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(Hypertension. 2001;37:535.)
© 2001 American Heart Association, Inc.

Scientific Contributions

Reactive Oxygen Species–Mediated Homologous Downregulation of Angiotensin II Type 1 Receptor mRNA by Angiotensin II

Toshihiro Ichiki; Kotaro Takeda; Tomotake Tokunou; Yuko Funakoshi; Kiyoko Ito; Naoko Iino; Akira Takeshita

From the Department of Cardiovascular Medicine, Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan.

Correspondence to Toshihiro Ichiki, MD, Department of Cardiovascular Medicine, Kyushu University Graduate School of Medical Sciences, 3-1-1 Maidashi, Higashi-ku, 812-8582 Fukuoka, Japan. E-mail ichiki{at}cardiol.med.kyushu-u.ac.jp

	Abstract

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Recent studies suggest a crucial role of reactive oxygen species (ROS) for the signaling of angiotensin (Ang) II through Ang II type 1 receptor (AT₁-R). However, the role of ROS in the regulation of AT₁-R expression has not been explored. In this study, we examined the effect of an antioxidant on the homologous downregulation of AT₁-R by Ang II. Ang II (10^-6 mol/L) decreased AT₁-R mRNA with a peak suppression at 6 hours of stimulation in rat aortic vascular smooth muscle cells. Preincubation of vascular smooth muscle cells with N-acetylcysteine (NAC), a potent antioxidant, almost completely inhibited the Ang II–induced downregulation of AT₁-R mRNA. The effect of NAC was due to stabilization of the AT₁-R mRNA that was destabilized by Ang II. The Ang II–induced AT₁-R mRNA downregulation was also blocked by PD98059, an extracellular signal–regulated protein kinase (ERK) kinase inhibitor. Ang II–induced ERK activation was inhibited by NAC as well as by PD98059. Exogenous H₂O₂ also suppressed AT₁-R mRNA. These results suggest that the production of ROS and the activation of ERK are critical for the downregulation of AT₁-R mRNA. The generation of ROS through stimulation of AT₁-R not only mediates signaling of Ang II but also may play a crucial role in the adaptation process of AT₁-R to the sustained stimulation of Ang II.

Key Words: receptor, angiotensin II • reactive oxygen species • protein kinases

	Introduction

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Angiotensin II (Ang II) plays an important role in the regulation of blood pressure, fluid homeostasis, and hypertrophy and/or hyperplasia of cardiovascular cells.¹ The biological function of Ang II is mediated by Ang II receptors located in the plasma membrane. Two isoforms of Ang II receptor have been cloned and designated type 1 receptor (AT₁-R)² and type 2 receptor.³ However, most of the cardiovascular effects of Ang II are ascribed to AT₁-R.

Downregulation of heterotrimeric G protein–coupled receptors by their own ligands is considered as an adaptation process of the receptors to prolonged agonist stimulation.^{4 5} It is known that conditions that activate the renin-angiotensin system in vivo downregulate Ang II receptors except in the adrenal cortex.^{6 7 8 9 10} Homologous downregulation of AT₁-R mRNA by Ang II is reported in vascular smooth muscle cells (VSMCs).¹¹ Ang II decreases the expression level of AT₁-mRNA via an increase in degradation rate. The accelerated degradation of AT₁-R mRNA is mediated by polyribosomal mRNA binding proteins induced by Ang II.¹² AT₁-R in VSMCs is also downregulated heterologously by fibroblast growth factor (FGF), platelet-derived growth factor (PDGF),¹³ and NO.¹⁴

Recent studies have shown a critical role of reactive oxygen species (ROS) in the signal transduction pathway of AT₁-R,¹⁵ as well as other growth factor receptors, such as PDGF¹⁶ and EGF.¹⁷ Treatment of VSMCs with Ang II for 4 hours increases production of superoxide (O₂^-) through membrane-bound NADH- and NADPH-driven oxidase.¹⁸ O₂^- mediates Ang II–induced hypertension because chronic infusion of liposome-encapsulated superoxide dismutase (SOD) strongly reduces the increased blood pressure induced by Ang II.¹⁹ H₂O₂ derived from NADH/NADPH oxidase activates p38 mitogen-activated protein kinase (MAPK) and is also important for Ang II–induced hypertrophy of VSMCs.²⁰ ROS plays an important role for the Ang II–induced activation of AP-1 that is a redox-sensitive transcription factor.¹⁵ However, the role of ROS generated by stimulation of AT₁-R in the regulation of AT₁-R expression has not been explored.

In the present study, we show that N-acetylcysteine (NAC), a potent antioxidant, almost completely inhibited Ang II–induced AT₁-R mRNA downregulation in VSMCs. This effect was due to stabilization of AT₁-R mRNA that is destabilized by Ang II stimulation.

	Methods

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Reagents
DMEM and FBS were purchased from GIBCO BRL. Ang II was purchased from Peptide Institute. PD98059, an extracellular signal-regulated protein kinase (ERK) kinase (MEK) inhibitor, was obtained from Research Biochemicals International. SB203580, an inhibitor of p38 MAPK, is a gift from SmithKline Beecham. Diphenylene iodonium (DPI) and NAC were obtained from Sigma Chemical Co.

[-³²P]dCTP and [¹²⁵I]Sar,¹Ile⁸-Ang II were obtained from DuPont NEN. Antibodies against phosphorylated forms of ERK (phospho-ERK) and ERK (recognizes both phosphorylated and nonphosphorylated forms) were obtained from New England Biolabs. Unless indicated otherwise, other chemical reagents were purchased from Wako Pure Chemicals.

Cell Culture
VSMCs were isolated from the thoracic aorta of Sprague-Dawley rats,²¹ maintained and used for the experiments as described previously.¹⁴ Passages between 5 and 12 were used for the experiments.

Northern Blot Analysis
Total RNA was prepared according to the acid guanidinium-phenol-chloroform extraction method.²² Northern blot analyses and quantification of the radioactivity of AT₁-R mRNA and 18S rRNA with a bioimaging analyzer were performed as described previously.¹⁴ To analyze the mRNA stability of AT₁-R, actinomycin D (5 µg/mL) was added after 3 hours of stimulation with Ang II and Northern blot analyses were performed.

Transfection of AT₁-R Gene Promoter- Luciferase Construct
A fusion DNA of AT₁-R gene promoter and luciferase (5 µg) that is described previously¹⁴ and LacZ expression plasmid (2 µg) were introduced to VSMCs according to DEAE dextran method and the manufacturer’s instructions (Promega). After 24 hours of transfection, the cells were stimulated with Ang II (10^-6 mol/L) for 24 hours. The luciferase activity was measured and normalized according to ß-galactosidase activity as described previously.¹⁴

Western Blot Analysis
VSMCs were lysed in a sample buffer (50 mmol/L NaCl, 30 mmol/L sodium phosphate, 50 mmol/L NaF, 5 mmol/L EDTA, 10 mmol/L Tris-HCl, pH 7.6, 1% Triton-X, 0.5% pepstatin A, 0.2 U/mL aprotinin, 5 mmol/L leupeptin, and 1 mmol/L PMSF) after stimulation. Protein concentration was quantified with BCA protein assay reagent (Pierce). Then, 20 µg of total protein was electrophoresed on 12% SDS-PAGE and transferred to polyvinylidine difluoride membrane (Immobilon; Millipore) electrophoretically (100 V, for 1 hour). Detection of phosphorylated ERK was performed with enhanced chemiluminescence according to the manufacturer’s instruction (Amersham Pharmacia Biotech). It has been shown that phosphorylation of MAPKs is associated with their activities.²³ Therefore, phosphorylation was considered as activation of MAPK. The membranes were stripped through incubation in a buffer containing 2% SDS, 100 mmol/L Tris-HCl, pH 7.4, and 100 mmol/L 2-mercaptoethanol at 70°C for 1 hour and reprobed with an antibody against ERK.

Estimation of AT₁-R Sites in VSMCs
The number of the AT₁-R binding sites in VSMCs was estimated by binding of [¹²⁵I]Sar,¹Ile⁸-Ang II as described previously¹⁴ in the presence or absence of NAC.

Statistical Analysis
Statistical analyses were performed with 1- or 2-way ANOVA and multiple comparison (Fisher’s) test if appropriate. A value of P<0.05 was considered significant.

	Results

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Downregulation of AT₁-R mRNA by Ang II
VSMCs were stimulated with Ang II (10^-6 mol/L), and the time course of AT₁-R mRNA expression was examined (Figure 1A). Ang II decreased AT₁-R mRNA expression with a peak suppression at 6 hours of stimulation. Ang II dose-dependently decreased the AT₁-R mRNA (Figure 1B).

View larger version (36K): [in this window] [in a new window]   Figure 1. Suppression of AT1-R mRNA by Ang II. VSMCs were stimulated (A) with Ang II (10-6 mol/L) for varying periods of time as indicated or (B) with varying concentrations of Ang II for 6 hours. Northern blot analyses of AT1-R mRNA and rRNA were performed. Left, Representative autoradiogram. Right, Radioactivity of the bands was measured with an imaging analyzer. The radioactivity of AT1-R mRNA was standardized with that of 18S rRNA. The ratio in the control was designated as 1. Results are expressed as mean±SEM (n=4). *P<0.01 vs control.

Effect of Antioxidants on Ang II–Induced AT₁-R mRNA Downregulation
Recent studies suggest that ROS play important roles in the signaling of AT₁-R.^{15 18 20 24} To examine the role of ROS in the homologous downregulation of AT₁-R mRNA, VSMCs were preincubated with NAC (10 mmol/L for 2 hours), a potent antioxidant, and stimulated with Ang II for 6 hours. NAC almost completely inhibited the Ang II–induced AT₁-R downregulation (Figure 2A). DPI was reported to inhibit NADH/NADPH oxidase and to reduce Ang II–induced H₂O₂ production.²⁰ DPI, however, failed to inhibit the Ang II–induced downregulation of AT₁-R mRNA.

View larger version (38K): [in this window] [in a new window]   Figure 2. Effect of antioxidants on the Ang II–induced AT1-R mRNA downregulation. VSMCs were preincubated with NAC (10 mmol/L for 2 hours) or DPI (10 µmol/L for 30 minutes). Then VSMCs were stimulated with Ang II (AII: 10-6 mol/L) for 6 hours. Northern blot analyses of AT1-R mRNA and rRNA were performed. Left, Representative autoradiogram. Right, Radioactivity of the bands was measured with an imaging analyzer. The radioactivity of AT1-R mRNA was standardized with that of 18S rRNA. The ratio in the control was designated as 1. Results are expressed as mean±SEM (n=4).

NAC Stabilized AT₁-R mRNA
Destabilization of AT₁-R mRNA was reported to be responsible for Ang II–induced AT₁-R mRNA downregulation.¹¹ We tested whether NAC affected the degradation rate of AT₁-R mRNA. As seen in Figure 3A, Ang II enhanced the degradation rate of AT₁-R mRNA and NAC inhibited the destabilization of AT₁-R mRNA induced by Ang II. Next we examined whether Ang II affected promoter activity of AT₁-R gene. The luciferase activity of luciferase gene driven by AT₁-R gene promoter was not changed significantly by Ang II stimulation (Figure 3B).

View larger version (19K): [in this window] [in a new window]   Figure 3. Effect of Ang II on the stability of AT1-R mRNA and AT1-R gene promoter activity. A, VSMCs were pretreated with or without NAC (10 mmol/L for 2 hours) and stimulated with Ang II (AII; 10-6 mol/L) for 3 hours. Then actinomycin D (5 µg/mL) was added and incubated for the indicated period. Northern blot analyses and an analysis of the radioactivity of the bands were performed as in Figure 1. The ratio of AT1-R mRNA/rRNA before actinomycin D addition in each group was designated as 1. Results are expressed as mean±SEM (n=4). The degradation rate did not differ statistically between the control group and the NAC plus Ang II group. P<0.01 vs control. P<0.01 vs AII+NAC (2-way ANOVA). B, AT1-R gene promoter-luciferase fusion DNA and LacZ expression plasmid were introduced to VSMCs. After 24 hours of transfection, the VSMCs were stimulated with Ang II (10-6 mol/L) for 24 hours, and luciferase and ß-galactosidase activities were measured.

To exclude the possibility that NAC inhibited the binding of Ang II to AT₁-R, binding of [¹²⁵I]Sar,¹Ile⁸-Ang II was examined in the presence or absence of NAC. Figure 4 shows that NAC did not affect the binding of Ang II to VSMCs.

View larger version (15K): [in this window] [in a new window]   Figure 4. Effect of NAC on Ang II binding to VSMCs. Binding of [125I]Sar,1Ile8-Ang II to VSMCs was determined in the presence or absence of NAC. A, Saturation curve. B, Scatchard plot analysis. Maximum binding and dissociation constant were similar between control and NAC-treated group.

H₂O₂ Suppressed AT₁-R mRNA
To confirm the role of ROS on AT₁-R expression, VSMCs were stimulated with H₂O₂ and the expression level of AT₁-R mRNA was examined. H₂O₂ suppressed AT₁-R mRNA in a time- and dose-dependent manner (Figure 5). The differential response of AT₁-R mRNA expression to H₂O₂ (300 µmol/L) between Figures 5A and 5B is probably due to the difference of the passage of VSMCs.

View larger version (30K): [in this window] [in a new window]   Figure 5. Suppression of AT1-R mRNA by H2O2. VSMCs were stimulated (A) with H2O2 (300 µmol/L) for varying periods of time as indicated or (B) with varying concentrations of H2O2 for 6 hours. Northern blot analyses of AT1-R mRNA and rRNA were performed. Left, Representative autoradiogram. Right, Radioactivity of the bands were measured with an imaging analyzer. The radioactivity of AT1-R mRNA was standardized with that of 18S rRNA. The ratio in the control was designated as 1. Results are expressed as mean±SEM (n=4). P<0.01 vs control.

ERK Is Involved in the AT₁-R mRNA Downregulation
ERK plays important roles for proliferation and differentiation of many cell types. It is well known that Ang II activates ERK in VSMCs.²⁵ To investigate the role of MAPK in Ang II–induced AT₁-R mRNA downregulation, VSMCs were pretreated with either PD98059 or SB203580 and then stimulated with Ang II for 6 hours. As shown in Figure 6, PD98059 inhibited the Ang II–induced AT₁-R mRNA downregulation, suggesting that ERK is involved in this process. SB203580 alone decreased AT₁-R mRNA level, and Ang II further decreased the AT₁-R mRNA level in the presence of SB203580. These data suggest that p38 MAPK may play a role for the basal expression of AT₁-R mRNA but not be involved in Ang II–induced downregulation.

View larger version (36K): [in this window] [in a new window]   Figure 6. Effect of MAPK inhibitors on Ang II–induced AT1-R mRNA downregulation. VSMCs were pretreated with PD98059 (30 µmol/L for 30 minutes) or SB203580 (10 µmol/L for 30 minutes) and stimulated with Ang II (AII: 10-6 mol/L) for 6 hours. Northern blot analyses (A) and an analysis of the radioactivity of the bands (B) were performed as in Figure 1. The ratio in the control was designated as 1. Results are expressed as mean±SEM (n=4). P<0.01 vs control. #P<0.05 vs SB203580.

Ang II induced phosphorylation of ERK with a peak at 3 minutes of stimulation as previously reported (data not shown and Eguchi et al²⁵ ). The Ang II–induced phosphorylation of ERK was blocked by preincubation with NAC as well as PD98059 (Figure 7A), whereas it was not significantly affected by DPI (Figure 7B) as reported previously.²⁰ H₂O₂ also activated ERK, and the activation was suppressed by PD98059 (Figure 7C).

View larger version (45K): [in this window] [in a new window]   Figure 7. Effect of PD98059, NAC, or DPI on Ang II- and H2O2-induced phosphorylation of ERK. VSMCs were pretreated (A) with PD98059 (30 µmol/L for 30 minutes) or NAC (10 mmol/L for 2 hours) or (B) with DPI (10 µmol/L for 30 minutes). Then VSMCs were stimulated with Ang II (AII: 10-6 mol/L) for 3 minutes. C, VSMCs were pretreated with PD98059 (30 µmol/L for 30 minutes) or SB203580 (10 µmol/L for 30 minutes) and then stimulated with H2O2 (300 µmol/L) for 10 minutes. Western blot analyses with an antibody against phosphorylated form of ERK (top: phospho-ERK) and an antibody against ERK (bottom) were performed. The same results were obtained in other independent experiments (n=4), and a representative autoradiogram is shown. IB indicates immunoblotting.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
We showed here that Ang II downregulated AT₁-R mRNA in an ROS- and ERK-dependent manner in rat VSMCs.

ROS have gained acceptance as a crucial molecule for the signal transduction of many receptors. H₂O₂ is an important signaling molecule of the PDGF receptor.¹⁶ The response of VSMCs to PDGF such as tyrosine phosphorylation, MAP kinase stimulation, and DNA synthesis was inhibited by blocking the production of H₂O₂ with NAC or catalase that catalyzes H₂O₂. Lo and Cruz²⁶ reported that basic FGF (bFGF)- and tumor necrosis factor –induced c-fos mRNA expression was strongly inhibited by NAC. In their report, DPI, an inhibitor of NADH/NADPH oxidase, also successfully inhibited the bFGF- and tumor necrosis factor –induced c-fos mRNA expression.

Recent studies suggest that ROS also play a crucial role in Ang II–induced hypertension. The infusion of Ang II to rats induced an activation of NADPH oxidase in aorta and high blood pressure accompanied by impairment of endothelial nitric oxide–dependent vasodilatation.¹⁹ This Ang II–induced high blood pressure was blocked by chronic infusion of liposome-encapsulated SOD, suggesting that Ang II–induced O₂^- is crucial for the vasoconstriction of resistant arteries. ROS are also important for the signaling of AT₁-R. Ang II–induced c-fos mRNA expression and AP-1 transcription factor activity were inhibited by NAC.¹⁵

Ushio-Fukai et al²⁰ also reported that DPI inhibited the Ang II–induced H₂O₂ production. Although NAC inhibited the Ang II–induced downregulation of AT₁-R mRNA, it was not inhibited by DPI. These data may suggest that other pathways than NADH/NADPH oxidase generated ROS in response to Ang II in our preparation. One possible molecule that is involved could be arachidonic acid. The release of arachidonic acid and subsequent metabolism by lipoxygenase pathway were reported to be crucial for VSMC growth on stimulation of G protein–coupled receptor.^{27 28} The possibility that ROS other than H₂O₂ induced by Ang II such as O₂^-18 or other metabolites induce the downregulation of AT₁-R mRNA is not excluded in the present study.

Recently, it was reported that activation of the thrombin receptor induced ROS generation through NADH/NADPH oxidase.²⁹ Also, differential effect of NAC and DPI on MAPK activation was reported as we observed in the Ang II–induced AT₁-R downregulation and ERK activation. NAC inhibited thrombin-induced activation of ERK, p38 MAPK, and Jun N-terminal kinase, whereas DPI inhibited only the thrombin-induced p38 MAPK activation. This study suggests that the broader inhibitory effects of NAC on MAPK activation may be due to glutathione, of which NAC increases its production by providing cysteine. Glutathiones not only scavenge ROS but also modulate intracellular levels of thiol.³⁰ Oxidation or reduction of thiol has been regarded as a critical regulatory mechanism of the activities of growth factors.^{31 32} In contrast, DPI strongly binds to flavoprotein and inhibits a variety of NAD(P)H-dependent oxidases.³³ Although DPI and NAC differentially inhibited thrombin-induced activation of MAPKs, both inhibited thrombin-induced proliferation of VSMCs.²⁹ Therefore, the differential antioxidant property between NAC and DPI may be a potential alternative explanation for the differential effect of these compounds on AT₁-R mRNA downregulation by Ang II. Further studies are necessary to clarify the NAC-sensitive and DPI-insensitive signaling pathway of AT₁-R.

Downregulation of the heterotrimeric G protein–coupled receptor is generally believed to be an adaptation process of these receptors to the prolonged ligand stimulation.^{4 5} The mechanism of this process is not clearly understood. In ß-adrenergic receptor, the induction of putative mRNA destabilizing factors that enhance the rate of degradation of receptor coding mRNA was proposed.^{34 35} A similar mRNA destabilizing factor was reported in Ang II–induced AT₁-R mRNA degradation.¹² Nickenig and Murphy¹² reported that polyribosomal mRNA binding protein that may enhance the decay of AT₁-R mRNA was induced by Ang II. Taken together, these previous reports and our data suggest that Ang II may induce this polyribosomal mRNA binding protein through an ERK-dependent pathway. However, the exact nature of this protein is still elusive.

It was also reported that Ang II decreased the transcription of AT₁-R gene.¹¹ However, the promoter activity of AT₁-R gene examined by luciferase assay was not affected by Ang II stimulation. Our result is consistent with a recent report³⁶ and suggests that the effect of Ang II on AT₁-R gene promoter activity, if any, is minimal.

A limitation of this study is that most of the experiments are conducted with chemical inhibitors such as PD98059 or NAC. We could not completely exclude the possible nonspecific effects of the drugs that we used. However, we carefully chose the concentration of these inhibitors based on several recent reports.^{20 23 25 26 29} Many studies, including these reports, have shown that these drugs are highly specific and sufficiently inhibit the target molecule at the concentration we used. Therefore, we believe that nonspecific effects are negligible. Studies with a dominant negative molecule may confirm our results.

ERK is a critical kinase that transduces mitogenic signals into the nucleus and leads to an activation of transcription factor and gene expression. The same signal transduction pathway activated through AT₁-R downregulates AT₁-R mRNA expression. The pathway between the production of ROS and the activation of ERK by Ang II is not clear at this point. However, this study shows that ROS and ERK are critical components for the homologous downregulation of AT₁-R mRNA by Ang II as well as for the signaling of AT₁-R.

	Acknowledgments

 
This study was supported in part by the Kaibara Morikazu Medical Research Promotion Foundation (Fukuoka, Japan), Uehara Memorial Foundation (Tokyo, Japan), and a Grant-in Aid for Scientific Research from the Ministry of Education, Science and Culture (11770355) (Japan).

Received October 25, 2000; first decision November 30, 2000; accepted December 14, 2000.

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