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25
Glucocorticoid Increases Angiotensin II Type 1
Receptor and Its Gene Expression
Atsuhisa Sato, Hiromichi Suzuki, Marohito Murakami, Yuichi Nakazato,
Yasushi Iwaita, Takao Saruta
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Abstract Recently, we demonstrated that glucocorticoid
potentiates inositol trisphosphate production evoked by angiotensin II in vascular smooth muscle cells. To clarify this
mechanism, we investigated the effects of dexamethasone on
the modulation of angiotensin II type 1 receptor and on
postreceptor mechanisms in vascular smooth muscle cells. The
number of angiotensin II type 1 receptors began to increase
after 12 hours' incubation with dexamethasone. After 48
hours, the B m value reached 27±3 fmol/mg protein in dexamethasone-treated cells and 15±3 fmol/mg protein in control
cells. However, binding affinity did not change. A glucocorticoid antagonist, RU 38486, completely blocked these effects of
dexamethasone. Also, to elucidate the effects of dexamethasone on postreceptor mechanisms, GTP analogue-induced
inositol trisphosphate production in permeabilized cells was
examined. Pretreatment with 1 ^tmol/L dexamethasone for 48
hours did not affect these inositol trisphosphate productions.
Moreover, dexamethasone had no effect on the level of G,^
protein. Furthermore, steady-state levels of angiotensin II type
1 receptor messenger RNA were increased 2.2±0.3-fold after
30 minutes' exposure to 1 /imol/L dexamethasone and
7.8±0.4-fold after 24 hours. We conclude that glucocorticoid
induced expression of the angiotensin II type 1 receptor gene
and resulted in an increase in the number of angiotensin II
type 1 receptors through the glucocorticoid-specific receptor,
without significant effect on postreceptor systems in vascular
smooth muscle cells. (Hypertension. 1994;23:25-3©.)
Key Words • dexamethasone • postreceptor signaling
systems • RNA, messenger • receptors, angiotensin •
muscle, smooth, vascular
H
modulates the function of G proteins in several other
kinds of cells.11'3
As to Ang II receptors, the development of nonpeptide Ang II antagonists has allowed the identification of
at least two different subtypes, Ang II type 1 (AT,)
receptor and Ang II type 2 (AT2) receptor.14-15 Recently, Murphy et al16 succeeded in complementary
DNA (cDNA) cloning of AT, receptor from rat
VSMCs. These significant advances have revealed that
the actions of Ang II on VSMCs are mediated primarily
through the AT! receptor.
In the present study, to clarify the mechanism for
glucocorticoid-enhanced Ang II receptor-mediated responses in VSMCs, the effect of glucocorticoid on AT,
receptor expression and the transcription of AT, receptor gene in VSMCs were examined. Furthermore, the
effect of glucocorticoid on postreceptor signaling systems in VSMCs was studied.
ypertension induced by glucocorticoid hormone
is not uncommon in clinical practice.1 Although the exact mechanisms of glucocorticoid-induced hypertension are still uncertain, enhancement of vascular responsiveness has been considered
one of the major contributing factors.2-3 Previously, we
demonstrated that the pressor responses to angiotensin
II (Ang II) and riorepinephrine are enhanced in patients with Cushing's syndrome4 and experimental models with glucocorticoid-induced hypertension.55
Very recently, we found that glucocorticoid potentiates inositol trisphosphate (IP3) production evoked by
Ang II in cultured vascular smooth muscle cells
(VSMCs),7 and these results showed that glucocorticoid
treatment would increase sensitivity to Ang II at the
cellular level. In that study, the action of glucocorticoid
was, in part, through prostaglandin synthesis inhibition;
however, the possibility that glucocorticoid directly
modulates Ang II receptor-mediated signaling pathways in VSMCs is suggested. This, together with other
experimental data, makes it conceivable that glucocorticoid increases transcription and expression of a1Badrenergic receptors8 and ft-adrenergic receptors in
smooth muscle cells910 and that glucocorticoid directly
Received March 26, 1993; accepted in revised form September
13, 1993.
From the Department of Internal Medicine, School of Medicine, Keio University, Tokyo, Japan.
Presented in part at the Council for High Blood Pressure
Research 46th Annual Fall Conference and Scientific Sessions in
Cleveland, Ohio, September 29-October 2, 1992, and published in
abstract form in Hypertension (1992;20:430).
Correspondence to Takao Saruta, MD, Department of Internal
Medicine, School of Medicine, Keio University, 35 Shinanomachi,
Shinjuku-ku, Tokyo, 160, Japan.
Methods
Materials
Reagents for these studies were purchased as follows:
dexamethasone; the peptidase inhibitors antipain, phosphoramidon, leupeptin, pepstatin, bestatin, amastatin, and bacitracin; and bovine serum albumin (BSA) were from Sigma
Chemical Co, St Louis, Mo. The nonpeptide AT, receptor
antagonist DuP 753 was a gift from Dr Smith (Du Pont de
Nemours Co, Wilmington, Del). PD 123319, an AT2 receptor
antagonist, was a gift from Dr Taylor (Parke-Davis Pharmaceutical Research, Warner-Lambert Co, Ann Arbor, Mich).
Ang II was from the Peptide Institute, Osaka, Japan. 125I-Ang
II and anti-G^ protein were from Du Pont NEN Research
Products, Boston, Mass. Fetal calf serum (FCS) was purchased
from GIBCO Laboratories, Grand Island, NY. Dulbecco's
modified Eagle medium (DMEM) and phosphate-buffered
saline (PBS) were from Nissui Pharmaceutical Co, Ltd, Tokyo,
Japan. RU 38486, a glucocorticoid antagonist, was kindly
provided by Roussel Uclaf, Paris, France.
26
Hypertension
Vol 23, No 1 January 1994
Cell Culture
VSMCs were prepared from the thoracic aorta of 6-week-old
Wistar-Kyoto rats by an enzyme method previously described7
and were cultured in DMEM containing 10% FCS, 100 U/mL
penicillin, and 100 /ig/mL streptomycin. The cultures were kept
at 37°C in a humid atmosphere consisting of 5% CO2 in air, and
the medium was changed every 3 or 4 days. After the cells
reached confluence, the medium was changed to culture media
containing various doses of dexamethasone or ethanol vehicle as
a control for various periods of time before the experiments.
Cells between the second and fourth passages were used for the
experiments. The cells had typical smooth muscle morphology,
including the presence of hills and valleys seen under a phasecontrast microscope. Cell viability was estimated by trypan blue
exclusion to be approximately 90%. We used dexamethasone
because of its more potent and pure glucocorticoid action than
the endogenous glucocorticoid.
Radioligand Receptor Binding Assay
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The specific binding of 1HI-Ang II to cell membrane receptors was determined by use of intact VSMCs. Confluent
monolayers of VSMCs grown in 24-well plates were rinsed
twice with PBS, pH 7.4. Equilibrium binding studies were
performed at room temperature for 60 minutes in PBS, pH 7.4,
containing increasing concentrations of 125I-Ang II, 1.6% heatinactivated BSA, and the peptidase inhibitors antipain, phosphoramidon, leupeptin, pepstatin, bestatin, and amastatin,
each at 1 Mg/mL, and bacitracin at 100 ftg/mL. Preliminary
experiments demonstrated that under these conditions, equilibrium was reached. After incubation, the cells were washed
three times with ice-cold PBS, pH 7.4. The attached cells were
dissolved with 1.0 mL of 0.2N NaOH and transferred to
polypropylene tubes. The dishes were rinsed with 1.0 mL of
distilled water, and the rinses were combined with the original
samples. Radioactivity in the dissolved cells was determined in
an autowell gamma system (Aloka Co, Tokyo, Japan). A
portion of the dissolved cells was used for determining the
protein content. Nonspecific binding was determined in the
presence of 10 /tmol/L unlabeled Ang II. Receptor density
(B,^ values) and dissociation constants (K,,) for 125I-Ang II
were calculated from Scatchard plots. Furthermore, to characterize Ang II receptor subtypes in VSMCs, monolayers of
VSMCs were also incubated with the fixed 12!I-Ang II and
increasing concentrations of Ang II, AT! receptor antagonist
DuP 753, and AT2 receptor antagonist PD 123319.
Isolation of Total Cellular RNA
The cells were washed three times with PBS and then lysed
in 4 mol/L guanidine isothiocyanate. Total cellular RNA was
isolated by the acid guanidinium/phenol/chlorofonn method.
After extraction with phenol/chloroform and precipitation
with ethanol, the RNA pellet was resuspended in 50 fiL of TE
buffer (Tris-HCl, EDTA), pH 7.4, and quantified by absorbance measurements at 260 nm. The integrity of the purified
RNA was determined by visualization of the 28S and 18S
ribosomal RNA bands after electrophoresis of each RNA
sample through a 1% agarose gel.
Northern Blot Hybridization
For Northern blot analysis, RNA (total cellular RNA, 20
/ig) was denatured with formamide and Formalin at 65°C for
10 minutes and fractionated by electrophoresis through a 1%
agarose gel. The RNA was transferred to a nylon filter
(blotting membrane Hybond-N was purchased from Amersham International, Ltd, Buckinghamshire, England). Prehybridization was conducted at 65°C for 2 hours in 5 x SSPE (1 x
SSPE=0.18 mol/L sodium chloride, 10 mmol/L sodium phosphate, and 1 mmol/L EDTA, pH 7.7), 5 x Denhardt's solution,
0.5% sodium dodecyl sulfate (SDS), and heat-denatured
salmon sperm DNA (1 mg/mL). A Kpn ISac I fragment that
encodes the rat AT, receptor, kindly provided by Dr Inagami
(Vanderbilt University, Nashville, Tenn), was used as a probe
for hybridization. The filters were then hybridized at 65°C for
at least 12 hours to the probe that was radiolabeled with
[a-^PJdCTP by the random primer synthesis method (Random Primer DNA Labeling Kit, TaKaRa Shuzo Co, Ltd,
Kyoto, Japan). After hybridization, the filter was washed twice
in 2x SSC (sodium chloride and sodium citrate buffer) and
0.1% SDS at 65°C and successively in l x SSC and 0.1% SDS
once and in 0.5 x SSC and 0.1% SDS once. The autoradiograms were scanned with a BAS 2000 (Fuji Film, Tokyo,
Japan) and quantified.17'18
Measurement of Inositol 1,4,5Trisphosphate Production
Production of IP3 in VSMCs was measured with a radioimmunoassay kit (TRK1000, Amersham), as previously described.7 To examine the effects of dexamethasone on postreceptor signaling systems, the accumulation of IP3 was
determined in permeabilized VSMCs by use of the nonhydrolyzable GTP analogue guanosine-5'-O-(3'-thiotriphosphate)
(GTP-yS) in dexamethasone-treated (1 /imol/L for 48 hours)
or nontreated cells. VSMCs were exposed to 200 U/mL a-toxin
(Life Technologies, Inc, Gaithersburg, Md), a transmembrane
pore-making exoprotein produced by Staphylococcus aureus,19
for 20 minutes at 37°C in buffer containing (mmol/L) glutamate potassium salt, 150; EGTA, 5; magnesium chloride, 1;
and 1,4-piperazine diethanesulfonic acid (PIPES), 10; pH 7.2,
adjusted with KOH. The cells were then rinsed with the above
medium and incubated with 100 fimo\fL GTP-yS for 20
minutes. IP3 production was measured with a competitive
binding assay that used a bovine-derived adrenal binding
protein specific for IP3.
Sodium Dodecyl Sulfate-Polyacrylamide Gel
Electrophoresis and Immunoblotting
The cells were washed three times with ice-cold PBS and
added sample buffer (60 mmol/L Tris-HCl, pH 6.8, 2% SDS,
10% glycerol, 5% 2-mercaptoethanol, 0.1% bromophenol
blue) and boiled for 5 minutes before resolution by SDSpolyacrylamide gel electrophoresis on gels containing 12%
acrylamide. The protein was loaded in 150-^tg aliquots to
achieve optimal blots. Low molecular weight protein standards
(Bio-Rad Laboratories, Hercules) were run in parallel with the
samples under study. After electrophoresis, proteins were
transferred to nitrocellulose membrane (TEF Co, Nagano,
Japan), which was then blocked overnight in blocking solution
(5% BSA in buffer comprising 150 mmol/L NaCl in 20 mmol/L
Tris-HQ, pH 7.5). After washing, the blot was incubated for 2
hours with the first antiserum (anti-Gqo) at a 1:1000 dilution in
0.05% Tween 20 in blocking solution. After a further wash, the
blot was incubated for 2 hours with the second antibody
(alkaline phosphatase-conjugated goat anti-rabbit IgG, BioRad Laboratories). After the final washes, the detection was
performed by the immunoblot assay procedure (Bio-Rad
Laboratories). Antiserum G,,, was raised against the C-terminal sequence of G^, and recognized the 42-kD forms of G,,,.
Protein Determination
The protein levels in VSMCs were determined by the
method of Pierce (Pierce Chemical Co, Rockford, III) using
BSA as a standard.
Statistical Analysis
The results are expressed as mean±SEM. Statistical significance was assessed by Student's t test. Values of P<£)5 were
considered to be significant.
Results
Effects of Dexamethasone on Angiotensin II
Type 1 Receptor
Fig 1 shows the competition curves with Ang II, DuP
753, and PD 123319 in 1 /imol/L dexamethasone-
Sato et al
100
o
o
Glucocorticoid and Angiotensin II Type 1 Receptor
27
2.0-
80
60
40
""5
in
0
20
i0"
l J
10"
1 l
10"
1 0
10"
9
10"
10"7 10"6
10'5
Concentration (mol/l)
Fo 1. Competition curves with angiotensin II (o), DuP 753 (•),
and PD 123319 (•) In dexamethasone-treated (—) or untreated
cells ( — ) . Trie data are based on the average of three independent (—) experiments.
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treated (48 hours) and untreated cells. In VSMCs,
Ang II inhibited the l23I-Ang II binding with an IQo of
2.0 nmol/L. DuP 753 produced a single displacement
curve (IQo=0.25 ^mol/L), but PD 123319 did not.
These findings clearly suggested that the AT, receptor
was dominantly present in VSMCs and are consistent
with earlier observations.15'20 The displacement curve
in dexamethasone-treated cells, expressed by percentage of control, showed that the characteristics of the
Ang II receptor were similar to those in the control
cells.
Next, to investigate the effect of dexamethasone on AT!
receptor, VSMCs were incubated with 1 /xmol/L dexamethasone for various periods before the cells were harvested. AT, receptor number, measured with the ligand
125
I-Ang II, was unchanged during the first several hours of
exposure to dexamethasone. The receptor number was
found to be increased after 12 hours' exposure to dexamethasone and progressively increased to 185.6±9.8% of
control at 48 hours (Fig 2). Fig 3 shows the typical binding
curves (A) and Scatchard plot (B) for l25I-Ang II binding
in dexamethasone-treated and control cells at 48 hours.
The B,,,,, value was 27±3 fmol/mg protein in dexamethasone-treated cells and 15 ±3 fmol/mg protein in control
cells. Conversely, the K,, of 125I-Ang n did not change
significantly (dexamethasone-treated cells, 1.2±0.3
nmol/L; control cells, 1.1 ±0.2 nmol/L).
As shown in Fig 4, VSMCs treated with different
concentrations of dexamethasone exhibited a dose-dependent increase in AT, receptor number. The increase
in AT, receptor density became apparent at as low as 10
nmol/L dexamethasone and became maximal at 1
^mol/L dexamethasone.
To demonstrate whether the effect of dexamethasone
was through the glucocorticoid receptor, VSMCs were
incubated with 10 ^mol/L RU 38486, a glucocorticoid
receptor-specific antagonist,21 with or without 1 /xmol/L
dexamethasone for 48 hours. Although RU 38486 alone
had no effect on the number of AT, receptors, it
completely blocked the effect of 1 /xmol/L dexamethasone (Fig 5). These results suggested that the effect of
dexamethasone on AT, receptor was through the glucocorticoid-specific receptor.
2
6
12
24
Time (hours)
48
FIG 2. Bar graph shows time course for the effect of dexamethasone on relative Increase in numbers of angiotensin II type 1
(AT,) receptors. Vascular smooth muscle cells were Incubated
for the Indicated time periods with 1 ^mol/L dexamethasone. AT,
receptor density was calculated from Scatchard plots. Data
represent mean±SEM of three independent experiments.
*P<.05 vs the value in the control experiment.
Effect of Dexamethasone on Angiotensin II Type 1
Receptor Messenger RNA Level
The steady-state AT, receptor mRNA level was measured by Northern blot analysis. Fig 6A shows Northern
blot analysis of total RNA from rat VSMCs. A single
band of 2.3 kb was observed in both dexamethasonetreated cells and control cells. The size of this mRNA
was consistent with the previous report.16 Fig 6B shows
the ethidium bromide staining of total RNA used for
Northern blot in Fig 6A. Both 28S and 18S ribosomal
RNA were present in similar amounts throughout the
gel. Fig 7 shows the relative increase in AT, receptor
mRNA calculated by the BAS 2000 system. After 30
minutes' treatment with dexamethasone, the abundance
of AT, receptor mRNA was 2.2±0.3 times control, and
it was 7.8 ±0.4 times control after 24 hours' exposure to
dexamethasone.
Effect of Dexamethasone on GTP
Analogue-Induced IP., Production
in Permeabilized Cells
To examine the effect of dexamethasone on Ang
H-mediated postreceptor signaling systems in VSMCs,
we first directly activated G proteins by GTPyS in
permeabilized cells. GTP-yS has been shown to stimulate IP3 accumulation and potentiate contraction of
permeabilized smooth muscle.1922 The basal intracellular IP3 content was 4.5±0.5 pmol/106 cells, consistent
with the previous study.7 After treatment with 1 /xmol/L
dexamethasone or ethanol vehicle for 48 hours, the cells
were stimulated with GTP-yS (100 /xmol/L for 20 minutes). As shown in Fig 8, increased accumulation of IP3
occurred in both dexamethasone-treated and control
cells. However, there was no significant difference of IP3
production between dexamethasone-treated cells and
control cells. It should be noted that in the experiments
conducted in the absence of a-toxin treatment, there
was no rise in IP3 levels in response to GTP-yS in both
groups (data not shown).
Effect of Dexamethasone on Gq Protein a-Subunit
Protein Level
To examine the effect of dexamethasone on Ang
II-mediated postreceptor signaling systems in VSMCs,
we next examined the effect of dexamethasone on Gq
protein a-subunit protein level by Western blot analysis.
28
Hypertension
Vol 23, No 1 January 1994
B
FIG 3. A typical binding curve (A) and
its Scatchard plot (B) for 128l-angiotensin II binding to vascular smooth muscle cells. The cells were exposed to 1
^mol/L dexamethasone (•) or ethano)
vehicle (o) for 48 hours. The maximum
binding for control was 16.5 fmol/mg
protein with a Kd of 1.15 nmol/L, and
the maximum binding for dexamethasone-treated cells was 30.5 fmol/mg
protein with a Kd of 1.10 nmol/L
' I ] Anglotensin II. n m d / l
r a l ] Angiotensin II bound,
fmol/mg. protein
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Fig 9 shows the results of Western blot analysis of the
proteins in 1 ^mol/L dexamethasone-treated cells and
control cells for various periods of time. No differences
were seen in Gqa protein level between dexamethasonetreated cells and control cells.
Effect of Dexamethasone on Cell Numbers
and Cell Protein
Dexamethasone had no significant effects on cell
numbers and cell protein in VSMCs incubated for 48
hours compared with control.
Discussion
In the present study, we investigated the effects of
glucocorticoid on AT, receptor, its gene expression, and
postreceptor signaling systems in VSMCs. Dexamethasone induced the gene expression of AT, receptor and
increased AT, receptor number through the glucocorticoid-specific receptor, without significant effect on postreceptor systems in VSMCs. These findings could potentially explain how glucocorticoid induces vascular
hypersensitivity to Ang II and are quite important in
elucidating the mechanism of increased vascular sensitivity that is found in patients with Cushing's syndrome.4
The radioligand binding study using the specific receptor antagonists DuP 753 and PD 123319 disclosed
that the actions of Ang II on VSMCs were mediated
primarily through the AT, receptor. Moreover, treatment with dexamethasone did not alter the characteristics of Ang II receptor subtypes. From the results of
the time course study, the number of AT, receptors was
increased after 12 hours' exposure to dexamethasone
and maintained its levels at 48 hours. This relatively
100
X3
If
C
long duration might account for protein synthesis, and
this behavior is consistent with the general mode of
action of steroid hormones. Furthermore, dexamethasone exhibited a dose-related increase in AT, receptor
numbers.
The effects of glucocorticoid on the regulation of Ang
II receptor have been demonstrated in in vivo and in
vitro studies. Douglas23 demonstrated that dexamethasone infusion produced downregulation of glomerular
Ang II receptor obtained from sodium-loaded rats.
Those results contradict our present findings. Since
glomerular mesangium and vascular smooth muscle
share many properties, similar actions of dexamethasone on Ang II receptor are expected. Although the
exact reasons for the difference between these studies
and our present findings remain to be determined,
several possible explanations can be suggested. The
dose of dexamethasone infused in sodium-loaded rats
was lower than in our in vitro study. In our experiment,
low doses of dexamethasone did not induce any significant changes in the number of AT, receptor. Second,
other factors, such as low levels of circulating Ang II, may
modulate the effects of glucocorticoid in sodium-loaded
rats. Furthermore, it is also possible that glucocorticoid
has tissue- and cell-specific regulation. Indeed, Chappell
et al24 showed that glucocorticoid downregulated both
AT, and AT2 receptors in pancreatic acinar cells.
Several recent studies have investigated the effects of
glucocorticoid on the mRNA of seven transmembrane-
i
80
60
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20
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-10
-9
-8
-7
-6
Log [dexamethasone] (moll)
FIG 4. Bar graph shows effect of various doses of dexamethasone on relative Increase in numbers of angiotensin II type 1
(AT,) receptors. Vascular smooth musde cells were exposed to
dexamethasone for 48 hours. Data are the average of two
independent experiments.
CONTROL
RUQMEIOumd/l DX 1jJmd/l
DX+RU38M6
Fra 5. Bar graph shows effect of RU 38486 on relative increase
in numbers of angiotensin II type 1 (AT,) receptors. Vascular
smooth muscle cells were incubated with 10 /imol/L RU 38486
with or without 1 timoi/L dexamethasone (DX) for 48 hours. Data
represent mean±SEM of three Independent experiments.
*P<.05 vs the value in the control experiment.
Sato et al Glucocorticoid and Angiotensin II Type 1 Receptor
A.
1 2
3
4
5
6
29
%of
basal IP3
-28S
-I8S
Alter Stimulation
200
0)
To
sz
a. 150
itol t:risp
V)
O
B.
100
50
w
o
-rRNA 28S
c
0
Baut
-rRNA I8S
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FIG 6. A, Northern Wot analysis of tot il RNA from cultured
vascular smooth muscle cells. The cells v ere incubated with or
without 1 nJnctf/L dexamethasone for the indicated time periods.
The 2.3-kb receptor messenger RNA is indicated by the arrow.
Each lane contains 20 fig total RNA. Lane 1, dexamethasone 30
minutes; lane 2, dexamethasone 1 hour; lane 3, dexamethasone
6 hours; lane 4, dexamethasone 12 hours; lane 5, dexamethasone 24 hours; lane 6, dexamethasone 0 hours (control). B,
Ethidlum bromide staining of total RNA used for Northern blot In
A; 28S and 18S ribosomal RNA (rRNA) are present in similar
amounts throughout the gel.
type receptors. Sakaue and Hoffman8 have clearly demonstrated that glucocorticoid regulated expression of
a,B-receptors by increasing the rate of transcription of
this gene. The pattern of change in expression of AT,
receptor mRNA by dexamethasone was similar to that
of the a1B-adrenergic receptor mRNA. Moreover, there
was a good correlation between the increase in the
expression of AT! receptor and the change in the
abundance of the receptor's mRNA, as observed in
a1B-receptors.
Compared with these similarities, the reported pattern
of change in /E^-adrenergic receptor gene is different.10
ft-Adrenergic receptor mRNA increased very rapidly,
within 1 to 2 hours, and returned slowly, by 10 hours, in
the continued presence of glucocorticoid. These different
DX1ymol/l
GIFTS lOOj/md/l
GTPyS+DX
FIG 8. Bar graph shows the effect of dexamethasone (DX) on
guanosine-5'-0-(3'-thlotriphosphate) (GTP-ySHnduced Inositol
trisphosphate (IP3) production in permeaWlized cells. Vascular
smooth muscle cells were incubated with or without 1 jimol/1.
dexamethasone for 48 hours. IP3 was measured after 20 minutes
of stimulation with 100 ^mol/L GTP-yS. Data represent
mean±SEM of three independent experiments. *P<.05 vs the
value in the control experiment.
regulatory mechanisms among AT,, a1B-, and ft-adrenergic receptor mRNA may be important, because these
receptors are closely involved in cardiovascular regulation in glucocorticoid-induced hypertension. From our
present study, it still remains unclear whether dexamethasone elevated transcription rate or increased the stability of ATi receptor gene product. Our data only presented the increased accumulation of AT, receptor
mRNA. For most steroid-regulated genes, gene expression is effected primarily at the transcription rate.25
Glucocorticoid has been shown, however, to have effects
on mRNA stability such as insulin mRNA in insulinoma
cells26 and interleukin-1/3 mRNA in monocytic cell
lines.27 Further studies will be needed.
Control
O 0.5
2
4
8
12 24 /Hours
8
12 24 /Hours
97.4
66
45
-•
31
DX
0.5
1
97.4
66
0C<
0
0.5
1
6
12
Time (hours)
FIG 7. Bar graph shows the time course of changes in the
abundance of angiotensin II type 1 (AT,) receptor messenger
RNA (mRNA). The cells were incubated with or without 1 jtmol/L
dexamethasone for the indicated time periods. Data represent
mean±SEM of three Independent experiments. *P<.05 vs the
value in the control experiment.
2
4
m
»
45
31
FIG 9. Western Wot analysis. The protein was prepared from
vascular smooth muscle cells incubated without (top) or with
(bottom) 1 fimoVL dexamethasone (DX) for the time shown.
Proteins were analyzed by Western blotting with rabbit antl-G^
and alkaline phosphatase-conjugated goat anti-rabbit IgG. Molecular size markers, in kilodaltons, are shown In the left lane.
30
Hypertension
Vol 23, No 1 January 1994
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We previously reported marked elevation of Ang IIinduced IP3 production in response to dexamethasone
exposure in VSMCs.7 These changes (threefold) were
significantly greater than could be explained by modest
increases in receptor density (twofold) and led us to test
the hypothesis that augmented signal transduction might
be partially explained by changes in postreceptor systems. Recent advances in the investigation of G protein
have demonstrated that the Ang II-linked G protein
concerned in IP3 production in VSMCs is G,.2829 First,
therefore, o-toxin-permeabilized VSMCs were used. In
these cells, GTP-yS induced an increase in IP3 production
without Ang II, and 48 hours' exposure to dexamethasone did not affect the IP3 production induced by
GTP-yS. It is therefore less likely that dexamethasone
exerts its action on IP3 production through postreceptor
signaling systems. However, GTP-yS might have the
capability of stimulating all kinds of G proteins other
than Gq in VSMCs. Then, we investigated the effect of
dexamethasone on G,,, protein level, and no significant
changes in Gqa protein level were found. The classification "G protein" is now used to refer to a family of G
proteins. In Gq protein, the interaction and regulation
with other G proteins, such as G,, G,, and so on, were not
fully understood. Therefore, it is clear that before we
conclude that there is no modulatory role of dexamethasone in postreceptor signaling systems, further precise
study would be needed.
In conclusion, we have first demonstrated that glucocorticoid induced the expression of the AT[ receptor
gene and resulted in an increase in the number of AT!
receptors through the glucocorticoid-specific receptor
in VSMCs. Moreover, we also demonstrated that glucocorticoid did not exert significant action on postreceptor
signaling systems. Taken together with the results of our
recent study,7 these results indicate that glucocorticoid
elevates AT, receptor numbers and also enhances receptor-mediated response, that is, Ang II-stimulated
IP3 production. This evidence makes clear the mechanisms of vascular hypersensitivity to Ang II in glucocorticoid-induced hypertension.
Acknowledgments
We would like to thank Dr Ronald D. Smith (Du Pont de
Nemours) and Dr Taylor (Parke-Davis Pharmaceutical Research) for their generous gift of the nonpeptide Ang II
antagonists DuP 753 and PD 123319 and Dr Inagami (Vanderbilt University) for his generous gift of rat AT, receptor
cDNA.
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3. Grunfeld JP, Eloy L. Glucocorticoids modulate vascular reactivity
in the rat. Hypertension. 1987;10:608-618.
4. Saruta T, Suzuki H, Handa M, Igarashi Y, Kondo K, Senba S.
Multiple factors contribute to the pathogenesis of hypertension in
Cushing's syndrome. J Chn Endocrinol Metab. 1986;62:275-279.
5. Handa M, Kondo K, Suzuki H, Saruta T. Dexamethasone hypertension in rats: role of prostaglandins and pressor sensitivity to
norepinephrine. Hypertension. 1984;6:236-241.
6. Nakamoto H, Suzuki H, Kageyama Y, Ohishi A, Murakami M,
Naito M, Saruta T. Characterization of alterations of hemody-
namics and neuroendocrine hormones in dexamethasone induced
hypertension in dogs. Clin Exp Hypertens A. 1991;13:587-606.
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A Sato, H Suzuki, M Murakami, Y Nakazato, Y Iwaita and T Saruta
Hypertension. 1994;23:25-30
doi: 10.1161/01.HYP.23.1.25
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