(Hypertension. 1998;32:886-895.)
© 1998 American Heart Association, Inc.
Scientific Contributions |
Expression of the Dopamine D3 Receptor Protein in the Rat Kidney
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.
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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 LTK- 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 LTK- cells, and in kidney and brain but not in noninfected Sf9 cell membranes or in D2 or D3 receptor transfected or nontransfected LTK- 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.
Key Words: dopamine • receptor, D3 • kidney • immunocytochemistry • glomerulus
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Introduction |
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The 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 pharmacological 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 11 12 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 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 17 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.
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Methods |
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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
288QPPSPGQTHGGLKR301 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 LTK- cell line
or infected with a baculovirus expression system into Spodoptera
fragiperda (Sf9) 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.
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Transfected or Infected Cells
A murine fibroblast LTK- 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 LTK-
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
LTK- 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 LTK- cell line.
Cell membranes from Sf9 cells stably infected with the rat D3 receptor and membranes from noninfected Sf9 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 (n=6) 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 µm),
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 avidin-biotin 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 (n=4) 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
-20°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.
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 µg/mL
[
1.5 µmol/L] leupeptin, and 10 µg/mL [1.5
µmol/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 Sf9 cell membranes or D2 or D3 receptor transfected or nontransfected LTK- 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 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
Transfected and nontransfected LTK- 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 LTK- cell membranes and infected
or noninfected Sf9 cell membranes was measured by specific
binding of
[125I]iodospiperone.25
Quinpirole (1 µmol/L) was used to determine specific
binding.
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Results |
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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 LTK- cells permanently transfected with
the full-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
LTK- parent cell line (Figure 2B), in
D3 receptor transfected cells treated with
preimmune serum, and in LTK- 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.
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Figure 3 shows a Western blot
analysis of the D3 receptor protein in
D3 receptor infected and noninfected Sf9
cell membranes, D2 or D3
receptor transfected and nontransfected LTK-
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 Sf9
cell membranes (lane 4) and in D3 receptor
transfected LTK- cells (lane 7), but not in
noninfected cell membranes (lane 3) or in nontransfected
LTK- 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 LTK- cells (lane 5). For the
infected Sf9 cell membranes, D3 receptor
binding density was 7.7 pmol/mg protein and for the transfected
LTK- cells, the receptor density was 1.4 pmol/mg
protein.
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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 sections 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.
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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.
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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).
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A renal staining pattern similar to that with the D3 receptor antibody directed toward the third extracellular loop of the receptor was obtained with antibody directed to the third intracellular domain of the receptor (data not shown).
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Discussion |
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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 cognitive 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 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 12 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 receptor-specific 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 immunogen) 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 Sf9 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 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 juxtaglomerular 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 receptor agonism in the inhibition of Na+/K+-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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Acknowledgments |
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We gratefully acknowledge the expert assistance of Ikuyo Yamaguchi who provided the transfected cells used in the present study.
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Footnotes |
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Reprint requests to Dr Robert M. Carey, Box 395, University of Virginia Health Sciences Center, Charlottesville, VA 22908.
Received March 9, 1998; first decision April 1, 1998; accepted July 23, 1998.
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References |
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