Subapical Localization of the Dopamine D3 Receptor in Proximal Tubules of the Rat Kidney
Departments of Pharmacology and Toxicology (AN,MR,BM,GL) and Anatomy (AM), University of Tübingen, Tübingen, Germany; Unité de Neurobiologie et Pharmacologie Moléculaire (INSERM U 109), Centre Paul Broca (JD,PS), Paris, France; and Department of Clinical Pharmacology, Central Hospital Bremen (BM), Bremen, Germany
Correspondence to: PD Gerd Luippold, Dept. of Pharmacology and Toxicology, Faculty of Medicine, University of Tübingen, Wilhelmstr. 56, D-72074 Tübingen, Germany. E-mail: gerd.luippold{at}uni-tuebingen.de
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Summary |
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Key Words: D3 receptor rat kidney immunofluorescence confocal laser scanning microscopy phalloidin adaptin
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Introduction |
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The effects of dopamine are mediated through G-protein-coupled receptors. The dopamine receptors identified thus far are divided into two subfamilies, D1-like (D1 and D5) and D2-like (D2, D3, and D4) based on differences in the amino acid sequences, pharmacological profiles, and signal transduction pathways (for review see Missale et al. 1998).
During recent years, increasing attention has been focused on the role of the D3R in the regulation of renal and cardiovascular function. The pharmacological activation of the D3R by 7-OH-DPAT induces hyperfiltration, diuresis, and an increase in absolute and fractional sodium excretion (Luippold et al. 1998a). In D3R(/) knockout mice the glomerular hyperfiltration due to amino acid load is abolished compared with wild-type mice (+/+) (Luippold et al. 2000
). In addition, the pathophysiological hyperfiltration in experimental diabetes mellitus is also reduced in diabetic D3R-deficient mice (Mühlbauer et al. 2002
).
An impaired D3R function may also be involved in the pathogenesis of hypertension. Disruption of the D3R gene in mice produced renal sodium retention and an increase in systemic blood pressure (Asico et al. 1998). Among Dahl rats, only the salt-sensitive strain develops hypertension due to a high-sodium diet. However, such salt sensitivity also was induced by a pharmacological inhibition of the D3R in the genetically salt-resistant rat strain (Luippold et al. 2001
).
Compared with a plethora of physiological findings concerning the D3R, only sparse information is available on the microanatomic localization of this receptor in the kidney. D3R mRNA was found in glomerular, tubular, and vascular fractions of rat kidneys (Gao et al. 1994; Luippold et al. 1998a
). However, these data supplied by RT-PCR provide only information about the regional distribution but not about precise cellular and subcellular histological localization of the D3R. Even with new technologies, such as laser capture microdissection in combination with RT-PCR, which allows detection of mRNA levels in specific cell types, information concerning the subcellular distribution is still limited. Autoradiographic studies, on the other hand, depend on selective pharmacological agonists for the respective receptor subtypes. Radioligand binding assays with [3H]-7-OH-DPAT, an agonist showing a good but not exclusive selectivity for D3R vs D2R, revealed an accumulation of the D3R within cortical proximal tubules and, to a lesser extent, in distal tubules and in the glomerular mesangium (Barili et al. 1997
).
The development of antibodies raised against selected sequences of dopamine receptor subtypes permits a more detailed characterization of the cellular and subcellular distribution of a particular receptor subtype. A first immunohistochemical investigation revealed the D3R in apical portions of proximal and distal tubular cells, in podocytes, renal arteries, and in cortical collecting ducts (O'Connell et al. 1998). These results did not completely correspond to D3R sites as previously defined by pharmacological and functional studies. Therefore, the present study was undertaken to gain further insights into the distribution of the D3R in the rat kidney.
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Materials and Methods |
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Antibody Preparation
The preparation of the immunizing peptide conjugates and the immunization procedure have already been described by Diaz et al. (2000). The polyclonal anti-D3R serum was produced in rabbits and was raised against a 15-residue peptide corresponding to epitopes in the third intracytoplasmic loop of the rat dopamine D3R. The immunizing peptide sequence showed no similarity with other dopamine receptor subtypes and did not reveal any other homology with a known protein (Diaz et al. 2000
).
Animal and Tissue Preparation
SpragueDawley rats (200350 g) were anesthetized by IP injection of thiopental sodium (Trapanal; Altana, Konstanz, Germany; 80 mg/kg bw). The rats were transcardially perfused (through the left ventricle) with 2050 ml of 0.85% NaCl solution (37C) followed by 400 ml of an ice-cooled fixative solution containing 2% paraformaldehyde (Merck; Darmstadt, Germany) in 0.1 M phosphate buffer (PB), pH 7.5. The kidneys were removed and cut into slices of 3-mm thickness. The slices were postfixed in the same fixative for a further 2 hr at 4C. Sometimes brains were also removed and processed in an identical manner. After cryoprotection in 30% sucrose solution (overnight at 4C) and washing in PB (three times for 5 min), the tissue pieces were embedded in Tissue-Tek (Sakura; Zoeterwoude, The Netherlands) followed by freezing in isopentane precooled by liquid nitrogen, transferred into liquid nitrogen, and stored at 80C until further use. Cryostat sections (1630 µm) were cut in transverse planes and collected on Superfrost Plus charged glass slides (R. Langenbrinck; Emmendingen, Germany).
Immunofluorescence Staining
The D3R immunoreactivity was detected by indirect immunofluorescence. Before the first immersion in buffer the tissue sections were encircled with the ImmunoPen (Calbiochem-Novabiochem; San Diego, CA) to form a hydrophobic barrier around them. Tissue Tek was removed from the sections by washing in TBS (0.05 M Tris buffer, pH 7.5, containing 150 mM NaCl) for 5 min. To prevent nonspecific background staining the sections were treated with a blocking solution (5% normal goat serum, 0.4% BSA, 0.1% gelatin, and 0.1% Tween-20 in TBS) for 1 hr at room temperature (RT). All incubation steps were carried out in a humidity chamber. After the blocking solution was blotted off the slices were exposed for 40 hr at 4C to the anti-D3R antibody [diluted 1:1000 in TBS containing 5% normal goat serum and 0.05% Tween-20 (TBS-NGST20)]. Then the sections were rinsed in TBS containing 0.1% gelatin and 0.05% Tween-20 (TBS-GT20) (three times for 5 min) and incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:200 in TBS-NGST20, 1 hr at RT in darkness) (Molecular Probes; Eugene, OR). Finally, the sections were washed twice in TBS-GT20 and once in TBS and were mounted in Fluorsave (Calbiochem-Novabiochem).
In some cases the kidney sections were applied to sodium dodecyl sulfate (SDS) before the immunostaining to reveal antigenic sites probably masked by the fixative. [0.51% SDS in PBS (0.01 M phosphate-buffered saline + 0.9% NaCl, pH 7.4)] followed by washing in PBS (three times for 5 min) according to Brown et al. (1996).
The immunostained sections were examined by the confocal laser scanning microscope ZEISS LSM 410 Invert or LSM 510 Axioplan (Carl Zeiss; Jena, Germany) [excitation: argon laser at 488 nm and heliumneon laser at 633 nm (double stainings); with appropriate filter sets to control for cross-bleeding].
Double Staining
Double staining was performed to examine the distribution of the D3R in more detail and in relation to specific cell compartments. Alexa Fluor 633phalloidin (Molecular Probes) stains F-actin of the cytoskeleton and was used to label the brush border of proximal tubules. After D3R labeling the sections were rinsed twice with PBS, incubated for 20 min at RT with Alexa Fluor 633phalloidin (1:40 in PBS, pH 7.4, containing 1% BSA), washed again, and mounted in Fluorsave.
Adaptins mediate the interaction between clathrin and membrane components and monoclonal anti-ß1/ß2-adaptins (Sigma-Aldrich; St Louis, MO) were applied to recognize clathrin-coated regions at the plasma membrane. Sections were incubated simultanously with a cocktail of antibodies against D3R and ß1/ß2-adaptins (1:100 in TBS-NGST20). Goat anti-mouse IgG conjugated with Cy5 (1:200 in TBS-NGST20, 1 hr at RT) (Jackson Immuno Research; West Grove, PA) was used as secondary antibody for anti-ß1/ß2-adaptins.
Specificity Tests
The specificity of the anti-D3R serum was checked by omission of the primary antibody and by preabsorption of the antiserum with the immunizing peptide Y15L corresponding to amino acids 319332 of the rat D3R (Diaz et al. 2000). The anti-D3R serum (1:1000) was incubated with the peptide at a concentration of 50 µg, 25 µg, or 8.5 µg/ml overnight at 4C. The latter value corresponds to a 10-fold excess of peptide antigen. After centrifugation the antiserum was applied to the immunostainings as described above. An immunohistochemical (IHC) analysis of brain regions known to express the D3R was also performed.
The specificity of the applied anti-D3R serum was also examined by Western blot analysis of kidney homogenates. Kidneys of nonperfused rats were dissected, minced, and homogenized in lysis buffer (20 mM Hepes, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 4 mM EGTA, 4 mM EDTA, 30 µg/ml aprotinin, 5 µg/ml leupeptin, 10 µg/ml trypsin inhibitor, and 1 mM dithiothreitol) followed by centrifugation (17.000 x g, 15 min, 4C). The supernatant was used for analysis. The protein concentration was determined according to the method of Bradford (1976) using a protein assay kit (Biorad; Munich, Germany) with BSA as standard. Samples were diluted 1:4 with Roti-Load sample buffer (Roth; Karlsruhe, Germany) and boiled for 2 min at 95C. Protein samples (20 µg) were separated by electrophoresis in 8% SDS-PAGE. For determination of molecular weight, a 10-kD protein ladder (Life Technologies; Eggenstein, Germany) was used. Thereafter, the fractionated proteins were electrophoretically transferred onto a nitrocellulose membrane. The membrane was blocked for 90 min with PBS containing 2% gelatin, 2% normal goat serum, 2% low-fat milk powder, and 0.02% Tween-20. Then the blot was incubated with the anti-D3R serum (1:1000) overnight at 4C, rinsed twice with PBS containing 0.02% Tween-20, and exposed to the secondary horseradish peroxidase-conjugated antibody (1:4000) (Santa Cruz Biotechnology; Santa Cruz, CA) for 1 hr at RT. The immunoreactive bands were visualized by enhanced chemiluminescence system onto a Hyperfilm-ECL (Amersham; Buckinghamshire, UK).
The specificity of the D3R antibody binding was confirmed by preabsorption of the antiserum with the immunizing peptide at a concentration of 8.5 µg/ml (working dilution 1:1000).
The anti-D3R serum was also extensively tested in the laboratory of P. Sokoloff (Diaz et al. 2000).
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Results |
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D3R staining was confined to the renal cortex. On no occasion did we locate the D3R in medullary regions. A general view of the D3R immunostaining in the renal cortex is presented in Figure 1A. The D3R was observed in distinct proximal tubular structures, especially in proximal convoluted tubules near to or in direct connection with the urinary pole of the glomerulus (S1 and S2 segments of proximal tubules) (Figure 1B). There was no detectable signal in other cell types, such as in distal tubules, glomeruli, renal vasculature, or cortical collecting ducts. Figures 1B and 2A demonstrate the dot-like distribution pattern of the D3R immunostaining. The fluorescent spots with a maximal diameter of 2 µm were restricted to the subapical portion of the proximal tubular cells. It appears that the receptors were located below or at the base of the brush border. Double labeling experiments with the F-actin marker phalloidin (Figure 2B) were performed to investigate whether D3R is a part of the brush border or is localized within the cytoplasm. The D3R and phalloidin signals were clearly separated from each other, suggesting that the D3R is accumulated within a cytoplasmic pool, probably corresponding to internalized apical receptors or a storage of newly synthesized D3R.
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Initial experiments were carried out to examine the distribution of the D3R in relation to endocytic compartments. Clathrin-coated vesicles are involved in the selective internalization of cell surface receptors, including G-protein-coupled receptors. Our interest focused on adaptin because this adaptor protein is known to mediate the interaction between clathrin and membrane components. The apical cell membrane was strongly stained by the anti-ß1/ß2-adaptins. However, the adaptin immunostaining did not correspond to the distribution of the D3R signal (Figures 2C and 2D). We therefore suspect that the D3R is present in vesicular structures without a clathrin coat. Further studies are required to clarify a possible association of the D3R with other compartments along the endocytic pathway.
Specificity
A variety of control incubations were performed, including replacement of the primary antibody by the diluent (TBS-NGST20) (data not shown) and preabsorption of the anti-D3R serum with the immunizing peptide (Figure 3D). Each test was carried out on consecutive sections from cortical regions of the same kidney to achieve a direct comparison with the normal D3R staining. The immunostaining could be completely suppressed by omission of the primary antibody and by all of the preabsorption treatments. The omission of the primary antibody was especially necessary because the proximal tubules are known for their strong autofluorescence with the 488-nm argon laser (Yip et al. 1998). In addition, the absence of staining exclude false-positive reactions possibly created by the secondary antibody. The complete lack of immunostaining after preabsorption (even at 8.5 µg/ml, corresponding to a 10-fold excess of peptide antigen) ensured that the immunizing peptide sequence of the D3R is really recognized by the anti-D3R serum and that the tissue immunostaining is a result of D3Rantibody binding.
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The anti-D3R serum was also tested by Western blotting using whole cell extracts of kidney homogenates (Figure 3A). The anti-D3R serum visualized a band of 85 kD, which is in good agreement with the results obtained in the laboratory of P. Sokoloff (Diaz et al. 2000
). In some of the immunoblots a very weak protein band of
62 kD was also detected, which might represent a degradation product. The immunodetection of the 85-kD band and of the weak 62-kD band was completely prevented by preabsorption of the anti-D3R serum with a 10-fold excess of the immunogen peptide. The Western blot analysis demonstrated that the antiserum recognized the D3R protein in the rat kidney. Furthermore, the specificity of the anti-D3R serum used was checked by Western blot analysis of membrane proteins of D2R- or D3R-expressing Chinese hamster ovary (CHO) cells. Strong immunofluorescence signals were detected on D3R-expressing CHO cells but not on wild-type or D2R-expressing cells (Diaz et al. 2000
).
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Discussion |
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Our specificity tests, i.e., omission of the primary antibody and the preabsorption controls, verified that the D3R fluorescence was not caused by an unspecific binding of the secondary antibody or by the autofluorescence described for proximal tubules (Maunsbach 1966; Hagege and Richet 1985
). This latter type of fluorescence appeared only at the basal pole of tubular cells. The immunostaining in the islands of Calleja, which are known to express the D3R, represents a further criterion for the specificity of the employed antiserum. In addition, studies of Diaz et al. (2000)
showed a close overlap between the D3R immunoreactivity, binding sites for [125I]-trans-7-OH-PIPAT, a D3R selective ligand, and the D3R mRNA distribution.
The Western blot result of an 85-kD protein band visualized by the anti-D3R serum is in good agreement with the data published by Nimchinsky et al. (1997), Diaz et al. (2000)
and Ladines et al. (2001)
. Because the band was not seen when the antiserum was preabsorbed with the immunizing peptide, there is every reason to believe that it represents the D3R. However, an additional 45-kD band was reported in Western blots of transfected CHO cells expressing the D3R (Diaz et al. 2000
) as well as in immunoblots of brush border membranes (Ladines et al. 2001
) or proximal tubular cells (Zeng et al. 2003
). The size of 45 kD corresponds to the molecular weight of the nonglycosylated form of the D3R, and the larger band at
85 kDa may represent either glycosylated forms or SDS and reduction-resistant dimers (Nimchinsky et al. 1997
; Diaz et al. 2000
). The appearance or absence of the 45-kD protein is probably caused by variations in the experimental conditions used for tissue preparation, which may result in deglycosylation. In the transfected CHO cells, the overexpressed D3R is largely present as the immature or nonglycosylated form (Diaz et al. 2000
). The very weak band at
62 kD, detected only in some of the blots, could correspond to the 57-kD band reported by O'Connell et al. (1998)
or the 60- kD band by Diaz et al. (2000)
.
A possible crossreactivity of the anti-D3R serum with the D2R was excluded by the lack of any immunofluorescence signal in D2R-expressing CHO-cells and by Western blot analysis of these cells. The peptide sequence selected for immunization reveals no homology with other dopamine receptor subtypes or known proteins in general (Diaz et al. 2000).
In the kidney, the immunostaining corresponds to D3R sites previously defined by radioligand binding assays and RT-PCR studies. Radioligand binding assays revealed a ligand accumulation primarily within cortical tubules and, to a lesser extent, to the glomerular mesangium (Barili et al. 1997). However, it should be mentioned that the D3R ligand 7-OH-DPAT used in that study also may interact with the D2R. RT-PCR studies demonstrated the expression of D3R in glomerular, tubular, and vascular fractions of the rat kidney (Gao et al. 1994
; Luippold et al. 1998a
) whereas, in the present study, the D3R signal was absent within glomeruli and the renal vasculature. Contaminations by other nephron elements during mRNA isolation could result in a false-positive localization by RT-PCR studies. Otherwise, masking effects by the fixative may hinder the recognition of the D3R epitope by the antibodies. It is known that staining of vascular and glomerular structures is generally much less intense in perfusion-fixed sections, most likely because these tissue compartments are more strongly fixed by vascular perfusion than extravascular tissues (Boivin et al. 2001
). Immunostaining on acetone-fixed cryostat sections of nonperfused kidneys exhibited no D3R sites at all (data not shown), indicating that a paraformaldehyde fixation is necessary for the epitope recognition by the employed antibody. An immersion fixation with paraformaldehyde instead of the vascular perfusion also did not reveal any D3R staining in the kidney.
The limited distribution of the D3R in the renal cortex, as found in our studies, was also shown earlier by O'Connell et al. (1998). However, O'Connell et al. described the D3R in distal tubules, cortical collecting ducts, podocytes, and the renal vasculature. The apparent differences in our study might be due to the applied anti-D3R sera directed against different epitopes. This could also include a different epitope accessibility by the fixation procedure. The sequence of our immunizing peptide contains a putative site for casein kinase 2. Phosphorylation by this or a similar enzyme activity at this site may hinder recognition by the antibody (Diaz et al. 2000
). In addition, since the D3R may exist as dimers (Nimchinsky et al. 1997
; Karpa et al. 2000
; Scarselli et al. 2001
), it is possible that the epitope responsible for the antibody recognition is impaired by such dimerization. However, the domains through which D3R interacts with each other still remain unknown. Several D3R-interacting proteins from the cytoplasm (Lin et al. 2001
; Binda et al. 2002
; Griffon et al. 2003
; Jeanneteau et al. 2004
) may also interfere with the antibody recognition and the precise subcellular site, such as the plasma membrane.
The difference between our results and those of O'Connell et al. (1998) is the cellular distribution of the D3R within the proximal tubular cells. Whereas the D3R labeling by O'Connell was confined to the apical region, we have reliably demonstrated D3R in a subapical cytoplasmic location as revealed by double staining with phalloidin. Possibly, the D3R exists at the cell membrane as well as in intracellular compartments. According to the methodical aspects concerning the application of different anti-D3R sera and different fixation procedures discussed above, only one of the D3R locations could be detected in the different studies. Regarding the differences between our and O'Connell's studies, it is also noteworthy that our antibody, but not that used by O'Connell et al. (1998)
, has received a strong validation by the fact that it does not generate any labeling in D3R knockout mice (Diaz et al. 2000
). Our present report is the first description of a D3R accumulation in a cytoplasmic pool in renal proximal tubular cells. An intracellular localization of D1R was previously demonstrated in proximal tubules (O'Connell et al. 1995
; Trivedi et al. 2002
), and the D3R occurred as a few cytoplasmic patches in rat neurons in addition to its primary localization at the plasma membrane (Diaz et al. 2000
). The functional significance of the intracellular localization of the D3R still remains speculative. Probably it reflects a recycling mechanism of internalized receptors or a storage compartment of newly synthesized D3R. In further studies it will be necessary to determine the conditions that induce an insertion of the intracellular D3R into the cell membrane. In this context the experiments by Brismar et al. (1998)
and Aperia (2000)
are of considerable interest. These authors pointed out that D1R can be recruited from intracellular compartments to the plasma membrane by D1R agonists, dopamine, and by the dopamine precursors L-DOPA (L-dihydroxyphenylalanine) and Glu-DOPA (
-L-glutamyl-L-DOPA). This receptor recruitment requires an intact microtubulin network and occurs via Golgi-derived vesicles (Kruse et al. 2003
). The studies were performed in cells from a proximal tubular-like cell line (LLCPK-1 cells). However, it has not been elucidated whether this phenomenon of recruitment also occurs in vivo under physiological conditions. The underlying molecular mechanisms are unknown, but D3R accessory proteins such as GIPC (Jeanneteau et al. 2004
), CLIC6 (Griffon et al. 2003
), 4.1N (Binda et al. 2002
), and
-filamin A (Lin et al. 2001
) may interfere with receptor trafficking and cell surface localization in the kidney. These proteins have been essentially described in the central nervous system and it is not known whether all are present in the kidney. Conceivably, the absence of one of these proteins in the kidney could alter the localization of the D3R in this structure with respect to that in other tissues. Furthermore, studies are required to demonstrate such a recruitment in the case of the D3R in the proximal tubule. Glomerular filtered L-DOPA is reabsorbed by proximal tubular cells and is converted to dopamine by the L-aromatic amino acid decarboxylase (Baines and Chan 1980
). Little is known about the mechanisms by which dopamine is stored in the proximal tubules or its secretion into the tubular lumen or peritubular interstitium. Since dopamine is rather unstable in a non-acidic environment, it is likely that dopamine in proximal tubules is intermediately taken up by vesicles. The possibility that dopamine binds to intracellular receptors in such vesicles cannot be ruled out (Aperia 2000
). In this regard, it is noteworthy that renal slices incubated with L-DOPA displayed a dopamine accumulation (Hagege and Richet 1985
) in the subapical region of proximal tubules comparable to our D3R staining. The dopamine accumulation shown by formaldehyde-induced fluorescence and our D3R labeling exhibit the same dot-like appearance.
The subapical localization and the spot-like distribution pattern of the D3R staining could also indicate that the D3R signal is a part of the vacuolar apparatus that is extensively developed in proximal tubular cells (Cui and Christensen 1993). The vacuolar apparatus is involved in endocytosis of glomerular filtered molecules connected with membrane recycling. It is characterized by the occurrence of small clathrin-coated vesicles (<0.5 µm in diameter) as well as larger endocytic vacuoles without a cytoplasmic coat (
1 µm in diameter) (Cui and Christensen 1993
). It should be mentioned that an exact size measurement of the fluorescent spots was complicated by the strong brightness of the Alexa Fluor 488 dye. Nevertheless, our stained vesicular compartments in the proximal tubules are also larger than classical clathrin-coated endocytic vesicles and differ from the D3R staining pattern in the brain. In the double labeling, the staining by anti-ß1/ß2-adaptins did not correspond to the distribution of the D3R signal, suggesting that the D3R is present in vesicular structures without a clathrin coat. However, clathrin and adaptin are necessary only for the rapid assembly process of endocytic vesicles, and afterward the clathrin shell is removed by an uncoating ATPase. Further studies are required to clarify a possible association of the D3R with other compartments along the endocytic pathway.
In conclusion, the D3R was identified, for the first time, in an intracellular compartment within proximal tubular cells of the kidney. This localization may result from endocytosis or reflect newly synthesized receptors. The way in which these morphological observations fit with the physiological effects of D3R is not yet understood. The involvement of the D3R in glomerular hyperfiltration due to amino acid load (Luippold et al. 2000) or diabetes mellitus (Mühlbauer et al. 2002
), as shown in functional studies by our group, cannot be explained by these immunocytochemical data. A reinsertion of the stained D3R into the apical or basolateral cell membrane of proximal tubules may participate in mediating the inhibitory effects of dopamine on sodium transport or in dopamine secretion into the tubular lumen or peritubular interstitium. Further studies will focus on D3R translocation and the conditions under which this occurs.
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Acknowledgments |
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Footnotes |
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Literature Cited |
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