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Synergy of aldosterone and high salt induces vascular smooth muscle hypertrophy through up-regulation of NOX1
Journal of Steroid Biochemistry & Molecular Biology 111 (2008) 29–36
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Synergy of aldosterone and high salt induces vascular smooth muscle hypertrophy through up-regulation of NOX1 ChunYuan Fan a,1,2 , Yasuyuki Kawai d,1 , Satoru Inaba a , Kenichiro Arakawa a , Masato Katsuyama c , Kouji Kajinami d , Toshihiro Yasuda b , Chihiro Yabe-Nishimura c , Tadashi Konoshita a , Isamu Miyamori a,∗ a
Third Department of Internal Medicine, 23-3 Matsuoka-Shimoaitsuki, Eiheiji, Fukui 910-1193, Japan Department of Biology, Faculty of Medical Sciences, University of Fukui, 23-3 Matsuoka-Shimoaitsuki, Eiheiji, Fukui 910-1193, Japan c Department of Pharmacology, Kyoto Prefectural University of Medicine, 465 Kajii-cho Kawaramachi-Hirokoji Kamigyo-ku, Kyoto 602-0841, Japan d Department of Cardiology, Kanazawa Medical University 1-1 Daigaku, Uchinada, Ishikawa 920-0293, Japan b
a r t i c l e
i n f o
Article history: Received 22 May 2007 Accepted 19 February 2008 Keywords: Aldosterone Salt NOX1 Superoxide
a b s t r a c t Aldosterone and excessive salt intake are obviously implicated in human arteriosclerosis. Aldosterone activates NADPH oxidase that induces superoxide production and cardiovascular cell hypertrophy. The activity of NADPH oxidase is influenced by the expression of its subunit, through which, vasoactive agents activate in the enzyme. Here, we show that aldosterone elicited overexpression of the NOX1 catalytic subunit of NADPH oxidase in the presence of high salt in A7r5 vascular smooth muscle cells. We also showed that NOX1 is a key subunit involved in physiological aldosterone-induced NADPH oxidase activation. Aldosterone dose-dependently increased NOX1 expression and NADPH activity, which subsequently caused superoxide over-production and A7r5 cell hypertrophy. However, aldosterone had little effect on any of NOX1, superoxide over-production and cell hypertrophy in NOX1 knock-down A7r5 cells. These results suggest that the aldosterone-induced effects are mainly generated through NOX1. Aldosterone-induced NOX1 over-expression was augmented by 145 mM sodium chloride, as compared with control medium containing 135 mM NaCl. However, NOX1 over-expression was not induced in the absence of aldosterone, even in the presence of 185 mM NaCl. The mineralocorticoid receptor antagonist, eplerenone, completely abolished NOX1 over-expression, indicating that aldosterone is essential for this process. © 2008 Elsevier Ltd. All rights reserved.
Aldosterone is an independent risk factor for arteriosclerotic cardiovascular diseases [1,2]. Excess aldosterone induces cardiovascular damage [3,4] and many clinical mega-trials have shown that a pharmacological mineralocorticoid receptor (MR) blockade prevents cardiovascular events [5,6]. However, epidemiological studies have revealed that a few inhabitants of northern Brazil and the Yanomamo Indians of southern Venezuela do not develop hypertension or other cardiovascular diseases despite having high plasma aldosterone concentrations [7,8]. These two groups consume a very low salt diet, which might be responsible for this effect. However, high salt intake can lead to cardiovascular diseases in both humans and other animals with a normal range of plasma aldosterone [9].
∗ Corresponding author. Tel.: +81 776 61 3111; fax: +81 776 61 8111. E-mail address: [email protected] (I. Miyamori). 1 These authors contributed equally to this work. 2 Present address: Department of Nephrology, State Key Laboratory of biotherapy, West China Medical School, Sichuan University, Chengdu, China. 0960-0760/$ – see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.jsbmb.2008.02.012
Therefore, recent studies have focused on aldosterone and high salt as a synergistic cause of cardiovascular disease [10–12]. Oxidative stress plays an important role in atherosclerosis. Reactive oxygen species (ROS) comprise the major origin of oxidative stress in the cardiovascular and renal parenchymal system, and they are attributed to aldosterone and salt [13–15]. The ROS, superoxide, is a univalent reduced form of oxygen [16,17] that is catalyzed mainly by reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase [18]. This enzyme consists of plasma membranespanning catalytic subunits (gp91phox , NOX1 or NOX4) and p22phox , as well as cytosolic regulatory subunits (p47phox , p67phox and Rac). The expression profiles of these subunits are diverse in cardiovascular cells [19], and NOX1, NOX4 and p22phox are also expressed in vascular smooth muscle cells (VSMCs) [20–22]. These subunits participate in VSMC damage as they produce superoxide. We previously showed that NOX1 is involved in prostaglandin F2␣ -induced hypertrophy of VSMCs [23], and other investigators have reported that angiotensin II-induced VSMCs hypertrophy is inhibited by antisense p22phox [24].
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The present study investigates the roles of NADPH oxidase activation, superoxide production, aldosterone and high salt in the process of VSMC hypertrophy. 1. Material and methods 1.1. Reagents Aldosterone was purchased from Across Organics (Geel, Belgium). Dihydroethidium (DHE) was obtained from Molecular Probes (Carlsbad, CA). Diphenyleneiodonium (DPI) chloride and other reagents were obtained from Sigma (St. Louis, MO). A 10 mM stock solution of eplerenone (Pfizer Inc., New York, NY) was prepared in DMSO according to the instructions provided with the product and diluted over 1 × 103 -fold with medium immediately before use. Culture medium, fetal bovine serum (FBS) and antibiotics were obtained from Invitrogen (Carlsbad, CA). [3 H]-Phenylalanine was purchased from American Radiolabeled Chemicals (St. Louis, MO). 1.2. Cell culture The A7r5 rat aortic smooth muscle cell line (American Type Culture Collection, Rockville, MD) was cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FBS in 5% CO2 and humidified air at 37 ◦ C, passaged by trypsinization and used between 5 and 20 passages. 1.3. Real-time PCR A7r5 cells were seeded in 10-cm dishes (1 × 106 cells/dish) and cultured for 24 h in DMEM supplemented with 10% FBS followed by a further 48 h without FBS. The cells were incubated with or without known inhibitors (10 mmol/L eplerenone, 10 mol/L PD98059, 10 mol/L SB203580, 5 mol/L SP600125, 50 mol/L PP2 or 10 mol/L GF109203x) for 1 h, followed by various concentrations of aldosterone and sodium for 24 h. Various sodium concentrations were obtained by adding sodium chloride to DMEM. Total RNA isolated from the cells using acid guanidinium thiocyanate/phenol/chloroform was amplified by real-time PCR using an ABI Prism 7000 Sequence Detection System (Applied Biosystems, Foster City, CA) with the SuperScript III Platinum SYBR Green One-Step qRT-PCR kit (Invitrogen). All samples were investigated in triplicate. The specific primers for NOX1, NOX4, p22phox and p47phox were as follows: NOX1 forward, 5 -CACGCTGAGAAAGCCATTGGATCAC-3 ; reverse, 5 -GGATGGAATAGGCTGGAGAGAACA-3 , NOX4 forward, 5 -TTGTTCCTCATGGTTACAGCTTCT-3 ; reverse, 5 -ATCCATGAAAATGTTCTGAGACGTA-3 , p22phox forward, 5 -TGGCCTGATCCTCATCACAG-3 ; reverse, 5 -AGGCACGGACAGCAGTAAGT-3 , p47phox forward, 5 -TCACCGAGATCTACGAGTTC-3 ; reverse, 5 -TCCCATGAGGCTGTTGAAGT-3 . The aberrant formation of primer dimers was monitored using dissociation curves. Amplified products were resolved by electrophoresis on 2% agarose gels to confirm a single band. Copy numbers were calculated from standard curves generated using authentic NOX1, NOX4, p22phox and p47phox cDNA templates. Data are expressed as copies/microgram of RNA or as levels relative to control (%).
eplerenone, 100 nmol/L DPI or 0.5 mmol/L apocynin) for 1 h, followed by 1 nmol/L aldosterone and 160 mmol/L sodium for a further 12 h. The cells were washed with phosphate buffered saline (PBS), and then DHE (10 mmol/L) in Hank’s balanced salt solution was added to the dishes. Cells were incubated in a light-protected humidified chamber at 37 ◦ C for 30 min and images were obtained using a confocal laser scanning microscope (Leica TCS SPII AOBS; Leica, Wetzlar, Germany). 1.5. Measurement of superoxide production A7r5 cells seeded in 6-well plates (2.5 × 105 cells/well) were cultured in serum-free DMEM for 48 h, inhibitors (10 mol/L eplerenone or 100 nmol/L DPI or 0.5 mmol/L apocynin) for 1 h, and 1 nmol/L aldosterone and 160 mmol/L sodium for 12 h. The cells were then harvested with trypsin, resuspended in Hank’s balanced salt solution and incubated for 30 min at 37 ◦ C with 5 mmol/L DHE. The ethidium fluorescence (excitation, 488 nm; emission, 610 nm) resulting from the specific oxidation of DHE by superoxide was measured using a flow cytometer (Beckman Coulter Epics XL, Miami, FL). The geometric mean of the ethidium fluorescence intensity was then analyzed. 1.6. Measurement of protein synthesis Protein synthesis was measured as [3 H]-phenylalanine incorporation. Quiescent confluent cells cultured in 6-well plates were incubated with [3 H]-phenylalanine (0.44 mCi/mL) plus 1 nmol/L aldosterone and 160 or 135 mmol/L sodium in the presence or absence of 10 mol/L eplerenone or 100 nmol/L DPI or 0.5 mmol/L apocynin for 24 h at 37 ◦ C. After three washes with ice-cold PBS, the cells were incubated with 5% trichloroacetic acid (TCA) on ice for 1 h, fixed with 95% ethanol and then solubilized in 0.15N NaOH. The amount of incorporated [3 H]-phenylalanine was determined by liquid scintillation spectrometry (Aloka, Tokyo, Japan). 1.7. Synthesis of anti-NOX1 ribozymes Hammerhead ribozymes against NOX1 mRNA were designed using the MFOLD program as follows. Rzm 168 is targeted at the GUU triplet located at nucleotides 166–168 and Rzm 243 is targeted at the GUA located at nucleotides 241–243 of the rat NOX1 mRNA sequence. Schematic diagrams of these ribozymes and the construction of plasmids for ribozyme expression have been described elsewhere [23,25,26]. 1.8. Establishment of clones stably expressing anti-NOX1 ribozymes Ribozyme expression plasmids (pPUR-KE containing the NOX1 ribozyme sequence) were transfected into A7r5 cells using the GenePORTER2 transfection reagent (Gene Therapy Systems, San Diego, CA). Stable transfectants were selected by single cell cloning in the presence of puromycin (10 mg/mL). For mock transfection, the pUR-KE vector was transfected and selected with puromycin. Ribozyme expression was verified as described [23,27].
1.4. DHE staining 1.9. Statistical analysis We evaluated intracellular superoxide production using the oxidative fluorescence emitter, DHE. A7r5 cells seeded in 10cm dishes (1 × 106 cells/dish) were cultured in serum-free DMEM for 48 h and then incubated with inhibitors (either 10 mol/L
Data are expressed as means ± S.E.M. and were analyzed using the unpaired Student’s t-test for comparisons between two groups and by one-way ANOVA followed by the Tukey–Kramer test for
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Fig. 1. NOX1 mRNA expression in A7r5 cells. Results are shown as real-time PCR data. (A) Expression of NOX families with appropriate concentrations of aldosterone in high sodium (160 mmol/L) medium. (B) NOX1 expression in control (135 mmol/L) and high sodium (145, 160, and 185 mmol/L). (C) NOX1 expression with appropriate aldosterone in normal sodium (135 mmol/L) medium. (D) NOX1 expression in normal and high sodium with 1 nmol/L aldosterone. (E) Time course of NOX1 expression with 1 nmol/L aldosterone and 160 mmol/L sodium. *P < 0.05 compared with control. Ald, aldosterone.
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multiple comparisons. A probability value below 0.05 was considered statistically significant. 2. Results 2.1. Aldosterone and high salt synergistically increases NOX1 expression in A7r5 cells We examined the expression of NADPH oxidase regulation by aldosterone and high salt in A7r5 cells. Expression of the NADPH oxidase subunits (NOX1, NOX4, p22phox and p47phox ) was estimated by measuring mRNA production using real-time RT-PCR. Incubation with aldosterone for 24-h dose-dependently augmented NOX1 mRNA in the presence of high sodium (160 mmol/L). The expression of mRNA increased only in the presence of 0.1 nmol/L aldosterone and reached a plateau at 1 nmol/L (Fig. 1A). The expression of mRNA NOX4, p22phox and p47phox was not altered under the same conditions (Fig. 1A). This aldosterone-induced stimulation of NOX1 mRNA was diminished at the physiological sodium concentration (135 mmol/L) (Fig. 1C). However, NOX1 mRNA did not respond in the absence of aldosterone even with high sodium (185 mmol/L) in the medium (Fig. 1B). This means that NOX1 is not altered only by physiological osmotic stress. Additionally, NOX1 mRNA was significantly up-regulated at physiological sodium concentrations (control: 135 mmol/L sodium) in the presence of only 1 nmol/L aldosterone and increased dependently with the sodium concentration (Fig. 1D). These results suggest that aldosterone combined with a high sodium concentration stimulates NOX1 expression in vascular smooth muscle cells. The NOX1 stimulation began after 1–3 h in the presence of aldosterone, indicating that this effect is brought about through nucleolar MR (Fig. 1E). 2.2. Eplerenone suppresses aldosterone and high salt-induced NOX1 expression in A7r5 cells We examined NOX1 suppression by the selective MR inhibitor, eplerenone, because biological NOX1 might be stimulated by aldosterone and salt through ligand binding to MR. We found that eplerenone abolished aldosterone-induced NOX1 mRNA. By contrast, eplerenone did not totally suppress NOX1 induced by aldosterone together with high salt (Fig. 2A). This finding suggested that the effects of aldosterone and a high sodium concentration are mediated not only by ligand binding to MR but also via MR independent pathways. Therefore, we screened several signal transduction pathways by inhibiting their related kinases. We added PP2 (src kinase inhibitor), GF10923x (PKC inhibitor), PD98059 (ERK1/2 inhibitor), SB203580 (MAPK inhibitor) and SP600125 (JNK inhibitor) together with aldosterone and sodium to the culture medium. The induction of NOX1 was completely abrogated by PP2 and GF10923x, but hardly suppressed by PD98059, SB203580 and SP600125 (Fig. 2B). 2.3. Effects of aldosterone and high salt on superoxide production To evaluate whether aldosterone and high salt induce superoxide production in A7r5 cells, intracellular superoxide production was measured by DHE staining and fluorescence microscopy. A 12h incubation with aldosterone and high sodium activated A7r5 cell superoxide production as indicated by increased intercellular red signals (Fig. 3A: a and b), whereas eplerenone reduced production to almost the control level (Fig. 3A: d). The NADPH oxidase inhibitors, DPI and apocynin, prospectively suppressed superoxide production (Fig. 3A: e and f). Flow cytometry showed obvious fluorescence, and both the MR antagonist and NADPH oxidase inhibitors
Fig. 2. Eplerenone suppresses aldosterone and salt-induced NOX1 expression. Results are shown as real-time PCR data. (A) NOX1 mRNA in 1 nmol/L aldosterone and/or high sodium (160 mmol/L) with or without 10 mmol/L eplerenone. (B) NOX1 mRNA expression after incubation with several kinase inhibitors. Bars represent means ± S.E.M. *P < 0.05 compared with control; # P < 0.05 compared with aldosterone and high salt.; † P < 0.05 compared with aldosterone. Ald, aldosterone.
suppressed superoxide production in VSMC incubated with aldosterone and high salt. 2.4. Effects of aldosterone and high salt on VSMC hypertrophy The hypertrophy of VSMC is the most important influence of aldosterone and the biomarker of such hypertrophy is cellular uptake of [3 H]-phenylalanine. The uptake of [3 H]-phenylalanine was higher in growth-arrested A7r5 cells incubated with aldosterone and high sodium for 24 h than with aldosterone alone (Fig. 4). The increased uptake was neutralized by eplerenone, DPI and apocynin. These results suggest that aldosterone and high sodium induce VSMC hypertrophy and that the process is mediated by aldosterone through MR and subsequent superoxide production by NADPH oxidase. 2.5. Effects of aldosterone and high salt in NOX1 mRNA knock-down cells To verify that NOX1 is a key regulator of aldosterone, high salt-induced superoxide production and VSMC hypertrophy, we constructed NOX1 mRNA knock-down A7r5 cells expressing spe-
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Fig. 3. Aldosterone and high salt-induced superoxide production in A7r5 cells. (A) Superoxide production is visualized by dihydroethidium (DHE) staining. Included reagent(s) are shown on the right of each image. (B) Quantitative DHE staining. Right panel, fluorescence intensity detected by flow cytometry. Mean values were calculated from six independent samples. Bars represent means ± S.E.M. *P < 0.05 compared with control; # P < 0.05 compared with aldosterone and high salt; † P < 0.05 compared with aldosterone. Ald, aldosterone; Ep, eplerenone; Apo, apocynin.
cific ribozymes. Two knock-down clones expressing ribozyme 168 (Rzm168) and ribozyme 243 (Rzm243) knocked NOX1 mRNA down to about a half of the respective levels of a mock-transfected A7r5 cell (Fig. 5A). Aldosterone and high salt-induced NOX1 expression in both Rzm168 and Rzm243 cells, but to significantly lower levels than in mock-transfected cells (Fig. 5A). Furthermore, aldosterone and high salt-induced [3 H]-phenylalanine uptake were diminished in Rzm168 and Rzm243 cells (Fig. 5B). Superoxide production in Rzm168 and Rzm243 cells was not affected by aldosterone and high salt (Fig. 6A). Flow cytometry showed that measurable peaks of superoxide were suppressed in both NOX1 knock-down clones (Fig. 6B). These results suggested that NOX1/NADPH oxidase is involved in superoxide production and VSMC hypertrophy via aldosterone and high salt. 3. Discussion We demonstrated here that a combination of aldosterone and high salt caused superoxide production and VSMC hypertrophy through the up-regulation of NOX1, a catalytic subunit of
superoxide-generating NADPH oxidase. NOX1 was up-regulated in VSMCs in the presence of a high salt concentration and of aldosterone, and naturalized by the MR antagonist, eplerenone. Superoxide production and VSMC hypertrophy were mediated by aldosterone and high salt, and the effects were counteracted by eplerenone and NOX1 knock-down. These data suggest that the combination of aldosterone and high salt caused VSMC hypertrophy in response to superoxide production by NADPH oxidase that is up-regulated through MR. Both aldosterone and salt play important roles in atherosclerotic cardiovascular disease. Mineralocorticoid receptor antagonists and NADPH oxidase inhibitors might help to reduce of the incidence of cardiovascular disease through inhibiting vascular hypertrophy. Among the NOX superfamily, NOX1, NOX4 and NOX5 are expressed in VSMCs [20–22] and NOX4 is considered the major catalytic subunit of NADPH oxidases in endothelial cells [28]. These subunits play important roles in vascular hypertrophy; for example, the catalytic subunit of NADPH oxidase, p22phox , is implicated in angiotensin II (AngII)-induced VSMC hypertrophy [24–29]. We found that aldosterone and high salt did not affect NOX4, p22phox and p47phox expression. However, NOX1 was stimulated and this
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Fig. 4. Aldosterone and high salt-induced [3 H]-phenylalanine uptake. Calculated [3 H]-phenylalanine uptake by cells (N = 6). Bars represent means ± S.E.M. *P < 0.05 compared with control; # P < 0.05 compared with aldosterone and high salt treated. Ald, aldosterone; Ep, eplerenone; Apo, apocynin.
Fig. 5. NOX1 expression and [3 H]-phenylalanine uptake by cells expressing ribozyme. (A) NOX1 mRNA in cell clones expressing ribozyme and incubated with aldosterone and high salt. NOX1 mRNA was measured by real-time RT-PCR. Data are expressed as copies/microgram RNA (N = 3–6 per group). (B) Uptake of [3 H]phenylalanine in two cell clones each expressing ribozymes (N = 3). Bars represent means ± S.E.M. *P < 0.05; compared with control mock-transfected cells. # P < 0.05 compared with mock-transfected, aldosterone and high salt-treated cells. Ald, aldosterone.
effect was blocked by the MR antagonist, eplerenone. Aldosterone and high salt were involved in superoxide production by VSMCs followed by hypertrophic change. The overexpression of NOX1 in smooth muscle cells potentiates AngII-induced hypertension and hypertrophy [30]. The present study found that aldosterone and high salt did not elicit excessive superoxide production in NOX1 knock-down cells. Therefore, NOX1 is the major NADPH oxidase subunit in vascular cells associated with superoxide production and this enzyme system is functionally important in aldosterone and high salt-induced VSMC hypertrophy. The local renin–angiotensin–aldosterone system might exacerbate the intracellular redox state, since both AngII and aldosterone mutually act upon local vascular cells [31–33]. We reported NOX1 does not participate in AngII-induced VSMC hypertrophy in mice [34]. Thus, we postulated that aldosterone-induced superoxide production under conditions of high salt is independent of AngII. In support of this notion, eplerenone abolished aldosterone and high salt-induced superoxide production as did NADPH oxidase inhibitors. This suggests that aldosterone individually activates NADPH oxidase followed by excessive superoxide production in VSMCs. We found that NOX1 expression was induced by aldosterone and high salt after 3 h, and that eplerenone abolished superoxide production and cell hypertrophy. Aldosterone binds intracellular MR and translocates it to the nucleus, where it binds to its ligand and interacts with the regulatory region of target gene promoters [35]. By contrast, aldosterone might have a non-genomic effect in VSMC that activated the Na/H antiporter within minutes [36]. Therefore our results suggest that the effects of aldosterone and high salt are elicited through the classical genomic pathway. The precise mechanism through which aldosterone and salt induces a pro-oxidative effect on VSMC hypertrophy remains uncertain. Some vasoactive agents are involved in cardiovascular damage through regulation of the intra/extracellular mineral balance. For example, AngII induces vascular hypertrophy by increasing the cytosolic free calcium concentration [37,38], and aldosterone up-regulates the Na+ /H+ exchanger that catalyzes the electroneutral countertransport of extracellular Na+ and intracellular protons [39,40]. However, aldosterone increases cellular sodium uptake through the Na+ /H+ exchanger when the extracellular Na+ concentration is high (146 mmol/L) [41]. Moreover, elevated intracellular Na+ activates Ca2+ inward flow through vascular Na+ /Ca2+ exchanger type-1 [42]. We speculate that aldosterone and a high extracellular sodium concentration activate intercellular Ca2+ uptake and initiate the transduction of intracellular signals related to NOX1 expression in VSMCs. Another important finding of our study was that NOX1 expression was maximal in VSMCs incubated with a physiological aldosterone concentration and high salt. In contrast, physiological aldosterone with a normal salt concentration induced little superoxide production and cell hypertrophy. These findings suggest that a high salt intake leads to the progression of cardiovascular disorders at a physiological concentration of aldosterone. A recent study has shown that aldosterone stimulates ROS production through NADPH oxidase activation in rat mesangial cells [43]. However, the effective aldosterone concentration (100 nmol/L) outside the physiological range, and that notion that salt could lower the threshold of vascular hypertrophy is intriguing. Because eplerenone incompletely inhibited aldosterone and high salt-induced NOX1 up-regulation in the present study, we examined other pathways that might be involved in NOX1 regulation. We found that the MAPK family was not involved in NOX1 regulation by aldosterone and high salt, whereas src and PKC abrogated the effect. Transactivation of epidermal growth factor (EGF) receptors is dependent on src followed by
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Fig. 6. Superoxide production in A7r5 cells expressing ribozyme. (A) Superoxide production visualized by dihydroethidium (DHE) staining. Left, control; right, cells incubated with aldosterone and high salt. (B) Quantitative DHE staining. Right panel, fluorescence intensity detected by flow cytometry. Mean values were calculated from six independent samples. Bars represent means ± S.E.M. *P < 0.05 compared with control; # P < 0.05 compared with aldosterone and high salt-treated. Ald, aldosterone.
phosphatidylinositol-3 kinase [44,45], and we previously reported that PKC␦ up-regulates NOX1 via EGF receptor transactivation, which results in PGF2␣-induced hypertrophy of VSMC [46]. These findings suggest that PKC and EGF receptors play important roles in NOX1 regulation induced by aldosterone and high salt. Acknowledgements This study was supported by a grant from the Japanese Ministry of Health, Labour and Welfare 2005. This work was supported in part by Health and Labor Sciences Research Grants for Disorders of Adrenocortical Hormone Production from the Ministry of Health, Labor, and Welfare, Japan.
References [1] M. Fritsch Neves, E.L. Schiffrin, Aldosterone: a risk factor for vascular disease, Curr. Hypertens. Rep. 5 (2003) 59–65. [2] M.R. Ward, P. Kanellakis, D. Ramsey, J. Funder, A. Bobik, Eplerenone suppress constrictive remodeling and collagen accumulation after angioplasty in porcine coronary arteries, Circulation 104 (4) (2001) 467–472. [3] G.P. Rossi, V. Di Bello, C. Ganzaroli, A. Sacchetto, M. Cesari, A. Bertini, D. Giorgi, R. Scognamiglio, M. Mariani, A.C. Pessina, Excess aldosterone is associated with alterations of myocardial texture in primary aldosteronism, Hypertension 40 (2002) 23–27. [4] G.P. Rossi, A. Sacchetto, P. Visentin, C. Canali, G.R. Graniero, P. Palatini, A.C. Pessina, Changes in left ventricular anatomy and function in hypertension and primary aldosteronism, Hypertension 27 (1996) 1039– 1045. [5] B. Pitt, F. Zannad, W. Remme, R. Cody, A. Castaigne, A. Perez, J. Palensky, J. Wittes, The effect of spironolactone on morbidity and mortality in patients with severe heart failure, N. Engl. J. Med. 341 (1999) 709–717.
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[6] B. Pitt, W. Remme, F. Zannad, J. Neaton, F. Martinez, B. Roniker, R. Bittman, S. Hurley, J. Kleiman, M. Gatlin, Eplerenone, a selective aldosterone blocker, in patients with left ventricular dysfunction after myocardial infarction, N. Engl. J. Med. 348 (2003) 1309–1321. [7] W.J. Oliver, E.L. Cohen, J.V. Neel, Blood pressure, sodium intake, and sodium related hormones in the Yanomamo Indians, a no-salt culture, Circulation 52 (1975) 146–151. [8] J.J. Mancilha-Carvalho, R. de Oliveria, R.J. Esposito, Blood pressure and electrolyte excretion in the Yanomamo Indians, an isolated population, J. Human Hypertens. 3 (1989) 309–314. [9] P.R. Conlin, Interactions of high salt intake and the response of the cardiovascular system to aldosterone, Cardiol. Rev. 13 (2005) 118–124. [10] K. Yoshida, S. Kim-Mitsuyama, R. Wake, Y. Izumiya, Y. Izumi, T. Yukimura, M. Ueda, M. Yoshiyama, H. Iwao, Excess aldosterone under normal salt diet induces cardiac hypertrophy and infiltration via oxidative stress, Hypertens. Res. 28 (2005) 447–455. [11] E.R. Blasi, R. Rocha, A.E. Rudolph, E.A. Blomme, M.L. Polly, E.G. McMahon, Aldosterone/salt induces renal inflammation and fibrosis in hypertensive rats, Kidney Int. 63 (2003) 1791–1800. [12] Y. Sun, J. Zhang, L. Lu, S.S. Chen, M.T. Quinn, K.T. Weber, Aldosterone-induced inflammation in rat heart. Role of oxidative stress, Am. J. Pathol. 161 (2002) 1773–1781. [13] A. Nishiyama, L. Yao, Y. Nagai, K. Miyata, M. Yoshizumi, S. Kagami, S. Kondo, H. Kiyomoto, T. Shokoji, S. Kimura, M. Kohno, Y. Abe, Possible contributions of reactive oxygen species and mitogen-activated protein kinase to renal injury in aldosterone/salt-induced hypertensive rats, Hypertension 43 (2004) 841–848. [14] A. Virdis, M.F. Neves, F. Amiri, E. Viel, R.M. Touyz, E.L. Schiffrin, Spironolactone improved angiotensin-induced vascular changes and oxidative stress, Hypertension 40 (2002) 504–510. [15] M. Iglarz, R.M. Touyz, E.C. Viel, F. Amiri, E.L. Schiffrin, Involvement of oxidative stress in the profibrotic action of aldosterone. Interaction with the renin–angiotensin system, Am. J. Hypertens. 17 (2004) 597–603. [16] W. Droge, Free radicals in the physiological control of cell function, Physiol. Rev. 82 (2002) 47–95. [17] B. Lassegue, R.E. Clempus, Vascular NAD(P)H oxidases: specific features, expression, and regulation, Am. J. Physiol. 285 (2003) R277–R297. [18] K.K. Griendling, D. Sorescu, M. Ushio-Fukai, NAD(P)H oxidase: role in cardiovascular biology and disease, Circ. Res. 86 (2000) 494–501. [19] I.R. Hanna, Y. Taniyama, K. Szocs, P. Rocic, K.K. Griendling, NAD(P)H oxidasederived reactive oxygen species as mediators of angiotensin II signaling, Antioxid. Redox. Signal. 4 (2002) 899–914. [20] B. Banfi, G. Molnar, A. Maturana, K. Steger, B. Hegedus, N. Demaurex, K.H. Krause, A Ca(2+) -activated NADPH oxidase in testis, spleen, and lymph nodes, J. Biol. Chem. 276 (2001) 37594–37601. [21] G. Cheng, Z. Cao, X. Xu, E.G. van Meir, J.D. Lambeth, Homologs of gp91phox: cloning and tissue expression of Nox3, Nox4, and Nox5, Gene 269 (2001) 131–140. [22] B. Lassegue, D. Sorescu, K. Szocs, Q. Yin, M. Akers, Y. Zhang, S.L. Grant, J.D. Lambeth, K.K. Griendling, Novel gp91(phox) homologues in vascular smooth muscle cells : nox1 mediates angiotensin II-induced superoxide formation and redox-sensitive signaling pathways, Circ. Res. 88 (2001) 888–894. [23] M. Katsuyama, C. Fan, C. Yabe-Nishimura, NADPH oxidase is involved in prostaglandin F2alpha-induced hypertrophy of vascular smooth muscle cells: induction of NOX1 by PGF2alpha, J. Biol. Chem. 277 (2002) 13438–13442. [24] M. Ushio-Fukai, A.M. Zafari, T. Fukui, N. Ishizaka, K.K. Griendling, p22phox is a critical component of the superoxide-generating NADH/NADPH oxidase system and regulates angiotensin II-induced hypertrophy in vascular smooth muscle cells, J. Biol. Chem. 271 (1996) 23317–23321. [25] S. Koseki, T. Tanabe, K. Tani, S. Asano, T. Shioda, Y. Nagai, T. Shimada, J. Ohkawa, K. Taira, Factors governing the activity in vivo of ribozymes transcribed by RNA polymerase III, J. Virol. 73 (1999) 1868–1877. [26] Y. Kato, T. Kuwabara, M. Warashina, H. Toda, K. Taira, Relationships between the activities in vitro and in vivo of various kinds of ribozyme and their intracellular localization in mammalian cells, J. Biol. Chem. 276 (2001) 15378–15385. [27] T. Kuwabara, M. Warashina, M. Orita, S. Koseki, J. Ohkawa, K. Taira, Formation of a catalytically active dimer by tRNA(Val)-driven short ribozymes, Nat. Biotechnol. 16 (1998) 961–965.
[28] T. Ago, T. Kitazono, H. Ooboshi, T. Iyama, Y.H. Han, J. Takada, M. Wakisaka, S. Ibayashi, H. Utsumi, M. Iida, Nox4 as the major catalytic component of an endothelial NAD(P)H oxidase, Circulation 109 (2004) 227–233. [29] D.S. Weber, P. Rocic, A.M. Mellis, K. Laude, A.N. Lyle, D.G. Harrison, K.K. Griendling, Angiotensin II-induced hypertrophy is potentiated in mice overexpressing p22phox in vascular smooth muscle, Am. J. Physiol. Heart Circ. Physiol. 288 (2005) H37–H42. [30] A. Dikalova, R. Clempus, B. Lassegue, G. Cheng, J. McCoy, S. Dikalov, A. San Martin, A. Lyle, D.S. Weber, D. Weiss, W.R. Taylor, H.H. Schmidt, G.K. Owens, J.D. Lambeth, K.K. Griendling, Nox1 overexpression potentiates angiotensin IIinduced hypertension and vascular smooth muscle hypertrophy in transgenic mice, Circulation 112 (2005) 2668–2676. [31] K. Mukai, M. Imai, H. Shimada, Y. Ishimura, Isolation and characterization of rat CYP11B genes involved in late steps of mineralo- and glucocorticoid syntheses, J. Biol. Chem. 268 (1994) 9130–9137. [32] C.E. Gomez-Sanchez, M.Y. Zhou, E.N. Cozza, H. Morita, M.F. Foecking, E.P. Gomez-Sanchez, Aldosterone biosynthesis in the rat brain, Endocrinology 138 (1997) 3369–3373. [33] J.S. Silvestre, V. Robert, C. Heymes, B. Aupetit-Faisant, C. Mouas, J.M. Moalic, B. Swynghedauw, C. Delcayre, Myocardial production of aldosterone and corticosterone in the rat: physiological regulation, J. Biol. Chem. 273 (1998) 4883– 4891. [34] K. Matsuno, H. Yamada, K. Iwata, D. Jin, M. Katsuyama, M. Matsuki, S. Takai, K. Yamanishi, M. Miyazaki, H. Matsubara, C. Yabe-Nishimura, Nox1 is involved in angiotensin II-mediated hypertension: a study in Nox1-deficient mice, Circulation 112 (2005) 2677–2685. [35] D.J. Mangelsdorf, C. Thummel, M. Beato, P. Herrlich, G. Schutz, K. Umesono, B. Blumberg, P. Kastner, M. Mark, P. Chambon, R.M. Evans, The nuclear receptor superfamily: the second decade, Cell 83 (1995) 835–839. [36] M. Christ, K. Douwes, C. Eisen, G. Bechtner, K. Theisen, M. Wehling, Rapid effects of aldosterone on sodium transporter in vascular smooth muscle cells, Hypertension 25 (1995) 117–123. [37] D.F. Liao, J.L. Duff, G. Daum, S.L. Pelech, B.C. Berk, Angiotensin II stimulates MAP kinase kinase kinase activity in vascular smooth muscle cells, Role Raf. Circ Res. 79 (1996) 1007–1014. [38] C. Peiro, J.L. Llergo, J. Angulo, J.M. Lopez-Novoa, A. Rodriguez-Lopez, L. Rodriguez-Manas, C.F. Sanchez-Ferrer, Effects of captopril, losartan, and nifedipine on cell hypertrophy of cultured vascular smooth muscle from hypertensive Ren-2 transgenic rats, Br. J. Pharmacol. 121 (1997) 1438–1444. [39] S. Ebata, S. Muto, K. Okada, J. Nemoto, M. Amemiya, T. Saito, Y. Asano, Aldosterone activates Na+ /H+ exchanger in vascular smooth muscle cells by nongenomic and genomic mechanism, Kidney Int. 56 (1999) 1400–1412. [40] M. Karmazyn, Q. Liu, X.T. Gan, B.J. Brix, L. Fliegel, Aldosterone increases NHE-1 expression and induces NHE-1-dependent hypertrophy in neonatal rat ventricular myocytes, Hypertension 42 (2003) 1171–1176. [41] M. Yamamuro, M. Yoshimura, M. Nakayama, K. Abe, M. Shono, S. Suzuki, T. Sakamoto, Y. Saito, K. Nakao, H. Yasue, H. Ogawa, Direct effects of aldosterone on cardiomyocytes in the presence of normal and elevated extracellular sodium, Endocrinology 147 (2006) 1314–1321. [42] T. Iwamoto, S. Kita, J. Zhang, M.P. Blaustein, Y. Arai, S. Yoshida, K. Wakimoto, I. Komuro, T. Katsuragi, Salt-sensitive hypertension is triggered by Ca2+ entry via Na+ /Ca2+ exchanger type-1 in vascular smooth muscle, Nat. Med. 10 (2004) 1193–1199. [43] K. Miyata, M. Rahman, T. Shokoji, Y. Nagai, G.X. Zhang, G.P. Sun, S. Kimura, T. Yukimura, H. Kiyomoto, M. Kohno, Y. Abe, A. Nishiyama, Aldosterone stimulates reactive oxygen species production through activation of NADPH oxidase in rat mesangial cells, J. Am. Soc. Nephrol. 16 (2005) 2906–2912. [44] M. Ushio-Fukai, K.K. Griendling, P.L. Becker, L. Hilenski, S. Halleran, R.W. Alexander, Epidermal growth factor receptor transactivation by angiotensin II requires reactive oxygen species in vascular smooth muscle cells, Arterioscler. Thromb. Vasc. Biol. 21 (2001) 489–495. [45] D. Bokemeyer, U. Schmitz, H.J. Kramer, Angiotensin II-induced growth of vascular smooth muscle cells requires an Src-dependent activation of the epidermal growth factor receptor, Kidney Int. 58 (2000) 549–558. [46] C.Y. Fan, M. Katsuyama, C. Yabe-Nishimura, PKC␦ mediates up-regulation of NOX1, a catalytic subunit of NADPH oxidase, via transactivation of the EGF receptor: possible involvement of PKCd in vascular hypertrophy, Biochem. J. 390 (2005) 761–767.
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Synergy of aldosterone and high salt induces vascular smooth muscle hypertrophy through up-regulation of NOX1 ChunYuan Fan a,1,2 , Yasuyuki Kawai d,1 , Satoru Inaba a , Kenichiro Arakawa a , Masato Katsuyama c , Kouji Kajinami d , Toshihiro Yasuda b , Chihiro Yabe-Nishimura c , Tadashi Konoshita a , Isamu Miyamori a,∗ a
Third Department of Internal Medicine, 23-3 Matsuoka-Shimoaitsuki, Eiheiji, Fukui 910-1193, Japan Department of Biology, Faculty of Medical Sciences, University of Fukui, 23-3 Matsuoka-Shimoaitsuki, Eiheiji, Fukui 910-1193, Japan c Department of Pharmacology, Kyoto Prefectural University of Medicine, 465 Kajii-cho Kawaramachi-Hirokoji Kamigyo-ku, Kyoto 602-0841, Japan d Department of Cardiology, Kanazawa Medical University 1-1 Daigaku, Uchinada, Ishikawa 920-0293, Japan b
a r t i c l e
i n f o
Article history: Received 22 May 2007 Accepted 19 February 2008 Keywords: Aldosterone Salt NOX1 Superoxide
a b s t r a c t Aldosterone and excessive salt intake are obviously implicated in human arteriosclerosis. Aldosterone activates NADPH oxidase that induces superoxide production and cardiovascular cell hypertrophy. The activity of NADPH oxidase is influenced by the expression of its subunit, through which, vasoactive agents activate in the enzyme. Here, we show that aldosterone elicited overexpression of the NOX1 catalytic subunit of NADPH oxidase in the presence of high salt in A7r5 vascular smooth muscle cells. We also showed that NOX1 is a key subunit involved in physiological aldosterone-induced NADPH oxidase activation. Aldosterone dose-dependently increased NOX1 expression and NADPH activity, which subsequently caused superoxide over-production and A7r5 cell hypertrophy. However, aldosterone had little effect on any of NOX1, superoxide over-production and cell hypertrophy in NOX1 knock-down A7r5 cells. These results suggest that the aldosterone-induced effects are mainly generated through NOX1. Aldosterone-induced NOX1 over-expression was augmented by 145 mM sodium chloride, as compared with control medium containing 135 mM NaCl. However, NOX1 over-expression was not induced in the absence of aldosterone, even in the presence of 185 mM NaCl. The mineralocorticoid receptor antagonist, eplerenone, completely abolished NOX1 over-expression, indicating that aldosterone is essential for this process. © 2008 Elsevier Ltd. All rights reserved.
Aldosterone is an independent risk factor for arteriosclerotic cardiovascular diseases [1,2]. Excess aldosterone induces cardiovascular damage [3,4] and many clinical mega-trials have shown that a pharmacological mineralocorticoid receptor (MR) blockade prevents cardiovascular events [5,6]. However, epidemiological studies have revealed that a few inhabitants of northern Brazil and the Yanomamo Indians of southern Venezuela do not develop hypertension or other cardiovascular diseases despite having high plasma aldosterone concentrations [7,8]. These two groups consume a very low salt diet, which might be responsible for this effect. However, high salt intake can lead to cardiovascular diseases in both humans and other animals with a normal range of plasma aldosterone [9].
∗ Corresponding author. Tel.: +81 776 61 3111; fax: +81 776 61 8111. E-mail address: [email protected] (I. Miyamori). 1 These authors contributed equally to this work. 2 Present address: Department of Nephrology, State Key Laboratory of biotherapy, West China Medical School, Sichuan University, Chengdu, China. 0960-0760/$ – see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.jsbmb.2008.02.012
Therefore, recent studies have focused on aldosterone and high salt as a synergistic cause of cardiovascular disease [10–12]. Oxidative stress plays an important role in atherosclerosis. Reactive oxygen species (ROS) comprise the major origin of oxidative stress in the cardiovascular and renal parenchymal system, and they are attributed to aldosterone and salt [13–15]. The ROS, superoxide, is a univalent reduced form of oxygen [16,17] that is catalyzed mainly by reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase [18]. This enzyme consists of plasma membranespanning catalytic subunits (gp91phox , NOX1 or NOX4) and p22phox , as well as cytosolic regulatory subunits (p47phox , p67phox and Rac). The expression profiles of these subunits are diverse in cardiovascular cells [19], and NOX1, NOX4 and p22phox are also expressed in vascular smooth muscle cells (VSMCs) [20–22]. These subunits participate in VSMC damage as they produce superoxide. We previously showed that NOX1 is involved in prostaglandin F2␣ -induced hypertrophy of VSMCs [23], and other investigators have reported that angiotensin II-induced VSMCs hypertrophy is inhibited by antisense p22phox [24].
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The present study investigates the roles of NADPH oxidase activation, superoxide production, aldosterone and high salt in the process of VSMC hypertrophy. 1. Material and methods 1.1. Reagents Aldosterone was purchased from Across Organics (Geel, Belgium). Dihydroethidium (DHE) was obtained from Molecular Probes (Carlsbad, CA). Diphenyleneiodonium (DPI) chloride and other reagents were obtained from Sigma (St. Louis, MO). A 10 mM stock solution of eplerenone (Pfizer Inc., New York, NY) was prepared in DMSO according to the instructions provided with the product and diluted over 1 × 103 -fold with medium immediately before use. Culture medium, fetal bovine serum (FBS) and antibiotics were obtained from Invitrogen (Carlsbad, CA). [3 H]-Phenylalanine was purchased from American Radiolabeled Chemicals (St. Louis, MO). 1.2. Cell culture The A7r5 rat aortic smooth muscle cell line (American Type Culture Collection, Rockville, MD) was cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FBS in 5% CO2 and humidified air at 37 ◦ C, passaged by trypsinization and used between 5 and 20 passages. 1.3. Real-time PCR A7r5 cells were seeded in 10-cm dishes (1 × 106 cells/dish) and cultured for 24 h in DMEM supplemented with 10% FBS followed by a further 48 h without FBS. The cells were incubated with or without known inhibitors (10 mmol/L eplerenone, 10 mol/L PD98059, 10 mol/L SB203580, 5 mol/L SP600125, 50 mol/L PP2 or 10 mol/L GF109203x) for 1 h, followed by various concentrations of aldosterone and sodium for 24 h. Various sodium concentrations were obtained by adding sodium chloride to DMEM. Total RNA isolated from the cells using acid guanidinium thiocyanate/phenol/chloroform was amplified by real-time PCR using an ABI Prism 7000 Sequence Detection System (Applied Biosystems, Foster City, CA) with the SuperScript III Platinum SYBR Green One-Step qRT-PCR kit (Invitrogen). All samples were investigated in triplicate. The specific primers for NOX1, NOX4, p22phox and p47phox were as follows: NOX1 forward, 5 -CACGCTGAGAAAGCCATTGGATCAC-3 ; reverse, 5 -GGATGGAATAGGCTGGAGAGAACA-3 , NOX4 forward, 5 -TTGTTCCTCATGGTTACAGCTTCT-3 ; reverse, 5 -ATCCATGAAAATGTTCTGAGACGTA-3 , p22phox forward, 5 -TGGCCTGATCCTCATCACAG-3 ; reverse, 5 -AGGCACGGACAGCAGTAAGT-3 , p47phox forward, 5 -TCACCGAGATCTACGAGTTC-3 ; reverse, 5 -TCCCATGAGGCTGTTGAAGT-3 . The aberrant formation of primer dimers was monitored using dissociation curves. Amplified products were resolved by electrophoresis on 2% agarose gels to confirm a single band. Copy numbers were calculated from standard curves generated using authentic NOX1, NOX4, p22phox and p47phox cDNA templates. Data are expressed as copies/microgram of RNA or as levels relative to control (%).
eplerenone, 100 nmol/L DPI or 0.5 mmol/L apocynin) for 1 h, followed by 1 nmol/L aldosterone and 160 mmol/L sodium for a further 12 h. The cells were washed with phosphate buffered saline (PBS), and then DHE (10 mmol/L) in Hank’s balanced salt solution was added to the dishes. Cells were incubated in a light-protected humidified chamber at 37 ◦ C for 30 min and images were obtained using a confocal laser scanning microscope (Leica TCS SPII AOBS; Leica, Wetzlar, Germany). 1.5. Measurement of superoxide production A7r5 cells seeded in 6-well plates (2.5 × 105 cells/well) were cultured in serum-free DMEM for 48 h, inhibitors (10 mol/L eplerenone or 100 nmol/L DPI or 0.5 mmol/L apocynin) for 1 h, and 1 nmol/L aldosterone and 160 mmol/L sodium for 12 h. The cells were then harvested with trypsin, resuspended in Hank’s balanced salt solution and incubated for 30 min at 37 ◦ C with 5 mmol/L DHE. The ethidium fluorescence (excitation, 488 nm; emission, 610 nm) resulting from the specific oxidation of DHE by superoxide was measured using a flow cytometer (Beckman Coulter Epics XL, Miami, FL). The geometric mean of the ethidium fluorescence intensity was then analyzed. 1.6. Measurement of protein synthesis Protein synthesis was measured as [3 H]-phenylalanine incorporation. Quiescent confluent cells cultured in 6-well plates were incubated with [3 H]-phenylalanine (0.44 mCi/mL) plus 1 nmol/L aldosterone and 160 or 135 mmol/L sodium in the presence or absence of 10 mol/L eplerenone or 100 nmol/L DPI or 0.5 mmol/L apocynin for 24 h at 37 ◦ C. After three washes with ice-cold PBS, the cells were incubated with 5% trichloroacetic acid (TCA) on ice for 1 h, fixed with 95% ethanol and then solubilized in 0.15N NaOH. The amount of incorporated [3 H]-phenylalanine was determined by liquid scintillation spectrometry (Aloka, Tokyo, Japan). 1.7. Synthesis of anti-NOX1 ribozymes Hammerhead ribozymes against NOX1 mRNA were designed using the MFOLD program as follows. Rzm 168 is targeted at the GUU triplet located at nucleotides 166–168 and Rzm 243 is targeted at the GUA located at nucleotides 241–243 of the rat NOX1 mRNA sequence. Schematic diagrams of these ribozymes and the construction of plasmids for ribozyme expression have been described elsewhere [23,25,26]. 1.8. Establishment of clones stably expressing anti-NOX1 ribozymes Ribozyme expression plasmids (pPUR-KE containing the NOX1 ribozyme sequence) were transfected into A7r5 cells using the GenePORTER2 transfection reagent (Gene Therapy Systems, San Diego, CA). Stable transfectants were selected by single cell cloning in the presence of puromycin (10 mg/mL). For mock transfection, the pUR-KE vector was transfected and selected with puromycin. Ribozyme expression was verified as described [23,27].
1.4. DHE staining 1.9. Statistical analysis We evaluated intracellular superoxide production using the oxidative fluorescence emitter, DHE. A7r5 cells seeded in 10cm dishes (1 × 106 cells/dish) were cultured in serum-free DMEM for 48 h and then incubated with inhibitors (either 10 mol/L
Data are expressed as means ± S.E.M. and were analyzed using the unpaired Student’s t-test for comparisons between two groups and by one-way ANOVA followed by the Tukey–Kramer test for
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Fig. 1. NOX1 mRNA expression in A7r5 cells. Results are shown as real-time PCR data. (A) Expression of NOX families with appropriate concentrations of aldosterone in high sodium (160 mmol/L) medium. (B) NOX1 expression in control (135 mmol/L) and high sodium (145, 160, and 185 mmol/L). (C) NOX1 expression with appropriate aldosterone in normal sodium (135 mmol/L) medium. (D) NOX1 expression in normal and high sodium with 1 nmol/L aldosterone. (E) Time course of NOX1 expression with 1 nmol/L aldosterone and 160 mmol/L sodium. *P < 0.05 compared with control. Ald, aldosterone.
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multiple comparisons. A probability value below 0.05 was considered statistically significant. 2. Results 2.1. Aldosterone and high salt synergistically increases NOX1 expression in A7r5 cells We examined the expression of NADPH oxidase regulation by aldosterone and high salt in A7r5 cells. Expression of the NADPH oxidase subunits (NOX1, NOX4, p22phox and p47phox ) was estimated by measuring mRNA production using real-time RT-PCR. Incubation with aldosterone for 24-h dose-dependently augmented NOX1 mRNA in the presence of high sodium (160 mmol/L). The expression of mRNA increased only in the presence of 0.1 nmol/L aldosterone and reached a plateau at 1 nmol/L (Fig. 1A). The expression of mRNA NOX4, p22phox and p47phox was not altered under the same conditions (Fig. 1A). This aldosterone-induced stimulation of NOX1 mRNA was diminished at the physiological sodium concentration (135 mmol/L) (Fig. 1C). However, NOX1 mRNA did not respond in the absence of aldosterone even with high sodium (185 mmol/L) in the medium (Fig. 1B). This means that NOX1 is not altered only by physiological osmotic stress. Additionally, NOX1 mRNA was significantly up-regulated at physiological sodium concentrations (control: 135 mmol/L sodium) in the presence of only 1 nmol/L aldosterone and increased dependently with the sodium concentration (Fig. 1D). These results suggest that aldosterone combined with a high sodium concentration stimulates NOX1 expression in vascular smooth muscle cells. The NOX1 stimulation began after 1–3 h in the presence of aldosterone, indicating that this effect is brought about through nucleolar MR (Fig. 1E). 2.2. Eplerenone suppresses aldosterone and high salt-induced NOX1 expression in A7r5 cells We examined NOX1 suppression by the selective MR inhibitor, eplerenone, because biological NOX1 might be stimulated by aldosterone and salt through ligand binding to MR. We found that eplerenone abolished aldosterone-induced NOX1 mRNA. By contrast, eplerenone did not totally suppress NOX1 induced by aldosterone together with high salt (Fig. 2A). This finding suggested that the effects of aldosterone and a high sodium concentration are mediated not only by ligand binding to MR but also via MR independent pathways. Therefore, we screened several signal transduction pathways by inhibiting their related kinases. We added PP2 (src kinase inhibitor), GF10923x (PKC inhibitor), PD98059 (ERK1/2 inhibitor), SB203580 (MAPK inhibitor) and SP600125 (JNK inhibitor) together with aldosterone and sodium to the culture medium. The induction of NOX1 was completely abrogated by PP2 and GF10923x, but hardly suppressed by PD98059, SB203580 and SP600125 (Fig. 2B). 2.3. Effects of aldosterone and high salt on superoxide production To evaluate whether aldosterone and high salt induce superoxide production in A7r5 cells, intracellular superoxide production was measured by DHE staining and fluorescence microscopy. A 12h incubation with aldosterone and high sodium activated A7r5 cell superoxide production as indicated by increased intercellular red signals (Fig. 3A: a and b), whereas eplerenone reduced production to almost the control level (Fig. 3A: d). The NADPH oxidase inhibitors, DPI and apocynin, prospectively suppressed superoxide production (Fig. 3A: e and f). Flow cytometry showed obvious fluorescence, and both the MR antagonist and NADPH oxidase inhibitors
Fig. 2. Eplerenone suppresses aldosterone and salt-induced NOX1 expression. Results are shown as real-time PCR data. (A) NOX1 mRNA in 1 nmol/L aldosterone and/or high sodium (160 mmol/L) with or without 10 mmol/L eplerenone. (B) NOX1 mRNA expression after incubation with several kinase inhibitors. Bars represent means ± S.E.M. *P < 0.05 compared with control; # P < 0.05 compared with aldosterone and high salt.; † P < 0.05 compared with aldosterone. Ald, aldosterone.
suppressed superoxide production in VSMC incubated with aldosterone and high salt. 2.4. Effects of aldosterone and high salt on VSMC hypertrophy The hypertrophy of VSMC is the most important influence of aldosterone and the biomarker of such hypertrophy is cellular uptake of [3 H]-phenylalanine. The uptake of [3 H]-phenylalanine was higher in growth-arrested A7r5 cells incubated with aldosterone and high sodium for 24 h than with aldosterone alone (Fig. 4). The increased uptake was neutralized by eplerenone, DPI and apocynin. These results suggest that aldosterone and high sodium induce VSMC hypertrophy and that the process is mediated by aldosterone through MR and subsequent superoxide production by NADPH oxidase. 2.5. Effects of aldosterone and high salt in NOX1 mRNA knock-down cells To verify that NOX1 is a key regulator of aldosterone, high salt-induced superoxide production and VSMC hypertrophy, we constructed NOX1 mRNA knock-down A7r5 cells expressing spe-
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Fig. 3. Aldosterone and high salt-induced superoxide production in A7r5 cells. (A) Superoxide production is visualized by dihydroethidium (DHE) staining. Included reagent(s) are shown on the right of each image. (B) Quantitative DHE staining. Right panel, fluorescence intensity detected by flow cytometry. Mean values were calculated from six independent samples. Bars represent means ± S.E.M. *P < 0.05 compared with control; # P < 0.05 compared with aldosterone and high salt; † P < 0.05 compared with aldosterone. Ald, aldosterone; Ep, eplerenone; Apo, apocynin.
cific ribozymes. Two knock-down clones expressing ribozyme 168 (Rzm168) and ribozyme 243 (Rzm243) knocked NOX1 mRNA down to about a half of the respective levels of a mock-transfected A7r5 cell (Fig. 5A). Aldosterone and high salt-induced NOX1 expression in both Rzm168 and Rzm243 cells, but to significantly lower levels than in mock-transfected cells (Fig. 5A). Furthermore, aldosterone and high salt-induced [3 H]-phenylalanine uptake were diminished in Rzm168 and Rzm243 cells (Fig. 5B). Superoxide production in Rzm168 and Rzm243 cells was not affected by aldosterone and high salt (Fig. 6A). Flow cytometry showed that measurable peaks of superoxide were suppressed in both NOX1 knock-down clones (Fig. 6B). These results suggested that NOX1/NADPH oxidase is involved in superoxide production and VSMC hypertrophy via aldosterone and high salt. 3. Discussion We demonstrated here that a combination of aldosterone and high salt caused superoxide production and VSMC hypertrophy through the up-regulation of NOX1, a catalytic subunit of
superoxide-generating NADPH oxidase. NOX1 was up-regulated in VSMCs in the presence of a high salt concentration and of aldosterone, and naturalized by the MR antagonist, eplerenone. Superoxide production and VSMC hypertrophy were mediated by aldosterone and high salt, and the effects were counteracted by eplerenone and NOX1 knock-down. These data suggest that the combination of aldosterone and high salt caused VSMC hypertrophy in response to superoxide production by NADPH oxidase that is up-regulated through MR. Both aldosterone and salt play important roles in atherosclerotic cardiovascular disease. Mineralocorticoid receptor antagonists and NADPH oxidase inhibitors might help to reduce of the incidence of cardiovascular disease through inhibiting vascular hypertrophy. Among the NOX superfamily, NOX1, NOX4 and NOX5 are expressed in VSMCs [20–22] and NOX4 is considered the major catalytic subunit of NADPH oxidases in endothelial cells [28]. These subunits play important roles in vascular hypertrophy; for example, the catalytic subunit of NADPH oxidase, p22phox , is implicated in angiotensin II (AngII)-induced VSMC hypertrophy [24–29]. We found that aldosterone and high salt did not affect NOX4, p22phox and p47phox expression. However, NOX1 was stimulated and this
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Fig. 4. Aldosterone and high salt-induced [3 H]-phenylalanine uptake. Calculated [3 H]-phenylalanine uptake by cells (N = 6). Bars represent means ± S.E.M. *P < 0.05 compared with control; # P < 0.05 compared with aldosterone and high salt treated. Ald, aldosterone; Ep, eplerenone; Apo, apocynin.
Fig. 5. NOX1 expression and [3 H]-phenylalanine uptake by cells expressing ribozyme. (A) NOX1 mRNA in cell clones expressing ribozyme and incubated with aldosterone and high salt. NOX1 mRNA was measured by real-time RT-PCR. Data are expressed as copies/microgram RNA (N = 3–6 per group). (B) Uptake of [3 H]phenylalanine in two cell clones each expressing ribozymes (N = 3). Bars represent means ± S.E.M. *P < 0.05; compared with control mock-transfected cells. # P < 0.05 compared with mock-transfected, aldosterone and high salt-treated cells. Ald, aldosterone.
effect was blocked by the MR antagonist, eplerenone. Aldosterone and high salt were involved in superoxide production by VSMCs followed by hypertrophic change. The overexpression of NOX1 in smooth muscle cells potentiates AngII-induced hypertension and hypertrophy [30]. The present study found that aldosterone and high salt did not elicit excessive superoxide production in NOX1 knock-down cells. Therefore, NOX1 is the major NADPH oxidase subunit in vascular cells associated with superoxide production and this enzyme system is functionally important in aldosterone and high salt-induced VSMC hypertrophy. The local renin–angiotensin–aldosterone system might exacerbate the intracellular redox state, since both AngII and aldosterone mutually act upon local vascular cells [31–33]. We reported NOX1 does not participate in AngII-induced VSMC hypertrophy in mice [34]. Thus, we postulated that aldosterone-induced superoxide production under conditions of high salt is independent of AngII. In support of this notion, eplerenone abolished aldosterone and high salt-induced superoxide production as did NADPH oxidase inhibitors. This suggests that aldosterone individually activates NADPH oxidase followed by excessive superoxide production in VSMCs. We found that NOX1 expression was induced by aldosterone and high salt after 3 h, and that eplerenone abolished superoxide production and cell hypertrophy. Aldosterone binds intracellular MR and translocates it to the nucleus, where it binds to its ligand and interacts with the regulatory region of target gene promoters [35]. By contrast, aldosterone might have a non-genomic effect in VSMC that activated the Na/H antiporter within minutes [36]. Therefore our results suggest that the effects of aldosterone and high salt are elicited through the classical genomic pathway. The precise mechanism through which aldosterone and salt induces a pro-oxidative effect on VSMC hypertrophy remains uncertain. Some vasoactive agents are involved in cardiovascular damage through regulation of the intra/extracellular mineral balance. For example, AngII induces vascular hypertrophy by increasing the cytosolic free calcium concentration [37,38], and aldosterone up-regulates the Na+ /H+ exchanger that catalyzes the electroneutral countertransport of extracellular Na+ and intracellular protons [39,40]. However, aldosterone increases cellular sodium uptake through the Na+ /H+ exchanger when the extracellular Na+ concentration is high (146 mmol/L) [41]. Moreover, elevated intracellular Na+ activates Ca2+ inward flow through vascular Na+ /Ca2+ exchanger type-1 [42]. We speculate that aldosterone and a high extracellular sodium concentration activate intercellular Ca2+ uptake and initiate the transduction of intracellular signals related to NOX1 expression in VSMCs. Another important finding of our study was that NOX1 expression was maximal in VSMCs incubated with a physiological aldosterone concentration and high salt. In contrast, physiological aldosterone with a normal salt concentration induced little superoxide production and cell hypertrophy. These findings suggest that a high salt intake leads to the progression of cardiovascular disorders at a physiological concentration of aldosterone. A recent study has shown that aldosterone stimulates ROS production through NADPH oxidase activation in rat mesangial cells [43]. However, the effective aldosterone concentration (100 nmol/L) outside the physiological range, and that notion that salt could lower the threshold of vascular hypertrophy is intriguing. Because eplerenone incompletely inhibited aldosterone and high salt-induced NOX1 up-regulation in the present study, we examined other pathways that might be involved in NOX1 regulation. We found that the MAPK family was not involved in NOX1 regulation by aldosterone and high salt, whereas src and PKC abrogated the effect. Transactivation of epidermal growth factor (EGF) receptors is dependent on src followed by
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Fig. 6. Superoxide production in A7r5 cells expressing ribozyme. (A) Superoxide production visualized by dihydroethidium (DHE) staining. Left, control; right, cells incubated with aldosterone and high salt. (B) Quantitative DHE staining. Right panel, fluorescence intensity detected by flow cytometry. Mean values were calculated from six independent samples. Bars represent means ± S.E.M. *P < 0.05 compared with control; # P < 0.05 compared with aldosterone and high salt-treated. Ald, aldosterone.
phosphatidylinositol-3 kinase [44,45], and we previously reported that PKC␦ up-regulates NOX1 via EGF receptor transactivation, which results in PGF2␣-induced hypertrophy of VSMC [46]. These findings suggest that PKC and EGF receptors play important roles in NOX1 regulation induced by aldosterone and high salt. Acknowledgements This study was supported by a grant from the Japanese Ministry of Health, Labour and Welfare 2005. This work was supported in part by Health and Labor Sciences Research Grants for Disorders of Adrenocortical Hormone Production from the Ministry of Health, Labor, and Welfare, Japan.
References [1] M. Fritsch Neves, E.L. Schiffrin, Aldosterone: a risk factor for vascular disease, Curr. Hypertens. Rep. 5 (2003) 59–65. [2] M.R. Ward, P. Kanellakis, D. Ramsey, J. Funder, A. Bobik, Eplerenone suppress constrictive remodeling and collagen accumulation after angioplasty in porcine coronary arteries, Circulation 104 (4) (2001) 467–472. [3] G.P. Rossi, V. Di Bello, C. Ganzaroli, A. Sacchetto, M. Cesari, A. Bertini, D. Giorgi, R. Scognamiglio, M. Mariani, A.C. Pessina, Excess aldosterone is associated with alterations of myocardial texture in primary aldosteronism, Hypertension 40 (2002) 23–27. [4] G.P. Rossi, A. Sacchetto, P. Visentin, C. Canali, G.R. Graniero, P. Palatini, A.C. Pessina, Changes in left ventricular anatomy and function in hypertension and primary aldosteronism, Hypertension 27 (1996) 1039– 1045. [5] B. Pitt, F. Zannad, W. Remme, R. Cody, A. Castaigne, A. Perez, J. Palensky, J. Wittes, The effect of spironolactone on morbidity and mortality in patients with severe heart failure, N. Engl. J. Med. 341 (1999) 709–717.
36
C. Fan et al. / Journal of Steroid Biochemistry & Molecular Biology 111 (2008) 29–36
[6] B. Pitt, W. Remme, F. Zannad, J. Neaton, F. Martinez, B. Roniker, R. Bittman, S. Hurley, J. Kleiman, M. Gatlin, Eplerenone, a selective aldosterone blocker, in patients with left ventricular dysfunction after myocardial infarction, N. Engl. J. Med. 348 (2003) 1309–1321. [7] W.J. Oliver, E.L. Cohen, J.V. Neel, Blood pressure, sodium intake, and sodium related hormones in the Yanomamo Indians, a no-salt culture, Circulation 52 (1975) 146–151. [8] J.J. Mancilha-Carvalho, R. de Oliveria, R.J. Esposito, Blood pressure and electrolyte excretion in the Yanomamo Indians, an isolated population, J. Human Hypertens. 3 (1989) 309–314. [9] P.R. Conlin, Interactions of high salt intake and the response of the cardiovascular system to aldosterone, Cardiol. Rev. 13 (2005) 118–124. [10] K. Yoshida, S. Kim-Mitsuyama, R. Wake, Y. Izumiya, Y. Izumi, T. Yukimura, M. Ueda, M. Yoshiyama, H. Iwao, Excess aldosterone under normal salt diet induces cardiac hypertrophy and infiltration via oxidative stress, Hypertens. Res. 28 (2005) 447–455. [11] E.R. Blasi, R. Rocha, A.E. Rudolph, E.A. Blomme, M.L. Polly, E.G. McMahon, Aldosterone/salt induces renal inflammation and fibrosis in hypertensive rats, Kidney Int. 63 (2003) 1791–1800. [12] Y. Sun, J. Zhang, L. Lu, S.S. Chen, M.T. Quinn, K.T. Weber, Aldosterone-induced inflammation in rat heart. Role of oxidative stress, Am. J. Pathol. 161 (2002) 1773–1781. [13] A. Nishiyama, L. Yao, Y. Nagai, K. Miyata, M. Yoshizumi, S. Kagami, S. Kondo, H. Kiyomoto, T. Shokoji, S. Kimura, M. Kohno, Y. Abe, Possible contributions of reactive oxygen species and mitogen-activated protein kinase to renal injury in aldosterone/salt-induced hypertensive rats, Hypertension 43 (2004) 841–848. [14] A. Virdis, M.F. Neves, F. Amiri, E. Viel, R.M. Touyz, E.L. Schiffrin, Spironolactone improved angiotensin-induced vascular changes and oxidative stress, Hypertension 40 (2002) 504–510. [15] M. Iglarz, R.M. Touyz, E.C. Viel, F. Amiri, E.L. Schiffrin, Involvement of oxidative stress in the profibrotic action of aldosterone. Interaction with the renin–angiotensin system, Am. J. Hypertens. 17 (2004) 597–603. [16] W. Droge, Free radicals in the physiological control of cell function, Physiol. Rev. 82 (2002) 47–95. [17] B. Lassegue, R.E. Clempus, Vascular NAD(P)H oxidases: specific features, expression, and regulation, Am. J. Physiol. 285 (2003) R277–R297. [18] K.K. Griendling, D. Sorescu, M. Ushio-Fukai, NAD(P)H oxidase: role in cardiovascular biology and disease, Circ. Res. 86 (2000) 494–501. [19] I.R. Hanna, Y. Taniyama, K. Szocs, P. Rocic, K.K. Griendling, NAD(P)H oxidasederived reactive oxygen species as mediators of angiotensin II signaling, Antioxid. Redox. Signal. 4 (2002) 899–914. [20] B. Banfi, G. Molnar, A. Maturana, K. Steger, B. Hegedus, N. Demaurex, K.H. Krause, A Ca(2+) -activated NADPH oxidase in testis, spleen, and lymph nodes, J. Biol. Chem. 276 (2001) 37594–37601. [21] G. Cheng, Z. Cao, X. Xu, E.G. van Meir, J.D. Lambeth, Homologs of gp91phox: cloning and tissue expression of Nox3, Nox4, and Nox5, Gene 269 (2001) 131–140. [22] B. Lassegue, D. Sorescu, K. Szocs, Q. Yin, M. Akers, Y. Zhang, S.L. Grant, J.D. Lambeth, K.K. Griendling, Novel gp91(phox) homologues in vascular smooth muscle cells : nox1 mediates angiotensin II-induced superoxide formation and redox-sensitive signaling pathways, Circ. Res. 88 (2001) 888–894. [23] M. Katsuyama, C. Fan, C. Yabe-Nishimura, NADPH oxidase is involved in prostaglandin F2alpha-induced hypertrophy of vascular smooth muscle cells: induction of NOX1 by PGF2alpha, J. Biol. Chem. 277 (2002) 13438–13442. [24] M. Ushio-Fukai, A.M. Zafari, T. Fukui, N. Ishizaka, K.K. Griendling, p22phox is a critical component of the superoxide-generating NADH/NADPH oxidase system and regulates angiotensin II-induced hypertrophy in vascular smooth muscle cells, J. Biol. Chem. 271 (1996) 23317–23321. [25] S. Koseki, T. Tanabe, K. Tani, S. Asano, T. Shioda, Y. Nagai, T. Shimada, J. Ohkawa, K. Taira, Factors governing the activity in vivo of ribozymes transcribed by RNA polymerase III, J. Virol. 73 (1999) 1868–1877. [26] Y. Kato, T. Kuwabara, M. Warashina, H. Toda, K. Taira, Relationships between the activities in vitro and in vivo of various kinds of ribozyme and their intracellular localization in mammalian cells, J. Biol. Chem. 276 (2001) 15378–15385. [27] T. Kuwabara, M. Warashina, M. Orita, S. Koseki, J. Ohkawa, K. Taira, Formation of a catalytically active dimer by tRNA(Val)-driven short ribozymes, Nat. Biotechnol. 16 (1998) 961–965.
[28] T. Ago, T. Kitazono, H. Ooboshi, T. Iyama, Y.H. Han, J. Takada, M. Wakisaka, S. Ibayashi, H. Utsumi, M. Iida, Nox4 as the major catalytic component of an endothelial NAD(P)H oxidase, Circulation 109 (2004) 227–233. [29] D.S. Weber, P. Rocic, A.M. Mellis, K. Laude, A.N. Lyle, D.G. Harrison, K.K. Griendling, Angiotensin II-induced hypertrophy is potentiated in mice overexpressing p22phox in vascular smooth muscle, Am. J. Physiol. Heart Circ. Physiol. 288 (2005) H37–H42. [30] A. Dikalova, R. Clempus, B. Lassegue, G. Cheng, J. McCoy, S. Dikalov, A. San Martin, A. Lyle, D.S. Weber, D. Weiss, W.R. Taylor, H.H. Schmidt, G.K. Owens, J.D. Lambeth, K.K. Griendling, Nox1 overexpression potentiates angiotensin IIinduced hypertension and vascular smooth muscle hypertrophy in transgenic mice, Circulation 112 (2005) 2668–2676. [31] K. Mukai, M. Imai, H. Shimada, Y. Ishimura, Isolation and characterization of rat CYP11B genes involved in late steps of mineralo- and glucocorticoid syntheses, J. Biol. Chem. 268 (1994) 9130–9137. [32] C.E. Gomez-Sanchez, M.Y. Zhou, E.N. Cozza, H. Morita, M.F. Foecking, E.P. Gomez-Sanchez, Aldosterone biosynthesis in the rat brain, Endocrinology 138 (1997) 3369–3373. [33] J.S. Silvestre, V. Robert, C. Heymes, B. Aupetit-Faisant, C. Mouas, J.M. Moalic, B. Swynghedauw, C. Delcayre, Myocardial production of aldosterone and corticosterone in the rat: physiological regulation, J. Biol. Chem. 273 (1998) 4883– 4891. [34] K. Matsuno, H. Yamada, K. Iwata, D. Jin, M. Katsuyama, M. Matsuki, S. Takai, K. Yamanishi, M. Miyazaki, H. Matsubara, C. Yabe-Nishimura, Nox1 is involved in angiotensin II-mediated hypertension: a study in Nox1-deficient mice, Circulation 112 (2005) 2677–2685. [35] D.J. Mangelsdorf, C. Thummel, M. Beato, P. Herrlich, G. Schutz, K. Umesono, B. Blumberg, P. Kastner, M. Mark, P. Chambon, R.M. Evans, The nuclear receptor superfamily: the second decade, Cell 83 (1995) 835–839. [36] M. Christ, K. Douwes, C. Eisen, G. Bechtner, K. Theisen, M. Wehling, Rapid effects of aldosterone on sodium transporter in vascular smooth muscle cells, Hypertension 25 (1995) 117–123. [37] D.F. Liao, J.L. Duff, G. Daum, S.L. Pelech, B.C. Berk, Angiotensin II stimulates MAP kinase kinase kinase activity in vascular smooth muscle cells, Role Raf. Circ Res. 79 (1996) 1007–1014. [38] C. Peiro, J.L. Llergo, J. Angulo, J.M. Lopez-Novoa, A. Rodriguez-Lopez, L. Rodriguez-Manas, C.F. Sanchez-Ferrer, Effects of captopril, losartan, and nifedipine on cell hypertrophy of cultured vascular smooth muscle from hypertensive Ren-2 transgenic rats, Br. J. Pharmacol. 121 (1997) 1438–1444. [39] S. Ebata, S. Muto, K. Okada, J. Nemoto, M. Amemiya, T. Saito, Y. Asano, Aldosterone activates Na+ /H+ exchanger in vascular smooth muscle cells by nongenomic and genomic mechanism, Kidney Int. 56 (1999) 1400–1412. [40] M. Karmazyn, Q. Liu, X.T. Gan, B.J. Brix, L. Fliegel, Aldosterone increases NHE-1 expression and induces NHE-1-dependent hypertrophy in neonatal rat ventricular myocytes, Hypertension 42 (2003) 1171–1176. [41] M. Yamamuro, M. Yoshimura, M. Nakayama, K. Abe, M. Shono, S. Suzuki, T. Sakamoto, Y. Saito, K. Nakao, H. Yasue, H. Ogawa, Direct effects of aldosterone on cardiomyocytes in the presence of normal and elevated extracellular sodium, Endocrinology 147 (2006) 1314–1321. [42] T. Iwamoto, S. Kita, J. Zhang, M.P. Blaustein, Y. Arai, S. Yoshida, K. Wakimoto, I. Komuro, T. Katsuragi, Salt-sensitive hypertension is triggered by Ca2+ entry via Na+ /Ca2+ exchanger type-1 in vascular smooth muscle, Nat. Med. 10 (2004) 1193–1199. [43] K. Miyata, M. Rahman, T. Shokoji, Y. Nagai, G.X. Zhang, G.P. Sun, S. Kimura, T. Yukimura, H. Kiyomoto, M. Kohno, Y. Abe, A. Nishiyama, Aldosterone stimulates reactive oxygen species production through activation of NADPH oxidase in rat mesangial cells, J. Am. Soc. Nephrol. 16 (2005) 2906–2912. [44] M. Ushio-Fukai, K.K. Griendling, P.L. Becker, L. Hilenski, S. Halleran, R.W. Alexander, Epidermal growth factor receptor transactivation by angiotensin II requires reactive oxygen species in vascular smooth muscle cells, Arterioscler. Thromb. Vasc. Biol. 21 (2001) 489–495. [45] D. Bokemeyer, U. Schmitz, H.J. Kramer, Angiotensin II-induced growth of vascular smooth muscle cells requires an Src-dependent activation of the epidermal growth factor receptor, Kidney Int. 58 (2000) 549–558. [46] C.Y. Fan, M. Katsuyama, C. Yabe-Nishimura, PKC␦ mediates up-regulation of NOX1, a catalytic subunit of NADPH oxidase, via transactivation of the EGF receptor: possible involvement of PKCd in vascular hypertrophy, Biochem. J. 390 (2005) 761–767.