AJH 2000;13:673– 677
Type 1A Dopamine Receptor Expression in
the Heart Is Not Altered in Spontaneously
Hypertensive Rats
Toshiyuki Matsumoto, Ryoji Ozono, Nobuo Sasaki, Tetsuya Oshima, Hideo Matsuura,
Goro Kajiyama, Robert M. Carey, and Masayuki Kambe
We have recently demonstrated that type 1A
dopamine (D1A) receptor is expressed in the rat
heart, but its function still remains unknown. In
the present study, we investigated possible
changes in the expression level and the
distribution of the cardiac D1A receptor in the
development of left ventricular hypertrophy in
spontaneously hypertensive rats/Izumo strain
(SHR/Izm) at the ages of 4, 8, and 20 weeks. We
examined D1A receptor protein distribution by
immunohistochemistry and gene expression by
competitive polymerase chain reaction (competitive
PCR). In SHR/Izm, compared with the age-matched
Wistar Kyoto rats/ Izmo strain (WKY/Izm), blood
pressure and heart/body weight ratio were
significantly increased at 8 and 20 weeks. By
immunohistochemistry, the D1A receptor was
localized in cardiomyocytes and vascular smooth
muscle cells of coronary arteries, but not in
interstitial fibrotic tissue. D1A receptor distribution
was not changed either by the strain or the age.
D
opamine, a well-known neurotransmitter, is
also recognized as an important modulator
of renal function through independent local
dopaminergic system.1 Dopamine receptors
are pharmacologically classified into two classes: D1-like
Received July 9, 1999. Accepted October 28, 1999.
From the Department of Clinical Laboratory Medicine (TM, RO,
TO, MK) and First Department of Internal Medicine (NS, HM, GK),
Hiroshima University School of Medicine, Hiroshima, Japan; De-
partment of Internal Medicine, University of Virginia Health Sci-
ences Center (RMC), Charlottesville, Virginia.
© 2000 by the American Journal of Hypertension, Ltd.
Published by Elsevier Science, Inc.
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Competitive PCR analysis showed that the D1A
receptor mRNA level was significantly higher at 4
weeks than at 8 and 20 weeks in both strains of
rats and that there was no significant difference in
D1A receptor mRNA between SHR/Izm and WKY/
Izm at any age ( 43.2 ⴞ 10.4 attomol ⴛ 10ⴚ3/L v
43.1 ⴞ 11.2 attomol ⴛ 10ⴚ3/L at 4 weeks, P ⴝ not
significant, 3.9 ⴞ 0.9 attomol ⴛ 10ⴚ3/L v 4.0 ⴞ 1.3
attomol ⴛ 10ⴚ3/L at 8 weeks, P ⴝ not significant,
3.0 ⴞ 1.2 attomol ⴛ 10ⴚ3/L v 1.9 ⴞ 1.6 attomol ⴛ
10ⴚ3/L at 20 weeks, P ⴝ not significant). These
results do not support the hypothesis that changes
in D1A receptor expression are associated with the
development of left ventricular hypertrophy in
SHR. Am J Hypertens 2000;13:673– 677 © 2000
American Journal of Hypertension, Ltd.
KEY WORDS: Dopamine 1A receptor, spontaneously
hypertensive rats, immunohistochemistry,
competitive polymerase chain reaction, left
ventricular hypertrophy.
receptors stimulate, but D2-like receptors inhibit ade-
nylyl cyclase. These classes are further divided by dif-
ference in the molecular sequences; the D1-like receptor
family includes D1A and D1B receptors and the D2-like
receptors D2, D3, and D4 in the rats.
This study was supported by a Grant-in Aid for Scientific Re-
search (No. 08457639 and No. 07407065).
Address correspondence and reprint requests to Toshiyuki Mat-
sumoto, Department of Clinical Laboratory Medicine, Hiroshima
University School of Medicine, 1-2-3 Kasumi, Minami-ku, Hiro-
shima, Japan 734-8551; e-mail: fwga3144@mb.infoweb.ne.jp
0895-7061/00/$20.00
PII S0895-7061(99)00270-8
674 MATSUMOTO ET AL
Our group2,3 has demonstrated that both the mRNA
and protein of D1A receptor are expressed in cardio-
myocytes and coronary arteries in rats and humans.
However, the functional role of the D1A receptors
remains unclear. Recently Pravenec et al4 have con-
ducted linkage analysis in a recombinant inbred strain
derived from spontaneously hypertensive rats (SHR)
and normotensive rats to map the quantitative trait
loci determining cardiac mass and blood pressure.
These investigators have reported that the genetic
marker for the D1A receptor on chromosome 17, inde-
pendently of blood pressure, was most tightly associ-
ated with the cardiac weight. However, it is not
known from these linkage analysis studies whether
the gene associated with the mechanism of left ven-
tricular hypertrophy is that of D1A receptor per se or
other genes nearby the D1A receptor locus.
In the present study, using a combination of immu-
nohistochemistry and competitive PCR, we investi-
gated the expression of the D1A receptor in left ven-
tricular hypertrophy in SHR to gain insight into the
relationship between the cardiac D1A receptor and left
ventricular hypertrophy.
METHODS
Animals SHR/Izmo strain (SHR/Izm) and their
normotensive controls, Wistar-Kyoto rats/Izmo strain
(WKY/Izm), at ages of 4, 8, and 20 weeks (n ⫽ 9 each
age and strain), were purchased from Disease Model
Cooperative Research Association (Kyoto, Japan). Sys-
tolic blood pressure was measured by the tail– cuff
method as described previously.5 Animals were
deeply anesthetized with pentobarbital sodium and
the hearts were freshly removed and weighed. The
heart weight (in milligrams) divided by the body
weight (in grams) was considered as a measure of
ventricular hypertrophy.
Histologic Analysis For histologic demonstration of
cardiac hypertrophy and remodeling, freshly removed
ventricles from 8- and 20-week-old SHR/Izm and
WKY/Izm were immersion fixed in phosphate buff-
ered 10% formaldehyde solution and embedded in
paraffin.6 Two-micrometer sections were cut and pro-
cessed according to Masson’s trichrome staining pro-
tocol.
Immunohistochemistry Immunohistochemistry for
the D1A receptor in rat hearts was performed using a
polyclonal rabbit antiserum against the synthetic pep-
tide (GSEETQPFC) corresponding to the third extra-
cellular domain of D1A receptor as previously de-
scribed.2 The selectivity of the antibody was fully
evaluated and published elsewhere.7
RNA Extraction Total RNA was isolated from the
tissue homogenates as described by Chomczynski and
AJH–JUNE 2000 –VOL. 13, NO. 6, PART 1
Sacchi8 using Trizol reagent (GIBCO BRL Life Technol-
ogies Inc.). The RNA was treated with DNase I (GIBCO
BRL) for 15 min at room temperature. The amount of
RNA was quantified by spectrophotometry.
Reverse Transcriptase Polymerase Chain Reaction of
D1A Receptors Reverse transcriptase polymerase
chain reaction (RT-PCR) was performed with a Gene
Amp RNA PCR kit (Perkin-Elmer Corp.) as previously
described.2,3,9 The D1A receptor was amplified with
sense primer 5⬘-GGCTAAGCCTGGTCAAGAAC-3⬘
(nucleotides 1 to 20), and antisense primer 5⬘-
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AGAGATGACAAAGAAGTTG-3⬘ (nucleotides 233 to
251). The oligonucleotide sequence of the PCR product
was analyzed by sequencer. (Auto sequencer, ABI 377
by Taq cycle method performed by TaKaRa Co.).
Competitive PCR For competitive PCR, we synthe-
thized DNA internal standard using PCR MIMIC Con-
struction Kit (CLONTECH) according to the manufac-
turer’s instruction.10 Briefly, the oligonucleotide
extensions that have the sequence complimentary to
the D1A primers were added to both ends of the DNA
fragment of erb gene (MIMIC DNA fragment, sup-
plied in the kit) by a two-step PCR amplification. The
MIMIC DNA fragment was nonhomologous to the
D1A receptor gene except for the primer portion lo-
cated on both ends and 450 bp in length. However, the
amplification efficiency of the D1A receptor and
MIMIC DNA fragment was similar. We used this
DNA fragment as a competitive template (CT) for D1A
receptor cDNA amplification.
After RT of the RNA sample, D1A receptor cDNA
was PCR amplified with the same primer set as pre-
viously in six tubes containing the same amount of the
RT product and one of the following of CT; 0.1, 1.0, 10,
100, 1000, 10000 attomol/mL (final concentrations).
Then, we determined the concentration of CT that
produced CT and target cDNA template bands of
equal intensity. Subsequently, for more precise quan-
tification, we set up a fine-tuned serial dilution of CT
(two-fold) and repeated the same procedure. The re-
action mixture was incubated at 95°C for 9 min (initial
melt), followed by 35 cycles of 95°C for 45 sec and
49°C for 45 sec. PCR was completed by a final exten-
sion at 72°C for 7 min. Analysis of serially diluted
samples confirmed that our competitive PCR is sensi-
tive to a minimum twofold difference in the D1A re-
ceptor cDNA.
Statistical Analysis All results are expressed as
mean ⫾ SD. Statistical analysis was performed with
the use of a statistical software package (Statview,
Abacus). Changes in parameters within one strain of
rats with different ages and comparisons between
SHR/Izm and WKY/Izm were analyzed by a two-
AJH–JUNE 2000 –VOL. 13, NO. 6, PART 1 D1A IN SHR HEART 675

TABLE 1. BODY WEIGHT (BW), HEART WEIGHT (HW), SYSTOLIC BLOOD PRESSURE (SBP), AND HEART
RATE (HR) IN 4-, 8-, AND 20-WEEK-OLD SHR AND WKY

4 Weeks 8 Weeks 20 Weeks

SHR WKY SHR WKY SHR WKY

BW (g) 110 ⫾ 8 108 ⫾ 10 219 ⫾ 5 242 ⫾ 7 368 ⫾ 9* 422 ⫾ 14

HW (mg) 434 ⫾ 8 389 ⫾ 10 757 ⫾ 10* 696 ⫾ 9 1501 ⫾ 18* 1216 ⫾ 48
HW/BW 4.1 ⫾ 0.2 3.5 ⫾ 0.3 3.6 ⫾ 0.1* 3.1 ⫾ 0.1 4.0 ⫾ 0.2* 3.0 ⫾ 0.2
SBP (mm Hg) 120 ⫾ 2 114 ⫾ 5 159 ⫾ 2* 114 ⫾ 4 205 ⫾ 4* 127 ⫾ 6
HR (beats/min) 337 ⫾ 6 401 ⫾ 10 380 ⫾ 9 372 ⫾ 5 394 ⫾ 8 370 ⫾ 10
* P ⬍ .01 v WKY at the same age and baseline.

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way ANOVA and the Scheffé’s F test. A value of P ⬍ in SHR/Izm and WHY/Izm, suggesting that the D1A
.05 was accepted as statistically significant. receptor is age-dependently regulated. However,
there was no significant difference in D1A receptor
RESULTS
expression between SHR/Izm and WHY/Izm at any
Hypertension, Cardiac Hypertrophy, and Remodel- age (Figure 2; SHR/Izm v WHY/Izm: 43.2 ⫾ 10.4 v
ing in SHR/Izm As shown in Table 1, systolic blood 43.1 ⫾ 11.2 at 4 weeks, P ⫽ not significant; 3.9 ⫾ 0.9 v
pressure and the ratio of heart weight to body weight 4.0 ⫾ 1.3 at 8 weeks, P ⫽ not significant; 3.0 ⫾ 1.2 v
were significantly increased in 8- and 20-week-old 1.9 ⫾ 1.6 at 20 weeks, P ⫽ not significant; all units in
SHR/Izm compared with their age-matched WKY/ attomol ⫻ 10⫺3/L).
Izm controls, whereas no difference was detected in
DISCUSSION
4-week-old animals. Histologically, the ventricles of 8-
and 20-week-old SHR/Izm showed an increased We studied the cardiac expression of D1A receptor
cross-sectional diameter of cardiomyocyte. Interstitial during the development of left ventricular hypertro-
and perivascular fibrosis and thickening of the vascu- phy in SHR/Izm. The SHR/Izm at ages of 8 and 20
lar smooth muscle cell layer of coronary arteries were weeks, but not at 4 weeks, showed an increased car-
evident only in ventricles of 20-week-old SHR/Izm diac mass and histologic signs of cardiac remodeling
(data not shown). compared with the age-matched WKY/Izm. Consis-
tent with our previous report,2 the gene and protein of
Light Microscopic Immunohistochemistry Light
microscopic immunohistochemical staining for the
D1A receptor was observed in cardiomyocytes and the
vascular smooth muscle layer of the coronary artery in
both SHR/Izm and WKY/Izm at all ages. The myo-
cardial staining was homogeneous. There was no dif-
ference in this distribution pattern of the staining be-
tween SHR/Izm and WHY/Izm at any age (Figure 1).
The preadsorption and preimmune serum controls for
all of these immunohistochemical studies were nega-
tive.
RT-PCR An amplification product of the predicted
size (251 bp) for the D1A receptor was identified in the
brain and ventricles of SHR/Izm and WKY/Izm at all
ages. No band was observed when PCR was per-
formed without RT. Analysis of the oligonucleotide
sequence showed that the PCR product was 100%
identical to oligonucleotides 1 to 251 of known rat D1A FIGURE 1. Light microscopic immunohistochemical staining
receptor cDNA (data not shown). for the D1A receptor in left ventricles of SHR/Izm (A, B) and
WKY/Izm (C, D) at 16 weeks of age. Positive staining of D1A is
Quantification of D1A receptor cDNA by Competi- observed in A and C. Positive staining is observed in coronary
tive PCR The ventricular D1A receptor cDNA was artery and cardiomyocyte (), but not in fibrous tissues (2).
quantified by competitive PCR. The D1A receptor ex- There is no difference in the distribution pattern of the positive
pression was significantly (P ⬍ .001) higher in 4-week- staining between SHR/Izm and WKY/Izm. B and D are before the
old animals than in 8- and 20-week-old animals both adminisration of immune serum.
676 MATSUMOTO ET AL
FIGURE 2. Bar graph showing the D1A receptor
expression in 4-, 8-, and 20-week-old SHR/Izm and
WKY/Izm quantified by competitive PCR. The D1A
receptor expression was markedly and significantly
decreased at 8 and 20 weeks compared with 4 weeks
(P ⬍ .001), whereas no significant difference be-
tween SHR/Izm and WKY/Izm was detected at any
age. *P ⬍ .001 v 4-week-old SHR or WKY. The
difference was analyzed by two-way ANOVA.
the D1A receptor are expressed in cardiomyocytes and
coronary arteries of both SHR/Izm and WKY/Izm.
However, we could not detect the expected difference
in the D1A receptor gene expression and the D1A re-
ceptor localization between the two strains of rats at
any ages of 4, 8, and 20 weeks, covering both early and
established phases of cardiac hypertrophy. Our data
do not support the hypothesis that changes in D1A
receptor expression or its localization is associated
with the development of left ventricular hypertrophy
in SHR/Izm.
In a gene mapping study using recombinant inbred
strain4 between SHR and Brown Norway rats, it has
been suggested that a chromosomal region containing
D1A receptor locus is tightly associated with blood
pressure and cardiac mass. In the present study, how-
ever, we could not detect any difference in the D1A
receptor mRNA expression between SHR/Izm and
WKY/Izm, indicating that the sequences of 5⬘-flank-
ing region and other noncoding regions controlling
the amount of D1A receptor mRNA may not be differ-
ent. It has also been found that there was no difference
in the coding region of the D1A receptor gene between
the two strains (Robin Felder, University of Virginia,
personal communication). Taken together, the D1A re-
ceptor gene, including both coding and noncoding
regions, is intact in SHR/Izm. The linkage analysis
may indicate that there is another gene strongly asso-
ciated with the cardiac hypertrophy near the D1A re-
ceptor locus. Our negative results could be attributed
to the fact that we used WKY rather than Brown
Norway rats. Additional investigations using several
different strains of rats may be necessary to address
this question.
Although our study failed to demonstrate the dif-
ference in the D1A receptor expression itself, it does
not exclude the possibility that the cardiac D1A recep-
AJH–JUNE 2000 –VOL. 13, NO. 6, PART 1
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tor is involved in the mechanism of left ventricular
hypertrophy. This is because many other molecules
other than D1A receptor influence the overall function
of D1A receptor. For example, the D1A receptor adeny-
lyl cyclase coupling is impaired without affecting the
receptor number.11 A recent study12 indicates that G
protein-coupled receptor kinase is important to deter-
mine D1A receptor responsiveness. Ganguly et al13
have previously reported that an increase in cardiac
mass in rats with aortic coarctation was attenuated by
systemic administration of SCH-23390, a D1-like re-
ceptor antagonist, indicating that the D1-like receptors
are playing a role to promote cardiomyocyte hyper-
trophy.
It is interesting that the expression level of the D1A
receptor decreased with age. A high level of receptor
expression in fetal or early stages of life and the de-
crease with the maturation is a characteristic feature of
genes associated with cell differentiation and matura-
tion.14 A number of receptors, including some sub-
types of the dopamine receptor family,15 are develop-
mentally regulated. It is also common that such
developmentally regulated receptors are reexpressed
in response to various stimuli. In the future the rela-
tionship between the D1A receptor and cardiac matu-
ration should be clarified.
In summary, we studied the cardiac D1A receptor
expression in the development of cardiac hypertrophy
in SHR/Izm. We could detect no difference in the
distribution or expression level of the D1A receptor in
the heart in comparison with WKY/Izm. These results
do not support the hypothesis that change in D1A
receptor expression is associated with the develop-
ment of left ventricular hypertrophy in SHR. How-
ever, further investigation on the function of D1A re-
ceptor is needed to determine whether the D1A
AJH–JUNE 2000 –VOL. 13, NO. 6, PART 1
receptor is involved in the mechanism stimulating left
ventricular hypertrophy.
REFERENCES
1. Jose PA, Eisner GM, Drago J, et al: Dopamine receptor
signaling defects in spontaneous hypertension. Am J
Hypertens 1996;9:400 – 405.
2. Ozono R, O’Connell DP, Vaughan C, et al: Expression
of the subtype 1A dopamine receptor in the rat heart.
Hypertension 1996;27:693–703.
3. Ozono R, O’Connell DP, Wang ZQ, et al: Localization
of the dopamine D1 receptor protein in the human
heart and kidney. Hypertension 1997;30:725–729.
4. Pravenec M, Gauguier D, Schott JJ, et al: Mapping of
quantitative trait loci for blood pressure and cardiac
mass in the rat by genome scanning of recombinant
inbred strains. J Clin Invest 1995;96:1973–1978.
5. Ono N, Oshima T, Ishida M, et al: Platelet Ca2⫹ is not
increased in stroke-prone spontaneously hypertensive
rats: comparative study with spontaneously hyperten-
sive rats. Hypertension 1996;27:1312–1317.
6. Ozono R, Wang ZQ, Moore AF, et al: Expression of the
subtype 2 angiotensin (AT2) receptor protein in rat
kidney. Hypertension 1997;30:1238 –1246.
7. O’Connell DP, Botkin SJ, Ramos SI, et al: Localization
of dopamine D1A receptor protein in rat kidneys. Am J
Physiol 1995;268:F1185–F1197.
D1A IN SHR HEART 677
8. Chomczynski P and Sacchi N: Single-step method of
RNA isolation by acid guanidinium thiocyanate- phe-
nol-chloroform extraction. Anal Biochem 1987;162:156 –
159.
9. Yamaguchi I, Jose PA, Mouradian MM, et al: Expres-
sion of dopamine D1A receptor gene in proximal tubule
of rat kidneys. Am J Physiol 1993;264:F280 –F285.
10. Nedelman J, Heagerty P, Lawrence C: Quantitative
PCR with internal controls. Comput Appl Biosci 1992;
8:65–70.
11. Ohbu K, Hendley ED, Yamaguchi I, et al: Renal dopa-
mine-1 receptors in hypertensive inbred rat strains
with and without hyperactivity. Hypertension 1993;21:
485– 490.
Downloaded from https://academic.oup.com/ajh/article/13/6/673/186768 by guest on 08 November 2022
12. Tiberi M, Nash SR, Bertrand L, et al: Differential regu-
lation of dopamine D1A receptor responsiveness by
various G protein-coupled receptor kinases. J Biol
Chem 1996;271:3771–3778.
13. Ganguly PK, Mukherjee K, Sahai A: Renal dopamine
receptors are involved in the development of cardiac
hypertrophy. Mol Cell Biochem 1995;144:81– 84.
14. Shanmugam S, Corvol P, Gasc JM: Angiotensin II type
2 receptor mRNA expression in the developing cardio-
pulmonary system of the rat. Hypertension 1996;28:91–
97.
15. Fishburn CS, Bedford M, Lonai P, et al: Early expres-
sion of D3 dopamine receptors in murine embryonic
development. FEBS Lett 1996;381:257–261.