(Hypertension. 1996;27:867-874.)
© 1996 American Heart Association, Inc.
Articles |
Regulation of Angiotensin II Receptor Subtypes by Dexamethasone in Rat Mesangial Cells
From INSERM 64, Hôpital Tenon (D.C., S.V., R.A.); INSERM 36, Collège de France (C.L.-C.), Paris; and Centre d'Etudes Nucleaires Saclay, Gif-sur-Yvette (P.P.), France.
Correspondence to Raymond Ardaillou, Hôpital Tenon, 4 Rue de la Chine, 75020 Paris, France.
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Abstract The objective of this study was to examine the role of dexamethasone on the expression of angiotensin II (Ang II) receptors in cultured rat mesangial cells. Dexamethasone caused concentration- and time-dependent decreases in 125I–[Sar1,Ala8]Ang II binding that were prevented by glucocorticoid receptor inhibition with mifepristone. A lag time of 24 hours and a dexamethasone concentration of at least 10 nmol/L were necessary for this effect to occur. Dexamethasone-induced reduction of 125I–[Sar1,Ala8]Ang II binding resulted from decreased Ang II type 1 (AT1) receptor density. No change in the apparent dissociation constant was observed. Dexamethasone also markedly inhibited Ang II–dependent inositol phosphate accumulation. Both reverse transcription–polymerase chain reaction and Northern blot analysis using specific short probes from the 3' noncoding region of the cDNA demonstrated the presence of AT1A and AT1B receptor mRNAs in rat mesangial cells, with a slight predominance of AT1B. Therefore, we studied the effect of dexamethasone on the expression of these two subtypes in rat mesangial cells. Dexamethasone produced a time-dependent decrease of AT1B receptor mRNA that was apparent after 6 hours of incubation, whereas AT1A receptor mRNA did not change. Mifepristone also suppressed the dexamethasone-induced decrease in AT1B receptor mRNA. In conclusion, glucocorticoids diminish Ang II receptor density at the mesangial cell surface through a mechanism that implies successive interaction with the glucocorticoid receptor and specific reduction in AT1B receptor mRNA expression. This differential regulation of both AT1 receptor subtypes might allow glucocorticoids to exert adjusted effects in their various target tissues.
Key Words: receptors, angiotensin II • rat • mesangial cells • dexamethasone • mifepristone
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Two major types of the Ang II receptor, AT1 and AT2, have been cloned in the rat. AT1 receptors mediate the main functions of Ang II in the adult animal, including arteriolar vasoconstriction, salt retention, and thirst. The AT1 receptor was found to have A and B subtypes of 359 amino acids each that share 96% identity. The AT1A and AT1B receptor genes are localized on chromosomes 17 and 2, respectively.1 AT1A and AT1B receptor cDNAs exhibit 92% identity in the nucleotide sequence within the coding region, whereas they are only 58% and 62% homologous within the 5' and 3' untranslated regions, respectively.2 3 Both subtypes individually expressed in Chinese hamster ovary (CHO) cells could not be pharmacologically distinguished by their ligand binding properties.4 Moreover, they were shown to be coupled to the same signaling pathways.5 Despite these homologies, AT1A and AT1B receptor subtypes exhibit disparate tissue-specific expression profiles. AT1A receptor mRNA is abundant in the liver, kidney, aorta, uterus, adrenals, ovary, spleen, and lung1 and is present in the hypothalamus and subfornical region of the brain.6 AT1B receptor mRNA is found mainly in the pituitary, adrenals, kidney, and uterus.6 7 Therefore, both subtypes are present in the adult rat kidney, with a predominance for the AT1A subtype. Of note, regulation of receptor expression appears to be different for the two AT1 subtypes. Bilateral nephrectomy decreased expression of liver AT1A mRNA but increased adrenal AT1B mRNA levels.8 In renovascular hypertension (two-kidney, one clip), adrenal AT1B receptor mRNA levels decreased, whereas AT1A levels did not change.9 AT1A receptor mRNA was increased in rat ventricles with myocardial infarction, whereas the AT1B receptor mRNA level was unaffected.10 Finally, there was an inverse relationship between the expression of the AT1A and AT1B subtypes in the whole kidney in response to a low sodium diet in the rat.11
Glucocorticoids control expression of a variety of receptors and enzymes. Functional glucocorticoid receptors are widely distributed, and glucocorticoid-responsive elements have been described in the promoter regions of many genes. However, the role of these hormones in the control of Ang II receptors is still subject to debate. In an early study, Douglas12 showed that in vivo administration of corticosteroids to rats decreased Ang II receptor density in isolated glomeruli. On the contrary, Sato et al13 reported that glucocorticoids increased the number of AT1 receptors as well as AT1 receptor mRNA expression in rat cultured vascular smooth muscle cells. Similar results were found by Schelling et al,14 who also reported a stimulatory effect of dexamethasone on Ang II–dependent smooth muscle cell hypertrophy. Both studies did not separate the effect of glucocorticoids on AT1A and AT1B receptors. Recently, Matsubara et al15 demonstrated that dexamethasone induced significant increases in AT1A receptor mRNA and AT1 receptor site density in rat cardiac fibroblasts and cardiomyocytes in culture, whereas AT1B receptor mRNA was not affected. Uno et al16 also observed that rat AT1A receptor gene expression was upregulated by dexamethasone at the levels of both mRNA and protein in rat cultured aortic smooth muscle cells. These findings suggest that the glucocorticoid-dependent Ang II receptor downregulation in rat glomeruli previously described12 could depend on a change in the regulation of AT1B expression. Since rat glomerular Ang II receptors are localized in mesangial cells,17 we examined the effect of dexamethasone on Ang II receptor number and AT1 receptor mRNAs (AT1A and AT1B) in cultured rat mesangial cells. The results demonstrate that dexamethasone markedly decreases AT1 receptor number in rat mesangial cells and that this effect is associated with the downregulation of AT1B receptor gene expression.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Materials
Reagents for these studies were obtained from the following sources: dexamethasone and [Sar1,Ala8]Ang II from Sigma Chemical Co; RPMI medium and cell culture supplies from Gibco; fetal calf serum and restriction enzymes from Boehringer Mannheim; 125I–[Sar1,Ala8]Ang II (74 TBq/mmol) from the Centre d'Etudes Nucléaires, Gif-sur-Yvette, France; and [3H]myo-inositol (2.7 TBq/mmol) from the Radiochemical Centre, Amersham. RU 38486, or mifepristone, a glucocorticoid receptor antagonist, was a gift from Roussel Uclaf. AT1A and AT1B cDNA probes were gifts from Dr K. Bernstein (Atlanta, Ga) and K. Sandberg (Bethesda, Md), respectively. GAPDH cDNA probe was prepared in the laboratory by a random primer technique with the full-length GAPDH insert.18 All other reagents were purchased from Sigma.
Mesangial Cell Culture
Isolation and characterization of rat glomerular
mesangial cells were performed as previously
described.17 Glomeruli were prepared by mechanical sieving
from the cortex of male Sprague-Dawley rats weighing 150 to
200 g. The procedure followed in the care and euthanasia of the study
animals was in accordance with the Declaration of Helsinki and the
Guide for the Care and Use of Laboratory Animals, National
Institutes of Health. Mesangial cells were cultured in
RPMI-1640 medium supplemented with glutamine (5 mmol/L), HEPES (15
mmol/L), penicillin (100 U/mL), streptomycin (100 µg/mL), and 10%
fetal calf serum in an atmosphere of 5% CO2/95%
air. After the cells had reached confluence, the medium was changed to
appropriate culture media containing various concentrations of
dexamethasone for various periods of time before the
experiments. Cells in primary culture were used for RNA extraction and
cells at the second passage for binding studies. They were routinely
identified by light microscopy and indirect
immunofluorescence staining. They had a stellate
appearance, overgrew each other, and showed a network of intracellular
fibrils of myosin. They were negative for anti–von
Willebrand factor, antiurokinase, and anticytokeratin
antibodies, which excluded any contamination by the two other
glomerular cell types, endothelial and
epithelial cells.
Binding Studies
Binding studies were performed with
125I–[Sar1,Ala8]Ang II to
maintain a low rate of degradation of the iodinated tracer
in the medium.19 Confluent monolayers of rat
mesangial cells grown in 24-well plates were rinsed three
times with 0.15 mol/L NaCl. Equilibrium binding experiments were
performed at room temperature for 30 minutes with increasing
concentrations of
125I–[Sar1,Ala8]Ang II in
500 µL of serum-free RPMI-1640 medium containing 2 mmol/L
CaCl2 and 0.2% bovine serum albumin. In other
experiments, the concentration of
125I–[Sar1,Ala8]Ang II
was fixed at 0.40 nmol/L. At the end of the incubation period, the
medium was removed, the cells were rinsed twice with 0.15 mol/L NaCl,
and then they were dissolved with 0.5 mL of 1 mol/L NaOH and
transferred to polypropylene tubes. 125I radioactivity in
the dissolved cells was determined with an automatic gamma counter
(LKB) with 60% efficiency. A fraction of the dissolved cells was used
for measurement of the protein content according to Lowry et
al,20 with bovine serum albumin as the standard.
Nonspecific binding was determined in the presence of 1 µmol/L
unlabeled hormone. Specific binding was calculated as the difference
between total and nonspecific binding and was expressed as femtomoles
of 125I–[Sar1,Ala8]Ang II
bound per milligram of protein. Bmax and
Kd values of Ang II receptors on
mesangial cells were calculated from the Scatchard plots
derived from the saturation binding experiments with the use of the
Ligand program.21
To evaluate the possible effect of dexamethasone on 125I–[Sar1,Ala8]Ang II internalization, we measured surface-bound radioactivity and intracellular radioactivity separately as previously described.22 Cells were exposed to 1 µmol/L dexamethasone for 48 hours before binding experiments. They were then incubated for 30 minutes with 150 pmol/L 125I–[Sar1,Ala8]Ang II. At the end of this period, the medium was aspirated and cells were exposed to a hypertonic acid solution (50 mmol/L glycine and 150 mmol/L NaCl, pH 3) for 10 minutes at 4°C. Radioactivity present in the medium was considered to represent 125I–[Sar1,Ala8]Ang II bound to cell surface receptors. Intracellular radioactivity remaining after acid treatment was determined after the cells had been dissolved in 1 mol/L NaOH.
Analysis of IPs
IPs were measured as described.23 After the cells
had reached subconfluence, culture medium was removed and cells were
placed in inositol-deficient Waymouth medium containing 1.1 MBq/mL
of [3H]myo-inositol (2 mL per well) for 48
hours at 37°C with or without 1 µmol/L dexamethasone.
After 15 minutes of preincubation with 10 mmol/L LiCl, 0.1 µmol/L Ang
II was added for 1 minute. The incubation was terminated by rapid
aspiration of the medium and addition of 2 mL ice-cold 5%
trichloroacetic acid. Cells were scraped away from the wells and washed
once more with 5% trichloroacetic acid, and the aqueous phase was
extracted in diethylether. Samples were adjusted to pH 7.0 with 50
mmol/L sodium tetraborate and then loaded onto 2-mL Dowex AG1-X8
anion-exchange resin columns (Bio-Rad). The columns were washed
with 10 mL of water and 10 mL of 5 mmol/L sodium tetraborate. IPs were
then eluted with 10 mL of increasing concentrations of ammonium formate
in 0.1 mol/L formic acid. Five milliliters of each collected fraction
was mixed with scintillation fluid and counted in a beta counter (LKB).
After correction of quenching, IP values were expressed as a percentage
of control (ratio of IP content in Ang II–exposed cells to IP content
in cells exposed only to buffer).
Isolation of Total Cellular RNA
After incubation in Petri dishes with or without
dexamethasone, the cells were washed with 0.15 mol/L NaCl.
Then total RNA was extracted by the phenol-chloroform method and
precipitated with 3 mol/L LiCl.24 RNA concentration was
determined from the absorbance reading at 260 nm. Total RNA (15 to 20
µg per lane) was then fractionated by electrophoresis in an agarose
gel. The integrity of the purified RNA was determined by visualization
of the 28S and 18S ribosomal bands. RNA was then transferred to a nylon
GeneScreen Plus membrane (New England Nuclear).
Northern Blot Hybridization
After prehybridization, the blot was hybridized for 16 hours at
42°C with a 32P-labeled cDNA probe specific for the rat
AT1A or AT1B receptor. These two specific cDNA
probes correspond to the 3' untranslated regions of the genes and
differ by 40%, whereas the coding sequences are 90% homologous. The
cDNA templates were derived for AT1A from clone pCa18b, a
gift of Dr K. Bernstein, and for AT1B from clone RAG 6
D4-60, a gift of Dr K. Sandberg. Both clones include the coding and
noncoding sequences. The cDNA probes were prepared as previously
described.25 Depending on the enzymes used for
linearization of the plasmid, either a long probe including the coding
and noncoding sequences or a short probe containing only the 3'
untranslated region was synthesized. The AT1A short probe
(0.7 kb) was obtained with Ase I and BamHI. The
AT1B probe (0.6 kb) was obtained with HindIII.
The sizes of the probes were controlled by 1% agarose gel
electrophoresis. After hybridization, the filters were washed three
times at 42°C for 20 minutes in 2x SSC (1x SSC is 0.15 mol/L NaCl
and 0.015 mol/L sodium citrate, pH 7.0) containing 0.1% sodium
dodecyl sulfate and exposed to Fuji x-ray film at
-80°C in the presence of intensifying screens.
The same filters were dehybridized by boiling for 15 minutes in 0.1x SSC containing 1% sodium dodecyl sulfate and rehybridized with a 32P-labeled GAPDH cDNA probe. Quantification of AT1A and AT1B receptors and GAPDH mRNA labeling were achieved by scanning of the films with a densitometric scanner (Appligene). The intensity of the AT1A and AT1B receptor mRNA signals was related to that of GAPDH, which is expressed constitutively in rat mesangial cells.
RT-PCR
Choice of Primers
Oligonucleotide primers were chosen in
homologous parts of the coding region of the rat AT1A and
AT1B receptor genes. The reverse primer "b" (5'-GCA
CAA TCG CCA TAA TTA TCC-3', position 739-719 bp) and the sense primer
"a" (5'-CAC CTA TGT AAG ATC GCT TC-3', position 295-314 bp) were
synthesized by a PCR-mate 391 device (Applied Biosystems) according to
Murphy et al.2
RT Reaction
Moloney murine leukemia virus reverse transcriptase (200 U,
Bethesda Research Laboratories) was used to synthesize (90 minutes,
37°C) single-stranded cDNA from rat liver (0.06 µg), pituitary
(0.35 µg), and mesangial cell (0.20 µg) total RNA in
the presence of 1x105 to 8x105 molecules of
the AT1 receptor mutant cRNA9 used as the
internal standard and of 0.4 µmol/L of the reverse primer (b) in 20
µL of 50 mmol/L Tris-HCl buffer (pH 8.3), 75 mmol/L KCl, 3 mmol/L
MgCl2, 2.5 mmol/L dNTP, 10 mmol/L dithiothreitol,
and 50 U of RNase inhibitor (Boehringer). The
reaction was stopped by heating of samples for 10 minutes at
70°C.
PCR Amplification
Double-stranded cDNAs were synthesized and amplified with
2.5 U of Taq polymerase (Boehringer) and 80 nmol/L
sense (a) and antisense (b) primers in 0.05 mL of 10 mmol/L Tris-HCl
buffer (pH 8.3), 50 mmol/L KCl, 2.0 mmol/L MgCl2,
0.5 mmol/L dNTP, 2 mmol/L dithiothreitol, and 0.01% gelatin for 30
cycles at 92°, 54°, and 72°C for 60, 60, and 90 seconds,
respectively. Since the coding regions of the rat AT1A and
AT1B receptor genes are composed of only one exon,
contamination of sample RNAs by genomic DNA was excluded by directly
subjecting the sample RNAs to PCR amplification without an RT step.
Samples in which a DNA PCR product was seen under these conditions
were eliminated. A trace amount of [
-3H]dCTP (3 µCi)
was included in the PCR reaction for quantification of the different
PCR products, taking into account the number of C residues
present in each fragment (AT1A, 117;
AT1B, 113; internal standard, 105). After PCR
amplification, the PCR products were submitted to EcoRI
digestion (2000 U/µL) for 90 minutes at 37°C so that
AT1A RNA PCR products could be distinguished from those
of AT1B. The efficiency of the digestion was verified in
each experiment by observation of a complete digestion of a PCR
product arising from the amplification of the AT1A
cDNA. The different PCR products were separated on 1.5% agarose
gels for visual verification and on 5% polyacrylamide gels for
precise quantification. The bands were excised, solubilized in 0.025
mol/L periodic acid at 50°C, and counted by liquid scintillation
spectrometry (Picofluor, DuPont–New England Nuclear).
The yield of each RT-PCR amplification reaction was evaluated by the radioactivity incorporated into the PCR product of the synthetic RNA (added in trace amounts to each wild-type RNA analyzed).
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Effect of Dexamethasone on 125I–[Sar1,Ala8]Ang II Binding to Rat Mesangial Cells
Exposure of rat mesangial cells to 1 µmol/L dexamethasone resulted in a decrease in 125I–[Sar1,Ala8]Ang II binding after a lag time of 24 hours (Fig 1
). No
decrease was observed after 3 or 6 hours of incubation. The decrease in
binding reached 35.9±4.2% and 51.2±5.6% after 24 and 48 hours,
respectively, of dexamethasone treatment (baseline,
18.3±1.1 fmol/mg). Twenty-four– or 48-hour incubation with
dexamethasone produced a concentration-dependent
decrease in
125I–[Sar1,Ala8]Ang II
binding, with a threshold (79.4±2.7% and 74.0±5.0% of baseline
after 24 and 48 hours of incubation, respectively) at 10 nmol/L
dexamethasone (baseline, 24.8±0.65 fmol/mg). The maximal
decrease was reached with treatment by 1 µmol/L
dexamethasone (58.7±4.7% and 47.5±7.0% of baseline
after 24 and 48 hours of incubation, respectively) (Fig 2).
|
|
To determine whether the dexamethasone-mediated
125I–[Sar1,Ala8]Ang II
binding decrease was due to decreased Bmax or
increased Kd, we performed saturation
binding experiments (Fig 3). Bound
125I–[Sar1,Ala8]Ang II
increased progressively with increasing concentrations of the tracer,
but a plateau was not constantly reached over the range of
concentrations studied. Scatchard analysis of the data (n=5)
demonstrated that Ang II binds to a single class of receptors with a
mean Bmax of 642±265 fmol/mg cell protein and a
Kd of 1.07±0.34 nmol/L. Preexposure of rat
mesangial cells to 1 µmol/L dexamethasone
during 24 hours resulted in a marked reduction in Ang II binding that
was due to a decrease in Ang II receptor Bmax
(379±144 fmol/mg cell protein), with no change in
Kd (1.21±0.41 nmol/L). Comparison of the
regression lines obtained in each experiment from the control and
experimental data indicated that the difference between
Bmax values (abscissa intercepts) was always
significant (P<.05). This change in
Bmax did not reflect changes in rat
mesangial cell protein content or cell number, as both of
these parameters were similar after a 48-hour exposure of
confluent rat mesangial cells to either control medium or 1
µmol/L dexamethasone. We also verified that
dexamethasone inhibited to the same extent the amount of
125I–[Sar1,Ala8]Ang II
bound at the cell surface receptors and the amount that had been
internalized. Indeed, 19% and 17% of these two fractions,
respectively, were inhibited by dexamethasone (1 µmol/L)
after 24 hours of treatment and 26% and 31%, respectively, after 48
hours of treatment.
|
For determination of whether the
dexamethasone-induced
125I–[Sar1,Ala8]Ang II
binding decrease was mediated by glucocorticoid receptors, Ang II
binding was measured after incubation with the glucocorticoid receptor
antagonist mifepristone.26 As shown in Fig 4, incubation with 1 µmol/L mifepristone for 24 hours
did not significantly modify
125I–[Sar1,Ala8]Ang II
binding (91.6±4.9% of baseline; baseline, 17.6±2.14 fmol/mg).
Dexamethasone (1 µmol/L) after 24 hours of incubation
induced a decrease in
125I–[Sar1,Ala8]Ang II
binding (59.0±7.2% of baseline) that was abolished by mifepristone
coincubation (92.1±9.7% of baseline).
|
To verify that the residual receptors after dexamethasone treatment kept the pharmacological characteristics of AT1 receptors, we performed competitive binding experiments with cells that had been treated during 48 hours with 1 µmol/L dexamethasone. Inhibitions of 70.9% and 84.2% of 125I–[Sar1,Ala8]Ang II binding (0.40 nmol/L) were observed in the presence of 1 and 100 nmol/L losartan, respectively. Inhibitions were of the same magnitude with unlabeled [Sar1,Ala8]Ang II (71.9% and 88.4% at 1 and 100 nmol/L, respectively), whereas PD 123319, an AT2 antagonist, had no inhibitory potency (105% and 108% of control at 1 and 100 nmol/L, respectively).
Effect of Dexamethasone on Ang II–Dependent IP
Stimulation in Rat Mesangial Cells
Stimulation of the G protein–coupled AT1
receptors is associated in mesangial cells with
phospholipase C activation and an increase in IP production by
hydrolysis of inositol phospholipids. IP3, which is
initially formed from phosphatidylinositol 4,5-bisphosphate breakdown,
undergoes phosphorylation and
dephosphorylation reactions that result in the
formation of a variety of compounds.27 Therefore, we
examined the effect of dexamethasone on this response to
Ang II. Fig 5 shows that 0.1 µmol/L Ang II rapidly
stimulated (within 1 minute) the production of the three IP
forms studied, IP1, IP2, and
IP3, which are characterized by their increasing
degrees of phosphorylation. Relative increases in
IP2 and IP3 were greater than the increase in
IP1. Dexamethasone preincubation slightly
diminished basal IP production and markedly inhibited IP
production in the presence of Ang II. Inhibition was observed
for the three IP forms but was greater for IP3
(P<.001) than for IP2 and IP1
(P<.05). We also studied the time course of
dexamethasone inhibition on Ang II–dependent IP
production. No effect was observed after 1 or 3 hours of
incubation. The decrease in IP formation reached 56% to 70% of
control after 6 hours of dexamethasone treatment. It was
greater for IP2 and IP3 (32% and 29% of
control, respectively) than for IP1 (68% of control) after
24 hours of treatment.
|
Distribution of AT1A and AT1B Receptor mRNA
Levels in Rat Mesangial Cells
Fig 6 shows the amplified cDNA products
generated from AT1A and AT1B receptor subtype
mRNAs in the rat liver, pituitary, and mesangial cells.
Only one of the dilutions in which the generation of the PCR
products was still in the exponential range is shown.
AT1A and AT1B receptor mRNA levels were both
expressed in rat mesangial cells, AT1B being
slightly but significantly predominant (42±2% and 58±2% for
AT1A and AT1B, respectively; n=4,
P<.05), whereas the liver contained only AT1A
receptor mRNA and the pituitary predominantly expressed the
AT1B receptor subtype mRNA (84±2%, n=10) as previously
described.9 25 The presence of the internal standard
allowed us to verify that the efficiency of the RT and PCR reactions in
the different preparations studied was similar. We also used the liver
and pituitary extracts to demonstrate that there was no
cross-hybridization of the two short cDNA probes considered
specific for AT1A and AT1B mRNA, respectively.
As expected, using Northern blot analysis, we found that the
liver expressed only AT1A, whereas the pituitary
expressed predominantly AT1B.
|
Effect of Dexamethasone on AT1A and
AT1B Receptor mRNA in Rat Mesangial
Cells
Since both AT1B and AT1A receptors are
expressed in rat mesangial cells, we questioned whether
regulation of the transcription of these two subtypes by
dexamethasone differed. To explore this question, we
measured AT1A and AT1B receptor mRNA levels by
Northern blot analysis with specific cDNA probes. As
shown in Fig 7 and the Table,
dexamethasone (1 µmol/L)-treated cells demonstrated
decreases in AT1B mRNA levels after 3 hours of incubation.
The decrease was maximal at 6 hours and was still present at 24
hours. In contrast, AT1A mRNA levels were not modified over
the same period of time. Mifepristone inhibited the effect of
dexamethasone (1 µmol/L during 6 hours) on
AT1B receptor mRNA (Fig 8). Mifepristone
alone had no significant effect (89±6.8% of control) but counteracted
the inhibitory effect of dexamethasone
(52.2±3.7% and 105.0±3.2% of control with dexamethasone
alone and dexamethasone plus mifepristone, respectively;
n=4, P<.01).
|
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
The results of our study indicate that cultured rat mesangial cell incubation with dexamethasone produced glucocorticoid-specific and concentration- and time-dependent decreases in 125I–[Sar1,Ala8]Ang II binding. Decreased binding was due to a reduction in the density of the receptor sites, with unchanged affinity. Dexamethasone affected cell surface–bound and internalized 125I–[Sar1,Ala8]Ang II to the same extent, indicating that there was no change in the internalization process of the Ang II–receptor complex, which is characteristic of this hormone.28 The dexamethasone effect on Ang II receptor number was shown to be glucocorticoid receptor specific because mifepristone blocked dexamethasone-induced changes in 125I–[Sar1,Ala8]Ang II binding. This drug acts by complexing with the glucocorticoid receptor, thereby preventing glucocorticoid binding and then activation of the glucocorticoid-responsive element responsible for stimulation of the transcription.26 The dexamethasone-dependent decrease in Ang II receptors was associated with an inhibition of the stimulatory effect of Ang II on IP accumulation, which also required a lag time of at least 6 hours. Activation of the AT1 receptors present in mesangial cells results in phospholipase C stimulation and an increase in IP production.19 29 The parallel effect of dexamethasone on 125I–[Sar1,Ala8]Ang II binding and IP accumulation suggests that both events were related. However, the decrease in Ang II Bmax was approximately 33%, whereas decreases in IP1, IP2, and IP3 accumulations reached 46%, 69%, and 77%, respectively. Schelling et al14 have recently demonstrated that dexamethasone induced an uncoupling of Ang II–dependent phospholipase C activation in rat vascular smooth muscle cells. This conclusion was based on the fact that in this preparation, dexamethasone upregulated Ang II binding and decreased IP formation. Therefore, it is possible that the inhibitory effect of dexamethasone on Ang II–dependent IP formation resulted from both the decrease in Ang II binding sites and a negative regulatory effect on Ang II receptor–phospholipase C coupling. Alternatively, the residual binding could be linked to another signaling system. There are many mechanisms by which dexamethasone could decrease the density of Ang II receptors at the mesangial cell surface: inhibition of transcription, the increase in mRNA degradation, alteration in the posttranscriptional stages of protein synthesis, and changes in the distribution of the receptor sites between cytosol and membrane or in the activity of the receptor itself. However, since dexamethasone modified Ang II receptor Bmax but not Kd and incubations of 6 hours or less with dexamethasone had no effect on Ang II binding, it was likely that the dexamethasone effect occurred via a decrease in protein synthesis. To test this hypothesis, we measured AT1 receptor mRNA levels by Northern blot analysis.
In previous reports,19 29 the majority of Ang II receptors in rat glomeruli and mesangial cells were identified as AT1 receptors on a pharmacological basis because they specifically bound the AT1 antagonists losartan and EXP 3174 but not the AT2 antagonist PD 123177. Only a small portion of approximately 14% of Ang II receptors in rat mesangial cells exhibited specific characteristics, including sensitivity to AT2 antagonists, suggesting that they represent an AT2 subtype.29 It was then demonstrated that the rat kidney and particularly the rat glomerulus contain both AT1 subtypes, AT1A and AT1B.25 30 31 It was thus necessary to examine AT1A and AT1B mRNA levels separately. For this purpose we used the RT-PCR technique and Northern blot analysis, with probes specific for each subtype. RT-PCR demonstrated the presence of AT1A and AT1B mRNAs in cultured rat mesangial cells, with a slight predominance for the latter. Both subtype mRNAs were also detected with the cDNA-specific probes. In support of their specificity is the fact that the short probes, which are less than 60% identical, did not cross-hybridize with the heterologous mRNA. This was demonstrated by the results of Northern blot analysis in tissues that express exclusively or predominantly one mRNA subtype, AT1A in the liver and AT1B in the pituitary gland. Moreover, these probes did not cross-hybridize in established CHO cells transfected with the AT1A and AT1B cDNA, respectively.31 Finally, radiolabeled riboprobes that were prepared from the same plasmids and with the same enzymes as those for the cDNA probes were recently used in an in situ hybridization study of the rat kidney and allowed the two AT1 subtypes to be distinguished.25
AT1A and AT1B receptor mRNAs were differently regulated by dexamethasone. Indeed, AT1B mRNA levels decreased with time, whereas AT1A levels remained unchanged. The fall in AT1B mRNA was apparent after 3 hours of incubation with dexamethasone and thus preceded the fall in 125I–[Sar1,Ala8]Ang II binding. This temporal relationship and the fact that mifepristone reversed the decrease in AT1B mRNA and Ang II binding site number as well strongly suggest that the events are related. Nevertheless, it still remains unclear whether dexamethasone inhibited the transcription rate and/or decreased the stability of the AT1B receptor gene product. Both effects have been previously described with glucocorticoids. For example, these agents selectively inhibited the transcription of the interleukin-1ß gene and decreased the stability of interleukin-1ß mRNA.32
Previous reports have concluded that glucocorticoids induced expression of the AT1 receptor gene and increased the number of AT1 receptors. These studies were performed on rat vascular smooth muscle cells with cDNA probes that did not distinguish the AT1A and AT1B mRNAs.13 14 Subsequently, it was shown that glucocorticoids specifically induced rat AT1A receptor gene expression. This was demonstrated on rat vascular smooth muscle cells with the use of AT1A and AT1B receptor cDNA probes prepared from the 3' untranslated regions16 and on rat cardiac fibroblasts and cardiac myocytes by quantitative PCR and nuclear run-off transcription assays.15 Moreover, nucleotide sequence analysis of the AT1A receptor gene revealed three glucocorticoid-responsive element sequences in the promoter region, two of which could be involved in the regulation of mRNA and receptor number.16 Significant differences in the regulatory regions of AT1A receptor and AT1B receptor genes that account for their different tissue-specific expression profiles have been described.33 34 The inhibitory effect of glucocorticoids on transcription has been related to the interaction of the glucocorticoid-receptor complex with other transcription factors sterically preventing them from occupying their target sites. Such a mechanism of interaction has been demonstrated with the transcriptional factor activator protein-1 (AP-1), a heterodimer of the oncogene products c-fos and c-jun. Indeed, inhibition of basal and induced transcription of the collagenase gene by glucocorticoids is exerted through interference with the action of AP-1.35 36 The model of direct interaction between the glucocorticoid-receptor complex and other transactivators involved in AT1B receptor gene regulation could explain the findings of the present study.
Our results show that AT1B receptor mRNA is abundantly expressed in rat mesangial cells so that its inhibition should be sufficient to result in the decrease of Ang II receptor number. The repartition of the two AT1 receptor subtypes in cultured rat mesangial cells indicates a slight preponderance for AT1B, contrary to what is observed in the whole rat kidney, where AT1A mRNA is largely predominant.11 In keeping with our results, Shanmugam et al,37 using the in situ hybridization technique, found AT1B mRNA in mesangial cells within the glomeruli of rat fetal kidneys, and Gasc et al,25 using the same method, reported that rat mesangial cells in the adult were more intensively labeled with the AT1B than AT1A probe. Quantitative RT-PCR analysis showed that AT1B receptor mRNA represented 27% of the total AT1 receptor mRNA in the whole rat kidney.9 Taken together, these results, as do ours, demonstrate a marked expression of the AT1B mRNA in rat mesangial cells. Therefore, it is logical to conclude that the fall by approximately 33% that we have observed in Ang II receptor number is likely to be related to the inhibition in AT1B mRNA expression.
The results of the present study indicate that dexamethasone can induce a variation of AT1B expression level in mesangial cells without affecting the expression of AT1A, whereas the contrary has been reported to occur in cardiac fibroblasts15 and vascular smooth muscle cells.13 This confirms the early data of Douglas12 in rat glomeruli and represents a supplementary example of the phenotypic differences between arterial smooth muscle cells and glomerular mesangial cells in the rat, although these two cell types possess contractile elements and have the same embryological origin. Moreover, the presence of two AT1 subtypes in the rat with disparate tissue expression and different controls represents an adjusted and efficient system that allows the effects of the regulatory factors to be differentiated according to the target cell.
|
Selected Abbreviations and Acronyms |
|---|
|
|
Acknowledgments |
|---|
This study was supported by a joint grant from the Institut National de la Santé et de la Recherche Médicale (INSERM) and Merck, Sharp & Dohme. The authors thank Pr Eric Clauser for his judicious advice and Aline Bidois and Nelly Knobloch for secretarial assistance.
Received February 14, 1995; first decision April 24, 1995; accepted December 5, 1995.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Szpirer C, Rivière M, Szpirer J, Levan G, Guo DF, Iwai N, Inagami T. Chromosomal assignment of human and rat hypertension candidate genes: type 1 angiotensin II receptor genes and the Sa gene. J Hypertens. 1993;31:1903-1906.
2. Murphy TJ, Alexander RW, Griendling KK, Runge MS, Bernstein KE. Isolation of a cDNA encoding the vascular type-1 angiotensin II receptor. Nature. 1991;351:233-236. [Medline] [Order article via Infotrieve]
3. Iwai N, Inagami T. Identification of two subtypes in the rat type I angiotensin II receptor. FEBS Lett. 1992;298:257-260. [Medline] [Order article via Infotrieve]
4. Chiu AT, Dunscomb J, Kosierowski J, Burton CRA, Santomenna LD, Corjay MH, Benfield P. The ligand binding signatures of the rat AT1A, AT1B and the human AT1 receptors are essentially identical. Biochem Biophys Res Commun. 1993;197:440-449. [Medline] [Order article via Infotrieve]
5.
Sandberg K, Hong J, Clark AJL, Shapira H, Catt
KJ. Cloning and expression of a novel angiotensin II
receptor subtype. J Biol Chem. 1992;267:9455-9458.
6. Kakkar SS, Riel KK, Neill JD. Differential expression of angiotensin II receptor subtype mRNAs (AT1A and AT1B) in the brain. Biochem Biophys Res Commun. 1992;185:688-692. [Medline] [Order article via Infotrieve]
7. Kakkar SS, Sellers JC, Devor DC, Musgrove LC, Neill JD. Angiotensin II type 1 receptor subtype cDNAs: differential tissue expression and hormonal regulation. Biochem Biophys Res Commun. 1992;183:1090-1096. [Medline] [Order article via Infotrieve]
8. Iwai N, Inagami T. Regulation of the expression of the rat angiotensin II receptor mRNA. Biochem Biophys Res Commun. 1992;182:1094-1099. [Medline] [Order article via Infotrieve]
9.
Llorens-Cortes C, Greenberg B, Huang H, Corvol
P. Tissular expression and regulation of type 1
angiotensin II receptor subtypes by quantitative reverse
transcriptase–polymerase chain reaction analysis.
Hypertension. 1994;24:538-548.
10. Nio Y, Matsubara H, Murasawa S, Kanasaki M, Inada M. Regulation of gene transcription of angiotensin II receptor subtypes in myocardial infarction. J Clin Invest. 1995;95:46-54.
11.
Du Y, Yao A, Guo D, Inagami T, Wang DH.
Differential regulation of angiotensin II receptor subtypes
in rat kidney by low dietary sodium.
Hypertension. 1995;25:872-877.
12.
Douglas JG. Corticosteroids
decrease glomerular angiotensin
receptors. Am J Physiol. 1987;252:F453-F457.
13. Sato A, Suzuki H, Murakami M, Nakazato Y, Iwaita Y, Saruta T. Glucocorticoid increases angiotensin II type 1 receptor and its gene expression. Hypertension. 1994;223:25-30.
14. Schelling JR, DeLuca DJ, Konieczkowski M, Marzu R, Sedor JR, Dubyak GR, Linas SL. Glucocorticoid uncoupling of angiotensin II-dependent phospholipase C activation in rat vascular smooth muscle cells. Kidney Int. 1994;46:675-682. [Medline] [Order article via Infotrieve]
15. Matsubara H, Kanasaki M, Murasawa S, Tsukaguchi Y, Nio Y, Inada M. Differential gene expression and regulation of angiotensin II receptor subtypes in rat cardiac fibroblasts and cardiomyocytes in culture. J Clin Invest. 1994;93:1592-1601.
16. Uno S, Guo DF, Nakajima M, Ohi H, Imada T, Hiramatsu R, Nakakubo H, Nakamura N, Inagami T. Glucocorticoid induction of rat angiotensin II type 1A receptor gene promoter. Biochem Biophys Res Commun. 1994;204:210-215. [Medline] [Order article via Infotrieve]
17. Foidart J, Sraer J, Delarue F, Mahieu P, Ardaillou R. Evidence for mesangial glomerular receptors for angiotensin II linked to mesangial cell contractility. FEBS Lett. 1980;121:333-339. [Medline] [Order article via Infotrieve]
18.
Fort P, Marty L, Piechaczyk M, El Subrouty S,
Dani C, Jeanteur P, Blanchard JM. Various rat adult tissues
express only one major mRNA species from the
glyceraldehyde-3-phosphate-dehydrogenase multigenic
family. Nucleic Acids Res. 1985;13:1431-1442.
19.
Chansel D, Czekalski S, Pham P, Ardaillou R.
Characterization of angiotensin II receptor subtypes in
human glomeruli and mesangial cells. Am J
Physiol. 1992;262:F432-F441.
20.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ.
Protein measurement with the Folin phenol reagent.
J Biol Chem. 1951;193:265-275.
21. Munson PJ, Rodbard D. Ligand: a versatile computerized approach for characterization of ligand-binding systems. Anal Biochem. 1980;107:220-239.[Medline] [Order article via Infotrieve]
22.
Chansel D, Bizet T, Vandermeersch S, Pham P,
Levy B, Ardaillou R. Differential regulation of
angiotensin II and losartan binding sites in
glomeruli and mesangial cells. Am J
Physiol. 1994;266:F384-F393.
23. Grandalano G, Biswas P, Ghosh D, Choudhury G, Abboud HE. Simvastatin inhibits PDGF-induced DNA synthesis in human mesangial cells. Kidney Int. 1993;44:503-508. [Medline] [Order article via Infotrieve]
24. He CJ, Rondeau E, Medcalf RL, Lacave R, Schleuning WD, Sraer JD. Thrombin increases proliferation and decreases fibrinolytic activity of kidney glomerular epithelial cells. J Cell Physiol. 1991;146:131-140. [Medline] [Order article via Infotrieve]
25.
Gasc JM, Shanmugam S, Sibony M, Corvol P. Tissue
specific expression of type 1 angiotensin II receptor
subtypes: an in situ hybridization study.
Hypertension. 1994;24:531-537.
26. Moguilewsky M, Philibert D. RU 38486: potent antiglucocorticoid activity correlated with strong binding to the cytosolic glucocorticoid receptor followed by an impaired activation. J Steroid Biochem. 1984;20:271-276. [Medline] [Order article via Infotrieve]
27. Shin SH, Fujiwara Y, Wada A, Takama T, Orita Y, Kamada T, Tagawa K. Angiotensin II-induced increase in inositol 1,4,5-trisphosphate in cultured rat mesangial cells. Biochem Biophys Res Commun. 1987;142:70-77. [Medline] [Order article via Infotrieve]
28. Ullian ME, Linas ST. Role of receptor cycling in the regulation of angiotensin II surface receptor number and angiotensin II uptake in rat vascular smooth muscle cells. J Clin Invest. 1989;84:840-846.
29.
Ernsberger P, Zhou J, Damon TH, Douglas JG.
Angiotensin II receptor subtypes in cultured rat renal
mesangial cells. Am J Physiol. 1992;263:F411-F416.
30. Ye MQ, Healy DP. Characterization of an angiotensin type-1 receptor partial cDNA from rat kidney: evidence for a novel AT1B receptor subtype. Biochem Biophys Res Commun. 1992;185:204-210. [Medline] [Order article via Infotrieve]
31.
Shanmugam S, Corvol P, Gasc JM. Ontogeny of the
two angiotensin II type I receptor subtypes in
rats. Am J Physiol. 1994;267:E828-E836.
32.
Lee SW, Tsou AP, Chan H, Thomas J, Petrie K, Eugui
EM, Allison AC. Glucocorticoids selectively inhibit the
transcription of the interleukin 1ß gene and decrease the stability
of interleukin 1ß mRNA. Proc Natl Acad Sci U S A. 1988;85:1204-1208.
33.
Murasawa S, Matsubara H, Urakami M, Inada M.
Regulatory elements that mediate expression of the gene for the
angiotensin II type 1A receptor for the
rat. J Biol Chem. 1993;268:26996-27003.
34. Guo DF, Inagami T. The genomic organization of the rat angiotensin II receptor AT1B. Biochim Biophys Acta. 1994;1218:91-94. [Medline] [Order article via Infotrieve]
35. Jonat C, Rahmsdorf HJ, Park KK, Sato ACB, Gebel S, Ponta H, Herrlich P. Antitumor promotion and antiinflammation: down-modulation of AP1 (Fos/Jun) activity by glucocorticoid hormone. Cell. 1990;62:1189-1204. [Medline] [Order article via Infotrieve]
36. Yang-Yen HF, Chambard JC, Sun YL, Smeal T, Schmidt TJ, Drouin J, Karin M. Transcriptional interference between c-jun and the glucocorticoid receptor: mutual inhibition of DNA binding due to direct protein-protein interaction. Cell. 1990;62:1205-1215. [Medline] [Order article via Infotrieve]
37.
Shanmugam S, Monnot C, Corvol P, Gasc JM.
Distribution of type 1 angiotensin II receptor subtype
messenger RNAs in the rat fetus.
Hypertension. 1994;23:137-141.
This article has been cited by other articles:
 |
|
 |
|
  I. G Bogdarina, P. J King, and A. J L Clark Characterization of the angiotensin (AT1b) receptor promoter and its regulation by glucocorticoids J. Mol. Endocrinol., August 1, 2009; 43(2): 73 - 80. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. R. Singh, K. M. Moritz, J. F. Bertram, and L. A. Cullen-McEwen Effects of dexamethasone exposure on rat metanephric development: in vitro and in vivo studies Am J Physiol Renal Physiol, August 1, 2007; 293(2): F548 - F554. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Y. Park, C. Y. Song, B. C. Kim, H. K. Hong, and H. S. Lee Angiotensin II mediates LDL-induced superoxide generation in mesangial cells Am J Physiol Renal Physiol, November 1, 2003; 285(5): F909 - F915. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Hus-Citharel, N. Bouby, J. Marchetti, D. Chansel, D. Goidin, D. Gourdji, P. Corvol, and C. Llorens-Cortes Desensitization of Type 1 Angiotensin II Receptor Subtypes in the Rat Kidney Endocrinology, November 1, 2001; 142(11): 4683 - 4692. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C.-B. Lanz, M. Causevic, C. Heiniger, F. J. Frey, B. M. Frey, and M. G. Mohaupt Fluid Shear Stress Reduces 11{beta}-Hydroxysteroid Dehydrogenase Type 2 Hypertension, January 1, 2001; 37(1): 160 - 169. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Schwobel, T. Fischer, B. Lanz, and M. Mohaupt Angiotensin II receptor subtypes determine induced NO production in rat glomerular mesangial cells Am J Physiol Renal Physiol, December 1, 2000; 279(6): F1092 - F1100. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Amiri and R. Garcia Regulation of angiotensin II receptors and PKC isoforms by glucose in rat mesangial cells Am J Physiol Renal Physiol, May 1, 1999; 276(5): F691 - F699. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Zhu, S. H. Zhang, C. Wagner, A. Kurtz, N. Maeda, T. Coffman, and W. J. Arendshorst Angiotensin AT1B Receptor Mediates Calcium Signaling in Vascular Smooth Muscle Cells of AT1A Receptor–Deficient Mice Hypertension, May 1, 1998; 31(5): 1171 - 1177. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||