Expression of the Dopamine D3 Receptor Protein in
the Rat Kidney
Damian P. O’Connell, Carl J. Vaughan, Anna M. Aherne, Suzanne J. Botkin, Zhi-Qin Wang,
Robin A. Felder, Robert M. Carey
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Abstract—The dopamine D3 receptor subtype was identified in rat kidney using both light microscopic immunohistochemistry
and electron microscopic immunocytochemistry. Antipeptide polyclonal antisera were directed to both extracellular and
intracellular regions of the native D3 receptor. Selectivity of the antipeptide antisera was validated by their ability to recognize
native receptor protein expressed in permanently transfected mouse LTK2 cells or Spodoptera fragiperda (Sf9) cell
membranes. Light microscopic immunohistochemical staining for the D3 receptor was observed only in the cortex. Specific
staining was present in proximal and distal tubules, cortical collecting ducts, glomeruli, and renal vasculature. Immunostaining
was observed predominantly in the apical portion of both the proximal and distal tubules. Renal arterial staining was
prominent in the medial and adventitial layers. Electron microscopic immunocytochemistry revealed immunogold particles in
arteriolar smooth muscle cells of the renal vasculature. In proximal and distal tubules and cortical collecting duct, immunogold
staining was localized to apical portions of tubule cells. D3 receptor immunogold staining in the glomeruli was clearly present
in podocytes. Western blot analysis demonstrated a single D3 receptor band in infected Sf9 cell membranes, in transfected
LTK2 cells, and in kidney and brain but not in noninfected Sf9 cell membranes or in D2 or D3 receptor transfected or
nontransfected LTK2 cells. The use of receptor subtype–selective antibodies allows for the tissue localization of specific
dopamine receptors that are not distinguished by current pharmacological or ligand-binding technology. The rat kidney
expresses the D3 receptor at sites previously deemed to have D2-like receptors. (Hypertension. 1998;32:886-895.)
Key Words: dopamine n receptor, D3 n kidney n immunocytochemistry n glomerulus
T
he dopaminergic system depends on the interaction of
dopamine with several specific receptors.1 In the late
1970s, 2 dopamine receptors, D1 and D2, were identified in
the brain.2 These receptors were characterized pharmacologically with D1- and D2-specific agonists and antagonists.
Dopamine receptors subsequently were localized in peripheral tissues such as blood vessels, kidney, and adrenal cortex
by ligand-binding studies and biological and biochemical
responses. Because structural similarities between central and
peripheral dopamine receptors were indeterminate, a separate
nomenclature for peripheral dopamine receptors was adopted:
D1-like and D2-like.3 D2-like receptors are located both
presynaptically and postsynaptically, with the presynaptic
receptor inhibiting catecholamine release from sympathetic
neurons.3,4
In recent years, multiple dopamine receptor subtypes have
been cloned and purified. Two D1-like receptor subtypes have
been identified (D1A and D1B in the rat, or D1 and D5 in
humans) and are coupled to the stimulation of adenylyl
cyclase. The D2 receptor has been found to exist in 2 isoforms
(D2Long and D2Short), both coupled to the inhibition of adenylyl
cyclase. The so-called D35 and D46 receptors are closely
related to, but distinct from, the D2 receptor. The pharmaco-
logical properties of D1-like receptors resemble most closely
those of the central nervous system D1A and D1B receptor
subtypes, whereas those of the D2-like receptors resemble the
properties of the central nervous system D2 receptor subclass.
Molecular techniques have revealed that the peripheral D1A
and central D1A receptors have identical mRNA nucleotide
sequences.7,8
We have demonstrated that the D1A dopamine receptor
subtype, cloned from the rat brain, is expressed in rat kidney
using antipeptide antisera directed to epitopes on the native
receptor.9 Such receptor subtype–selective antibodies permit
identification of specific receptors that remain indistinguishable when studied with agonist- and/or antagonist-based
radioligand, biochemical, or functional studies. Indeed, the
majority of D2-like receptor ligands in use have high affinities
for the D2, D3, and D4 receptor subtypes, rendering specific
identification impossible. Studies investigating the peripheral
localization of D2-like receptors with such ligands have
revealed receptor(s) in renal vessels (both adventitial and
endothelial cell layers), glomeruli, and cortical and medullary
tissue.10 –13 A novel D2-like receptor has been described in the
inner medullary collecting duct.13 In rabbit renal artery,14 rat
glomeruli,12 and rat renal cortex,15 D2-like receptor activation
Received March 9, 1998; first decision April 1, 1998; revision accepted July 23, 1998.
From the Departments of Pharmacology, University College Cork, Ireland (D.P.O’C., C.J.V., A.M.A.); and Departments of Medicine (S.J.B., Z.-Q.W.,
R.M.C.) and Pathology (R.A.F.), University of Virginia Health Sciences Center, Charlottesville, Va.
Reprint requests to Dr Robert M. Carey, Box 395, University of Virginia Health Sciences Center, Charlottesville, VA 22908.
© 1998 American Heart Association, Inc.
Hypertension is available at http://www.hypertensionaha.org
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Figure 1. Two-dimensional representation of the rat dopamine D3 receptor amino acid sequence. The presented results are those
obtained using antibodies to an epitope on the third extracellular loop, outlined in black with an asterisk, except for those in which the
designated peptide sequence on the third intracellular loop was used as the immunogen.
results in inhibition of adenylyl cyclase activity. Of the
cloned dopamine receptors, mRNA of the D1A, D2Long, and D3
receptor subtypes has been identified in the kidney.5,16 –18
The present study used specific antibodies directed against
synthetic peptides that correspond to epitopes in the putative
amino acid sequence of the rat D3 receptor to characterize the
regional distribution of this dopamine receptor subtype in the
rat kidney. This characterization was achieved by a combination of light microscopic immunohistochemistry, electron
microscopic immunocytochemistry, and Western blot
analysis.
Methods
Antisera
Polyclonal antisera were raised against synthetic peptide sequences
derived from the predicted rat dopamine D3 receptor amino acid
sequence, corresponding to epitopes located on extracellular and
intracellular portions of the receptor (Figure 1). The specific sequences were 404CHVSPELYR412 on the third extracellular loop and
288
QPPSPGQTHGGLKR301 on the third intracellular loop. KLH was
conjugated to the amino terminus, and prebled New Zealand White
rabbits were repetitively immunized with the KLH-peptide immunogen until maximum titers of antibody were obtained. Antibody
production was monitored by means of solid-phase enzyme-linked
immunoabsorbent assays (ELISA), in which the ELISA plates were
coated with peptide and demonstrated final titers of 104 to 106. The
antibodies were affinity purified as follows. The D3 receptor synthetic peptide (Research Genetics) was immobilized to AminoLink
coupling gel (Pierce Chemical Co) via sodium cyanoborohydride.
Once the column had been prepared, crude D3 receptor antiserum
was first IgG-purified via protein A column, as previously described.19 The IgG-rich fraction of D3 receptor antiserum was bound
to the D3 receptor affinity column. The column was washed to
remove all other IgGs. The D3 receptor affinity-purified antibody
was then eluted with 0.1 mol/L glycine, pH 2.5. The protein
concentration of the affinity-purified D3 receptor antibody was
determined to be 0.38 mg/mL by Bradford protein assay (Bio-Rad).
The specificity of both polyclonal antipeptide antisera was verified
by (1) attenuation of the ELISA response after preincubation of the
purified antipeptide antibody with its immunization peptide and (2)
the ability of the antipeptide antibody to recognize the native D3
receptor that had been stably transfected and expressed in a murine
fibroblast LTK2 cell line or infected with a baculovirus expression
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Renal D3 Dopamine Receptor
Figure 2. Light photomicrographs of D3 receptor
transfected LTK2 cells. A, Cells demonstrating
positive staining with antisera against the third
extracellular epitope of the D3 receptor (1:1000
dilution). B, Nontransfected parent cell line. Original magnification 31600.
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system into Spodoptera fragiperda (Sf 9) insect cells. Figures were
prepared from experiments using antibodies directed against the third
extracellular loop, except for when the antibody to the third intracellular loop was used.
Transfected or Infected Cells
A murine fibroblast LTK2 cell line was stably transfected with a
full-length rat D2 or D3 receptor cDNA using a modified calcium
phosphate method.20 The cDNA was subcloned in the expression
vector pRc/CMV (Invitrogen Corp) at the XbaI site. Both transfected
and nontransfected LTK2 cells had 10 mmol/L butyrate added to the
medium 48 hours before the experiment to enhance D2 or D3 receptor
gene expression.20 Nontransfected LTK2 cells were used as controls.
Parallel cell cultures were grown on poly-L-lysine– coated glass
well slides and subsequently fixed for 30 minutes in 4% paraformaldehyde in PBS at room temperature. The cells then were permeabilized with 0.001% saponin and, following standard blocking procedures, were incubated with the purified antipeptide antibody diluted
1:3000 in Tris/saline/azide (TSA) for 16 hours at room temperature.
Positive staining was visualized using an avidin-biotin immunoperoxidase reaction (Vectastain ABC Elite Kit, Vector Laboratory).
Controls for the procedure included (1) substitution of preimmune
sera, (2) omission of primary antibody, and (3) incubation of
antipeptide antibody with nontransfected parent LTK2 cell line.
Cell membranes from Sf 9 cells stably infected with the rat D3
receptor and membranes from noninfected Sf 9 cells were obtained
from Research Biochemicals Inc. These cell membranes have been
characterized pharmacologically by Boundy et al.21
Light Microscopic Immunohistochemistry
Male Wistar-Kyoto rats (n56) weighing between 250 to 500 g were
anesthetized with sodium pentobarbital (50 mg/kg IP). The animals
underwent a perfusion regimen as previously described.9 Briefly, the
animals were perfused through the left ventricle with a regimen of
0.9% saline followed by 1% sucrose in 0.12 mol/L sodium phosphate
and 4% paraformaldehyde in 0.12 mol/L sodium phosphate. After
perfusion, tissues were removed, the kidneys were decapsulated and
bisected, the brains were dissected, and all tissues were post-fixed in
4% paraformaldehyde in 0.12 mol/L phosphate for 1 hour and
cryoprotected overnight at 4°C in 30% sucrose in 0.12 mol/L sodium
phosphate. Frozen sections were cut (10 to 14 mm), mounted on
positively charged slides, and processed in an identical manner for
immunohistochemistry, as previously described.22 The sections were
incubated with the D3 receptor primary antibody diluted 1:1500 to
1:4000. Immunoreactive D3 receptor was detected with an avidinbiotin immunoperoxidase reaction (Vectastain ABC Kit, Vector
Laboratory). Slides were lightly counterstained with hematoxylin.
Controls included (1) omission of primary antibody, (2) replacement
of primary antibody with preimmune serum and with normal rabbit
IgG at the same protein concentration as the antibody, (3) preadsorption of primary antibody against the peptide-KLH conjugate, and
(4) preadsorption of primary antibody against the pure peptide
immunogen; in all but control 3, immunoreactivity was abolished.
Electron Microscopic Immunocytochemistry
Male Wistar-Kyoto rats (n54) weighing between 250 to 500 g
were anesthetized with sodium pentobarbital and transcardially
perfused, as previously described.9 The perfusion regimen consisted of a prefixative solution containing (per liter distilled
water) 9 g NaCl, 25 g polyvinylpyrrolidone, 0.25 g heparin, and
5 g procaine (pH 7.35), followed by a fixative solution of either
a combination of 0.1% glutaraldehyde and 2% paraformaldehyde
or 4% paraformaldehyde alone in 0.05 mol/L sodium cacodylate
buffer (pH 7.3). The kidneys were removed, decapsulated, hemisected, and subdivided into regions containing tissue from the
cortex, juxtamedullary cortex, and medulla. Each region was
minced into 1-mm3 pieces and post-fixed for a further 2 to 3
hours. Tissue samples were dehydrated and then infiltrated in
mixtures of absolute ethanol in Lowicryl K4 M resin. Subsequently, the tissue samples were embedded in pure Lowicryl K4
M using gelatin capsules and were polymerized under UV light
(360 nm) for 30 to 48 hours at 220°C. Ultrathin silver-cream
sections (60 to 70 nm) were cut and mounted on 200-mesh thin
bar nickel grids. Throughout immunolabeling, the rinsing steps
were performed by floating the grids on droplets on the covers of
microtest plates and placing the covers on a magnetic stirrer.22
Nonspecific binding in tissue sections was blocked for 15 minutes
with a combination of 1% ovalbumin and 2% nonfat dry milk in
Trizma, pH 7.2. The tissue then was incubated for 2 hours in one
of the following, which were at a 1:100 dilution in 1% ovalbumin
in Trizma, pH 8.3 (as previously used for similar experiments9,23):
(1) primary antibody, (2) primary antibody preadsorbed with the
peptide-KLH conjugate, (3) primary antibody preadsorbed with
peptide (immunogen), or (4) preimmune serum or normal rabbit
IgG. After a washing in 1% ovalbumin in Trizma, the grids were
incubated in 10-nm protein-A gold containing cold-water fish
gelatin for 30 minutes. After a wash sequence, the grids were
dried, counterstained in saturated uranyl acetate (2 minutes) and
lead acetate (30 seconds), and mounted on Formvar support.
Sections were examined in a Zeiss 10 CA transmission electron
microscope at 60 kV.
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889
soaked in 5% nonfat dry milk in Tween 20 solution (0.05% Tween
20, 10 mmol/L Tris-HCl [pH 7.2], 250 mmol/L NaCl) for 1 hour,
incubated with the antisera against the third extracellular epitope of
the D3 receptor (1:5000 dilution in Tween 20 solution) for 1 hour,
and reacted with a peroxidase-labeled secondary antibody (1:10 000
dilution) for 1 hour. Specific bands were visualized with chemiluminescence (ECL Western Blotting Detecting Kit, Amersham).
D3 Receptor Binding Studies
Figure 3. Western blot analysis. Lane 1 shows adult rat brain;
lane 2, adult rat kidney; lane 3, noninfected Sf 9 cell membranes; lane 4, D3 receptor infected Sf 9 cell membranes; lane 5,
D2 receptor transfected LTK2 cells; lane 6, nontransfected LTK2
cells; and lane 7, D3 receptor transfected LTK2 cells.
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Western Blot Analysis of D3 Receptor
Protein Expression
The kidneys and brains of adult Sprague-Dawley rats were dissected,
minced, and homogenized with Tissuemizer (Tekmar Corp) in
Buffer A (10% glycerol, 20 mmol/L Tris-HCl [pH 7.3], 100 mmol/L
NaCl, 2 mmol/L PMSF, 2 mmol/L EDTA, 2 mmol/L EGTA,
10 mmol/L sodium orthovanadate, 10 mg/mL ['1.5 mmol/L] leupeptin, and 10 mg/mL ['1.5 mmol/L] aprotinin; Sigma). The
homogenate was centrifuged at 30 000g for 30 minutes at 4°C. The
pellet was resuspended in Buffer B (Buffer A with 1% NP-40;
Sigma), stirred for 1 hour at 4°C and centrifuged again at 30 000g for
30 minutes at 4°C. The supernatant was used for analysis.
For control samples, D3 receptor infected or noninfected Sf 9 cell
membranes or D2 or D3 receptor transfected or nontransfected LTK2
cells were processed in the same way. The samples were analyzed
with SDS–polyacrylamide gel electrophoresis (5% acrylamide stacking gel and 8% running gel) in a standard protocol as described.24
The resolved proteins were transferred by elecroblotting (15 V for 20
minutes, Trans Blot SD DNA, Bio-Rad) onto a nitrocellulose sheet
(BA-S 83, Schleicher and Schull). The nitrocellulose sheet then was
Transfected and nontransfected LTK2 cell membranes were prepared
by lysis of cell monolayers in 1 mmol/L Tris-Cl, pH 7.5, for 15
minutes. The cell lysates then were scraped from the dish and
centrifuged at 40 000g for 15 minutes. The pellet was resuspended in
TME buffer (75 mmol/L Tris, pH 7.5, 12.5 mmol/L MgCl2,
1.5 mmol/L EDTA). Radioligand binding activity of the transfected
or nontransfected LTK2 cell membranes and infected or noninfected
Sf 9 cell membranes was measured by specific binding of [125I]iodospiperone.25 Quinpirole (1 mmol/L) was used to determine specific
binding.
Results
The immunohistochemical detection of receptor-specific protein allows the anatomic localization of this receptor to be
defined precisely. The synthetic peptides that were used as
immunogens in the production of antisera to the D3 receptor
were specifically chosen because of their predicted antigenicity and uniqueness in terms of absence of sequence homology
with other known proteins, including all receptors in any
species (Sequence Similarity Search, National Center for
Biotechnology Information, National Institutes of Health).26
The antibodies, which were directed to epitopes on the both
the putative third extracellular and intracellular loops of the
rat D3 receptor, produced specific renal staining.
Specificity of the antisera to D3 receptor protein was
validated by light microscopic immunohistochemistry studies
of the LTK2 cells permanently transfected with the full-
Figure 4. Low-power light photomicrographs of rat renal cortical tissue demonstrating positive immunostaining for D3 receptor. A, Positive staining was present in glomeruli (arrowheads), proximal tubules (arrows), and distal tubules (open arrows). B, Preadsorption control showing absence of staining. C, Positive immunostaining was confined to the cortex but absent in the medulla. Original magnification 3160.
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Figure 5. A, Immunostaining in proximal tubules
(arrows) and distal tubules (open arrows) was confined to the apical portions of the tubular cells. B,
Preadsorption control showed absence of staining. C, High-power view of immunostaining in
proximal tubules. The intensity of staining was
localized to the brush border of tubular cells. D,
Preadsorption control showed absence of staining. G indicates glomeruli. Original magnification:
A and B, 3320; C and D, 3640.
length cDNA of the D2 or D3 receptor, as previously described.17 D3 receptor immunoreactivity was detected in the
cytoplasm and cell membranes of the transfected cells (Figure
2A). Immunostaining was absent in the nontransfected LTK2
parent cell line (Figure 2B), in D3 receptor transfected cells
treated with preimmune serum, and in LTK2 cells transfected
with the D2 receptor. These results confirm that antipeptide
antiserum to the D3 receptor specifically recognizes the
appropriate peptide antigen expressed in both experimental
tissue and transfected cells. Our controls confirm that the
staining reaction is not an artifact created by the manner of
tissue processing or false-positive reactions produced in the
rabbit sera before the inoculation with KLH-conjugated
peptide.
Figure 3 shows a Western blot analysis of the D3 receptor
protein in D3 receptor infected and noninfected Sf 9 cell
membranes, D2 or D3 receptor transfected and nontransfected
LTK2 cells, and adult rat kidney and brain. A band of the
appropriate predicted size ('57 kDa) for the D3 receptor was
detected in D3 receptor infected Sf 9 cell membranes (lane 4)
and in D3 receptor transfected LTK2 cells (lane 7), but not in
noninfected cell membranes (lane 3) or in nontransfected
LTK2 cells (lane 6). A specific band of the same molecular
weight was detected in the rat kidney (lane 2) and brain (lane
1). No band was detected in D2 receptor transfected LTK2
cells (lane 5). For the infected Sf 9 cell membranes, D3
receptor binding density was 7.7 pmol/mg protein and for the
transfected LTK2 cells, the receptor density was 1.4 pmol/mg
protein.
Light microscopic immunohistochemical staining for the
D3 receptor was pronounced in the renal cortex. Staining was
present in the proximal tubules (PT) and distal tubules (DT)
(Figure 4A), the cortical collecting ducts (CCD), glomeruli,
and renal parenchymal vasculature (Figure 4A). Staining was
present in the cortex, but there was a line of demarcation with
absence of staining in the medulla (Figure 4C). In PT and DT
(Figure 5A and 5C) epithelia, immunostaining was observed
in discrete tubular cells exclusively in the apical portion of
these cells. Renal arterial staining was prominent in the
medial and adventitial layers (Figure 6A). Consecutive sec-
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Figure 6. A, High-power magnification of renal artery showing
all 3 layers: adventitia (AD), media (M), and intima (I). The immunostaining was most pronounced in the media. B, Low-power
magnification of immunostaining in rat nucleus accumbens
(asterisk) showing uniform positive staining for D3 receptor.
Original magnification: A, 31600; B, 3160.
tions processed with preimmune serum, normal rabbit serum
at the same dilution as the anti-D3 receptor serum, or immune
serum preadsorbed against the pure peptide immunogen did
not produce renal staining, whereas preadsorption of the
antibody against the conjugated carrier protein KLH did not
alter the immunohistochemical staining patterns described
previously. Figure 4B and 4D and Figure 5B and 5D are
typical examples of the renal cortex exposed to preadsorbed
antisera.
The distribution of D3 antisera immunostaining also was
examined in rat brain sections processed for light microscopic
immunohistochemistry. Positive staining was obtained in the
nucleus accumbens, islands of Calleja, and olfactory tubercle.
These regions of the brain are known to express the D3
receptor. Figure 6B shows an example of discrete positive
immunostaining in the nucleus accumbens.
Figure 7 shows heavy D3 receptor immunostaining in the
CCD. Figure 8 depicts D3 receptor immunostaining in the
glomerulus. Figure 8A and 8B depicts the heavy staining
pattern of the glomerulus, in which glomerular podocytes are
clearly labeled. Figure 8C demonstrates light staining in the
macula densa and absence of staining in the juxtaglomerular
cells of the afferent arteriole; Figure 8D is a preadsorption
control and shows no D3 receptor labeling.
November 1998
891
Figure 7. Light photomicrographs of the rat renal cortex depicting heavy immunostaining for the D3 receptor in the cortical collecting ducts (A) and absence of staining in a preimmune control
(B). Original magnification 3640.
Electron microscopic immunocytochemistry, as demonstrated by presence of 10-nm protein-A gold particles, further
defined the intracellular sites of expression of renal D3
receptor protein. Glomerular D3 receptor immunoreactivity
was confined to glomerular podocytes (Figure 9A). Staining
was present not only on the membranes of podocytes but also
on vesicles budding off these membranes, in the intracellular
space, and at the attachment of the podocytes to the foot
processes. In the PT, staining was membrane-bound in the
apical region of the PT cells (Figure 10A). Control sections
from identical cortical regions incubated with serum preadsorbed with D3 receptor immunogen demonstrated minimal
background staining (Figures 9B and 10B). In the CCD,
staining was present on the apical membranes of intercalated
cells (Figure 10C). Control sections stained with preimmune
serum, or immune serum preadsorbed with immunogen, had
no such immunogold labeling (Figure 10D). In the renal
artery, electron microscopic immunocytochemistry revealed
immunogold particles located predominantly in arteriolar
smooth muscle cells (Figure 9C). In the DT, D3 receptor
signal also was present on the apical membranes (data not
shown). In contrast with the D1A receptor,9 D3 receptor
immunoreactivity was not evident in the juxtaglomerular
cells (Figure 9D).
A renal staining pattern similar to that with the D3 receptor
antibody directed toward the third extracellular loop of the
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Renal D3 Dopamine Receptor
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Figure 8. A and B, Light photomicrographs of a
glomerulus in the rat renal cortex showing heavy
immunostaining for the D3 receptor. Panel B
shows uniform staining in a podocyte (arrowhead).
C, High-power magnification of the juxtaglomerular
apparatus, which demonstrated absence of staining in the juxtaglomerular cells (arrows) and light
staining in the macula densa (MD). D, Preadsorption control showing absence of staining. Original
magnification: A, 3640; B through D, 31600.
receptor was obtained with antibody directed to the third
intracellular domain of the receptor (data not shown).
Discussion
The dopamine D3 receptor was the second receptor of the
D2-like family to be identified and cloned. Using probes
derived from the D2 receptor sequence, the rat D3 receptor
was isolated by screening cDNA and genomic libraries with
a combination of reverse transcription and polymerase chain
reaction (RT-PCR). An amino acid sequence of 446 amino
acids was deduced from the open reading frame of the clone.5
The cellular expression of the mRNA encoding the D3
receptor has been mapped in the rat brain by in situ hybridization histochemistry. Highest levels of expression are observed in the caudate putamen, nucleus accumbens, islands of
Calleja, hypothalamus, and olfactory tubercle. Although these
central areas contain both D3 and D2 receptor mRNA, there is
no overlap of D3 receptor with D2 receptor mRNA at the
cellular level, suggesting discrete receptor expression. This
pattern of distribution has led to speculation that the D3
receptor subtype may mediate dopaminergic control of cog-
nitive and emotional function in the brain.5 In this regard, it is
interesting to note that pharmacological studies have demonstrated dopamine as having a 20-fold higher affinity at the D3
than the D2 receptor.5
Peripheral mapping of the D3 receptor has been very
limited. Sokoloff et al5 reported low copy expression of D3
receptor mRNA in kidney as revealed by Northern blotting.
More recently, Gao et al16 demonstrated kidney D3 receptor
mRNA expression using RT-PCR from isolated mRNA. As
these authors pointed out, however, this technique precludes
precise anatomic localization and may also suffer from
cross-contamination of mRNA from different nephron elements. Barili et al27 localized a putative D3 receptor to renal
glomeruli and PT and DT by 7-OH-DPAT binding. However,
7-OH-DPAT also interacts with the D2 receptors, albeit at
higher concentrations than required for D3 receptor binding.
The present study is the first to our knowledge to define the
site-specific localization of D3 receptor protein in whole
kidney sections and corroborates and extends the previous
reports that have suggested the renal presence of the D3
receptor. Moreover, the sites of D3 receptor protein as
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Figure 9. Electron micrographs demonstrating D3 receptor immunostaining. A,
Glomerular podocyte and foot processes
showing 10-nm gold staining (arrows) in
glomerular podocyte (P). B, Preadsorbed
control showing absence of staining in
podocyte. C, Immunoreactivity in vascular smooth muscle cells (arrows) from a
renal cortical artery. D, The absence of
immunoreactivity is evident in renincontaining granules (G) of the juxtaglomerular cells, a site previously noted to
contain D1A receptor immunoreactivity.17
Original magnification: A, 365 000; B
and C, 331 500; D, 320 000.
demonstrated in the present study correspond closely to renal
D2-like receptor binding sites, including glomeruli, PT, and
renal arteries, as previously defined by pharmacological
binding, biochemical, and functional studies.5,11–13,16,27 Use of
these studies to define the localization of receptor subtypes is
made difficult by the relative inability of available D2-like
receptor ligands to distinguish between the D2-like receptor
subtypes. Such difficulties are avoided by the use of receptorspecific antibodies.
One significant difference between our results and those in
the literature is the cellular distribution of the D3 receptor. A
radioligand-binding study showed the presence of D2-like
receptors in both brush border and basolateral membranes of
the canine cortical tubules.11 Our study shows only apical
binding in these cells. Also, Huo et al13 described a D2-like
(DA2K) receptor in the rat renal medulla, whereas in our study
the D3 receptor was absent in the medulla. The identity of the
DA2K receptor and its relationship to the D3 receptor are
unclear.
The specificity of the antibody staining demonstrated in the
present study was confirmed by the use of appropriate
controls (including preadsorption with the pure peptide im-
munogen) in the immunostaining procedures and by the
results of the D2 and D3 receptor transfection/infection studies
in which only the D3 receptor transfected cells or the infected
cell membranes were labeled by immunocytochemistry
and/or Western blot analysis. For Western blot analysis, a
band of appropriate predicted size ('57 kDa) was identified
in the D3 receptor infected Sf 9 cell membranes and in D3
receptor transfected cells. This band was similar to that
reported by Boundy et al.21 The staining patterns were
identical for the 2 different antisera (directed against both
extracellular and intracellular domains) used in the studies of
both native tissue and transfected cells, thereby excluding a
nonspecific or spurious signal. In the kidney, a single band of
approximately 57 kDa was detected by Western blot. D3
receptor immunostaining in the nucleus accumbens of the
brain and a specific D3 receptor band on Western blot from
the brain served as positive controls.
Quinpirole (LY 171555) is a dopamine-2 receptor agonist
that has been used in radioligand and functional studies.
Quinpirole has a KiD2/KiD3 ratio of 113 in the rat and thus is
relatively selective for the D3 receptor.28 Previous experiments conducted by our research group in a conscious
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Figure 10. Electron micrographs demonstrating D3 receptor immunostaining.
A, Apical brush border of proximal
tubule showing immunostaining in villi
(V). B, Preadsorption control for A. C,
Positive immunostaining in apical region
of an intercalated cell of the cortical collecting duct. D, Preadsorption control for
C. Original magnification: A and B,
363 750; C and D, 375 600.
experimental animal model have demonstrated that intrarenal
infusion of quinpirole produced antidiuresis, antinatriuresis,
and a decrease in renal hemodynamic function.29 Other
studies using quinpirole have revealed changes in renal blood
flow and superficial nephron glomerular filtration rate.30 The
present study confirms the presence of the D3 receptor in
renal tubular, glomerular, and vascular sites, supporting the
possible role of this dopamine receptor in modulating renal
function.
The distribution of the D3 receptor differs significantly
from the distribution of the D1A receptor as previously
reported by our group.9,17 In the glomerulus, strong D3
receptor immunoreactivity was demonstrated and localized to
podocytes. No staining was observed in mesangial cells. In
the case of the D1A receptor, no glomerular immunoreactivity
was detected. Whereas the D1A receptor had a strong immunoreactive signal in the juxtaglomerular cells, the D3 receptor
has no detectable signal in these cells in situ. We have
previously reported the presence of D3 receptor mRNA in rat
juxtaglomerular cells in primary culture.31 The reasons for the
discrepancy between the present results and our previous
study are unclear. One possibility is that the primary juxtaglo-
merular cell culture has up to 5% contamination with renal
tubular cells, and we may have been detecting D3 receptor
mRNA in these contaminating cells. Another possibility is
that our immunohistochemical technique is not sensitive
enough to detect the D3 receptor signal, which is present in
low copy, while PCR can detect the message. A third
possibility is that the expression of the D3 receptor increases
with culture.
With respect to the subcellular distribution of the D3
receptor, while the D1A receptor had both an apical and a
basolateral localization in PT and DT cells, the D3 receptor
labeling was confined to the apical region of these tubular
cells. Whereas the D1A receptor labeling in the CCD was
relatively weak, CCD labeling of the D3 receptor is comparatively strong. Immunoreactivity for both of these subtypes
was evident in vascular smooth muscle cells in different
orders of renal vessels. These differences conform to the
respective differences in the distribution of D1- and D2-like
receptors using older classification systems. Furthermore, the
presence of both D1A and D3 receptors in similar locations
provides morphological support for recent experimental evidence of an obligatory synergistic role of D1-like and D2-like
O’Connell et al
receptor agonism in the inhibition of Na1/K1-ATPase activity
in the proximal tubule.32
As described previously for the D1A receptor in heart and
kidney,9,17 the present study identified the D3 receptor in cell
membranes, vesicles budding off of membranes, and in the
cytoplasm of renal cells. The function of intracellular receptors is not clear, but their presence there may signify a
receptor cycling mechanism in which receptors may be
internalized and subsequently reinserted into the cell membrane. Further work is required to elucidate this possibility.
In conclusion, the dopamine D3 receptor protein, previously localized only to the central nervous system, is present
in the rat kidney in sites previously labeled as D2-like. The
distribution of the D3 receptor is distinctively different from
that of the D1A receptor. The results suggest that at least some
of the peripheral dopamine D2-like receptors correspond
structurally to the central dopamine D3 receptor.
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Acknowledgment
We gratefully acknowledge the expert assistance of Ikuyo Yamaguchi
who provided the transfected cells used in the present study.
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Expression of the Dopamine D3 Receptor Protein in the Rat Kidney
Damian P. O'Connell, Carl J. Vaughan, Anna M. Aherne, Suzanne J. Botkin, Zhi-Qin Wang,
Robin A. Felder and Robert M. Carey
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Hypertension. 1998;32:886-895
doi: 10.1161/01.HYP.32.5.886
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