potential mechanisms by which PKA activation leads to recruitment of D 1A receptors to the plasma membrane. The first is that PKA-dependent phosphorylation of (a) protein(s) potentiates the interaction of D 1A receptor-carrying cargo vesicles with the microtubulin network and subsequently leads to recruitment of D 1A receptors. This argument can be supported by the observation that disrupting the microtubulin network by nocodazole prevents L -dopa-induced translocation of D 1A 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 D 1A 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 D 1A receptors to the plasma membrane. The second possibility is that PKA phosphorylation of a D 1A receptor-interacting protein (DRiP), which retains D 1A receptors in intracellular compartments, is necessary for D 1A receptor recruitment. At the least, overexpression of one such DRiP, DRiP-78, resulted in the retention of D 1A receptors in the endoplasmic reticulum ( 4 ). Because D 1A 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 D 1A receptors in proximal tubules. However, it is possible that PKA-dependent phosphorylation of other DRiP(s) results in release and consequent translocation of D 1A receptors to the plasma membrane. Both of these possibilities are interesting, and additional detailed studies are required to identify the mechanism for the D 1A recruitment process.

In summary, we have shown that dopamine recruits D 1A receptors to Na-K-ATPase-rich caveolar plasma membrane via the D 1 -like receptor-cAMP pathway in proximal tubules of Sprague-Dawley rats. Furthermore, the recruited D 1A 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 D 1A receptors will enable us to investigate the dopamine-induced recruitment of D 1A receptors in various models, such as obesity and aging. It is likely that a defect in D 1A receptor recruitment may be one of the causative factors for the reported diminished natriuretic and diuretic response to dopamine in these conditions.* h9 \& r4 q: l" a
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GRANTS: N# g) _9 ?/ _" [: s- v4 [7 w/ S9 V

This study was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-58743.: T4 u- b) a) C' c# C
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ACKNOWLEDGMENTS

Present address of V. A. Narkar: Integrative Biology and Pharmacology, Univ. of Texas Medical School-Houston, 6431 Fannin St., Houston, TX 77030.
      【参考文献】
Aperia AC. Intrarenal dopamine: a key signal in the interactive regulation of sodium metabolism. Annu Rev Physiol 62: 621-647, 2000.' v  A- y4 Z1 Y
% u$ P1 ^' u. t/ v# m
. n$ U2 g1 y3 [( _

Beach RE and DuBose TD. Adrenergic regulation of Na  , K -ATPase activity in proximal tubules of spontaneously hypertensive rats. Kidney Int 38: 402-408, 1990.# J0 ]! z! i& {# c- |! i+ U$ N7 S
$ H6 d+ O4 E% _, z6 z

) y& p% y" F  G4 J; T) W
Bergstrand H, Kristoffersson J, Lundquist B, and Schurmann A. Effects of antiallergic agents, compound 48/80, and some reference inhibitors on the activity of partially purified human lung tissue adenosine cyclic 3',5'-monophosphate and guanosine cyclic 3',5'-monophosphate phosphodiesterases. Mol Pharmacol 13: 38-43, 1977.' }8 j+ J( K- _4 q# m: x
3 {" ^7 T' _' E% }1 C5 N

+ Y% P# J0 d6 O8 t2 s
Bermak JC, Li M, Bullock C, and Zhou QY. Regulation of transport of the dopamine D 1 receptor by a new membrane-associated ER protein. Nat Cell Biol 3: 492-498, 2001.
$ P* U9 K, ?1 C2 F
" B4 S! M, c. @4 u( s* f4 D6 Z% Z
' e) Z) C, u2 z9 D: u) x" y/ u
Bowman EJ, Siebers A, and Altendorf K. Bafilomycins: a class of inhibitors of membrane ATPases from microorganisms, animal cells, and plant cells. Proc Natl Acad Sci USA 85: 7972-7976, 1988.

$ Q9 O# B. J. Q4 A% p

Breton S, Lisanti MP, Tyszkowski R, McLaughlin M, and Brown D. Basolateral distribution of caveolin-1 in the kidney. Absence from H -ATPase-coated endocytic vesicles in intercalated cells. J Histochem Cytochem 46: 205-214, 1998.. b7 U% l- i2 j

0 w! g$ W! A* y8 ?1 P& H) {2 ]. D
# a+ l  V1 A: g% I( N  S
Brismar H, Agren M, and Holtback U. -Adrenoceptor agonist sensitizes the dopamine-1 receptor in renal tubular cells. Acta Physiol Scand 175: 333-340, 2002.
# ?+ r* l) ], L2 u5 k4 _4 W0 m
" A7 |8 E* g, v
. m: w/ R& V$ z9 ?) x
Brismar H, Asghar M, Carey RM, Greengard P, and Aperia A. Dopamine-induced recruitment of dopamine D 1 receptors to the plasma membrane. Proc Natl Acad Sci USA 95: 5573-5578, 1998.8 M) p) n: e" w, o2 ?4 p2 T% \
2 V! D7 E* l6 ?2 B8 Q

8 N; U6 Q  T+ {( k/ [2 A5 T' a3 t
Chen CJ, Beach RE, and Lokhandwala MF. Dopamine fails to inhibit renal tubular sodium pump in hypertensive rats. Hypertension 21: 364-372, 1993.# ]. A3 R$ o; v8 O) n4 t

  @: n% M- N; {8 o
0 V4 v1 F+ |) h  N/ ?* o
Efendiev R, Budu CE, Cinelli AR, Bertorello AM, and Pedemonte CH. Intracellular Na regulates dopamine- and angiotensin II-receptors availability at the plasma membrane, and their cellular responses in renal epithelia. J Biol Chem 278: 28719-28726, 2003.
$ ~3 Y/ w& @/ J: I
  `7 r& ]! l- \) B: h
, F! M1 X# N/ w) s. ~3 ~
Engelman JA, Wycoff CC, Yasuhara S, Song KS, Okamoto T, and Lisanti MP. Recombinant expression of caveolin-1 in oncogenically transformed cells abrogates anchorage-independent growth. J Biol Chem 272: 16374-16381, 1997.8 Z! N+ g; R2 ?( K% d6 Q" d
* Z: e& I% J' u0 ~$ i" K7 x0 N
; d$ h% C& e& }; y3 j/ Z
$ G+ {: G7 l6 E6 O) h+ A/ r
Felder CC, Blecher M, and José PA. Dopamine-1-mediated stimulation of phospholipase C activity in rat renal cortical membranes. J Biol Chem 264: 8739-8745, 1990.1 F6 F4 K) n% M

& H' w$ C0 I; l1 T+ e' W% N

Felder CC, Campbell T, Albrecht FE, and José PA. Dopamine inhibits Na/H exchanger activity in renal BBMV by stimulation of adenylate cyclase. Am J Physiol Renal Fluid Electrolyte Physiol 259: F297-F303, 1990.2 H# o! Q. Z! T/ K8 o8 E
% G& \  ]# g( v

* ]) K* P( V4 a" B
Gesek FA, Wolff DW, and Strandhoy JW. Improved separation method for rat proximal and distal renal tubules. Am J Physiol Renal Fluid Electrolyte Physiol 253: F358-F365, 1987." O3 A% z2 r( K

- ^5 f$ E6 w) G* Z/ N

Henomatsu N, Yoshimori T, Yamamoto A, Moriyama Y, and Tashiro Y. Inhibition of intracellular transport of newly synthesized prolactin by bafilomycin A 1 in a pituitary tumor cell line, GH3 cells. Eur J Cell Biol 62: 127-139, 1993.; J' }8 w5 F5 e1 R' K
, p& [3 o8 J6 Q# t5 H( o
) `. u: F4 E1 f9 U- E

Holtback U, Brismar H, DiBona GF, Fu M, Greengard P, and Aperia A. Receptor recruitment: a mechanism for interactions between G protein-coupled receptors. Proc Natl Acad Sci USA 96: 7271-7275, 1999.

Hussain T, Abdul-Wahab R, and Lokhandwala MF. Bromocriptine stimulated Na,K-ATPase in renal proximal tubules via cAMP pathway. Eur J Pharmacol 321: 259-263, 1997.! ^2 u8 U+ `; l1 E) |
3 T: b2 A1 T3 e! U2 A$ F6 e2 n
# y9 a/ a6 j" p
4 B$ I% T6 e8 ~8 V- V* D
Hussain T, Becker M, Beheray S, and Lokhandwala MF. Dopamine fails to inhibit Na,H-exchanger in proximal tubules of obese Zucker rats. Clin Exp Hypertens 23: 591-601, 2001.& j4 w, V3 r. J2 w1 r6 M

/ L9 R5 \0 _3 ?5 j5 u

Hussain T and Lokhandwala MF. Renal dopamine DA 1 receptor coupling with G S and G q/11 proteins in spontaneously hypertensive rats. Am J Physiol Renal Physiol 272: F339-F346, 1997.

Hussain T and Lokhandwala MF. Renal dopamine receptors and hypertension. Exp Biol Med 228: 134-142, 2003." E4 I( k% h+ U- v
3 P" M; E! s" `& q* y& r/ J  b

3 d5 {7 v- h2 F
Jin LQ, Wang HY, and Friedman E. Stimulated D 1 dopamine receptors couple to multiple G proteins in different brain regions. J Neurochem 78: 981-990, 2001." V' p; K. J' w5 a0 ?
' p& G. ^( W5 d$ g

Jose PA, Eisner GM, and Felder RA. Renal dopamine receptors in health and hypertension. Pharmacol Ther 80: 149-182, 1998.
( O4 c. k( Y  C
8 R# T, u" r; K' G6 d
  \+ s& F" B9 x# }# Q. k+ A4 i
Jose PA, Raymond JR, Bates MD, Aperia A, Felder RA, and Carey RM. The renal dopamine receptors. J Am Soc Nephrol 2: 1265-1278, 1992.  c2 I1 @; `! F( z! w
2 s, h5 Z. u6 d0 q/ f' C! h
, k" \' [5 e5 g( q
! i9 i9 S; `/ F7 r0 B
Kruse M, Adachi S, Scott L, Holtback U, Greengard P, Aperia A, and Brismar H. Recruitment of renal dopamine 1 receptors requires an intact microtubulin network. Pflügers Arch 445: 534-539, 2003.
% r9 b1 [) F% D! o1 b$ Y  [9 j

Li S, Galbiati F, Volonte D, Sargiacomo M, Engelman JA, Das K, Scherer PE, and Lisanti MP. Mutational analysis of caveolin-induced vesicle formation. Expression of caveolin-1 recruits caveolin-2 to caveolae membranes. FEBS Lett 434: 127-134, 1998.

Maquoi E, Peyrollier K, Noel A, Foidart JM, and Frankenne F. Regulation of membrane-type 1 matrix metalloproteinase activity by vacuolar H -ATPases. Biochem J 373: 19-24, 2003.( P  j$ }0 [  j
, Z& o6 Q! u8 u
6 v8 i% m/ o) j8 z$ i
; j) J' h0 v  A
Mellman I, Fuchs R, and Helenius A. Acidification of the endocytic and exocytic pathways. Annu Rev Biochem 55: 663-700, 1986.
6 y5 m, W( O' ~' h+ w6 ^

Nabi IR and Le PU. Caveolae/raft-dependent endocytosis. J Cell Biol 161: 673-677, 2003.* B/ z! o1 o' u, a) \1 P
  Z& A1 A1 D/ E. T+ Y3 [
, i' T; i) ]" I6 V. F: F

Narkar VA, Hussain T, and Lokhandwala MF. Activation of D 2 -like receptors causes recruitment of tyrosine-phosphorylated NKA 1 -subunits in kidney. Am J Physiol Renal Physiol 283: F1290-F1295, 2002." k2 y8 E4 f3 \1 }( X2 @7 ?) ^

- {  p% `' w* F1 j. L

Niznik HB, Liu F, Sugamori KS, Cardinaud B, and Vernier P. Expansion of the dopamine D 1 receptor gene family: defining molecular, pharmacological, and functional criteria for D 1A, D 1B, D 1C, and D 1D receptors. Adv Pharmacol 42: 404-408, 1998." C  e& z% w' ]" }% F

. w5 `- d  W1 q- w8 q5 a

O'Connell DP, Botkin SJ, Ramos SI, Sibley DR, Ariano MA, Felder RA, and Carey RM. Localization of dopamine D 1A receptor protein in rat kidneys. Am J Physiol Renal Fluid Electrolyte Physiol 268: F1185-F1197, 1995.

Ostrom RS. New determinants of receptor-effector coupling: trafficking and compartmentation in membrane microdomains. Mol Pharmacol 61: 473-476, 2002.

6 s( i% z0 w. L; a/ E0 n

Ostrom RS, Gregorian C, Drenan RM, Xiang Y, Regan JW, and Insel PA. Receptor number and caveolar co-localization determine receptor coupling efficiency to adenylyl cyclase. J Biol Chem 276: 42063-42069, 2001.5 ]0 |; u/ c/ a3 O! W3 ?  b
7 o( P; u) L7 o! a/ D% l# U0 Q9 w

! a* _. `$ T  n2 E! X. F
Ostrom RS, Post SR, and Insel PA. Stoichiometry and compartmentation in G protein-coupled receptor signaling: implications for therapeutic interventions involving G s. J Pharmacol Exp Ther 294: 407-412, 2000.' B  w/ Q1 U& X+ y
/ Z  x! T' n: M( D

Ozono R, O'Connell DP, Vaughan C, Botkin SJ, Walk SF, Felder RA, and Carey RM. Expression of the subtype 1A dopamine receptor in the rat heart. Hypertension 27: 693-703, 1996.
' E  e8 e& [9 L

5 K1 B1 _$ B" E" ]3 E3 E
Qanbar R and Bouvier M. Role of palmitoylation/depalmitoylation reactions in G protein-coupled receptor function. Pharmacol Ther 97: 1-33, 2003.

: l# K4 P. q, b$ Y

Ricci A, Escaf S, Vega JA, and Amenta F. Autoradiographic localization of dopamine D 1 receptors in the human kidney. J Pharmacol Exp Ther 264: 431-437, 1993.

' r7 u0 K& m5 t& R8 U
1 V7 m* w3 t" i% ~
Sabourin T, Bastien L, Bachvarov DR, and Marceau F. Agonist-induced translocation of the kinin B 1 receptor to caveolae-related rafts. Mol Pharmacol 61: 546-553, 2002.

" M7 v9 Z7 y4 I/ y9 i/ @
! j1 _5 O7 W2 o- m/ X( d6 ^
Sacktor B, Rosenbloom IL, Liang CT, and Cheng L. Sodium gradient- and sodium plus potassium gradient-dependent L -glutamate uptake in renal basolateral membrane vesicles. J Membr Biol 60: 63-71, 1981.8 C6 c7 ^7 k# Z- z# p6 M3 A

0 S/ o  G; d; h9 i) `
Scherer PE, Lewis RY, Volonte D, Engelman JA, Galbiati F, Couet J, Kohtz DS, van Donselaar E, Peters P, and Lisanti MP. Cell-type and tissue-specific expression of caveolin-2. Caveolins 1 and 2 co-localize and form a stable hetero-oligomeric complex in vivo. J Biol Chem 272: 29337-29346, 1997.
# e  w8 U% L# g7 D
. Z( O* Y) n+ b, e- J5 A
1 y2 [2 q5 I( I3 S% @- q
Schoonderwoert VT, Holthuis JC, Tanaka S, Tooze SA, and Martens GJ. Inhibition of the vacuolar H -ATPase perturbs the transport, sorting, processing and release of regulated secretory proteins. Eur J Biochem 267: 5646-5654, 2000.
# M. K" u' R; P8 S. b2 R' b

& }; |# _5 S9 B9 s- q
Soares-da-Silva P and Fernandes MH. Regulation of dopamine synthesis in the rat kidney. J Auton Pharmacol 10: S25-S30, 1990.' a8 p7 L0 M8 M% u
5 r# N- h( Y' H/ S- x

Ungar D and Hughson FM. Snare protein structure and function. Annu Rev Cell Dev Biol 19: 493-517, 2003.
5 Z6 U  Z3 P: m* D

Yang J, Gillingham AK, Hodel A, Koumanov F, Woodward B, and Holman GD. Insulin-stimulated cymosely alkalinization facilitates optimal activation of glucose transport in cardiomyocytes. Am J Physiol Endocrinol Metab 283: E1299-E1307, 2002.

- \2 ~2 `' |6 M. T8 Q- |

Yoshimori T, Yamamoto A, Moriyama Y, Futai M, and Tashiro Y. Bafilomycin A1, a specific inhibitor of vacuolar-type H -ATPase, inhibits acidification and protein degradation in lysosomes of cultured cells. J Biol Chem 266: 17707-17712, 1991.1 L( X* p% A9 h, {
2 V( p& W& m. ^" E; N

- M6 ~8 G* S, L4 U% W% A7 P' l
Yu P, Yang Z, Jones J, and Jose PA. D 1 dopamine receptor signaling involves caveolin-2 in rat renal proximal tubule cells (Abstract). FASEB J 17: A637, 2003.

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