Published online before print June 19, 2008, doi: 10.1161/CIRCRESAHA.108.179408
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© 2008 American Heart Association, Inc.
Cellular Biology |
Role of Oxidant Stress on AT1 Receptor Expression in Neurons of Rabbits With Heart Failure and in Cultured Neurons
From the Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha.
Correspondence to Irving H. Zucker, PhD, Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, 985850 Nebraska Medical Center, Omaha, NE 68198-5850. E-mail izucker{at}unmc.edu
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Abstract |
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We have previously reported that the expression of Angiotensin II (Ang II) type 1 receptors (AT1R) was increased in the rostral ventrolateral medulla (RVLM) of rabbits with chronic heart failure (CHF) and in the RVLM of normal rabbits infused with intracerebroventricular (ICV) Ang II. The present study investigated whether oxidant stress plays a role in Ang II–induced AT1R upregulation and its relationship to the transcription factor activator protein 1 (AP1) in CHF rabbits and in the CATHa neuronal cell line. In CATHa cells, Ang II significantly increased AT1R mRNA by 123±11%, P<0.01; c-Jun mRNA by 90±20%, P<0.01; c-fos mRNA by 148±49%, P<0.01; NADPH oxidase activity by 126±43%, P<0.01 versus untreated cells. Tempol and Apocynin reversed the increased expression of AT1R mRNA, c-Jun mRNA, c-fos mRNA, and superoxide production induced by Ang II. We also examined the effect of ICV Tempol on the RVLM of CHF rabbits. Compared to vehicle treated CHF rabbits, Tempol significantly decreased AT1R protein expression (1.6±0.29 versus 0.88±0.16, P<0.05), phosphorylated Jnk protein (0.4±0.05 versus 0.2±0.04, P<0.05), cytosolic phosphorylated c-Jun (0.56±0.1 versus 0.36±0.05, P<0.05), and nuclear phosphorylated c-Jun (0.67±0.1 versus 0.3±0.08, P<0.01). Tempol also significantly decreased the AP-1–DNA binding activity in the RVLM of CHF rabbits compared to the vehicle group (9.14x103 versus 41.95x103 gray level P<0.01). These data suggest that Ang II induces AT1R upregulation at the transcriptional level by induction of oxidant stress and activation of AP1 in both cultured neuronal cells and in intact brain of rabbits. Antioxidant agents may be beneficial in CHF and other states where brain Ang II is elevated by decreasing AT1R expression through the Jnk and AP1 pathway.
Key Words: angiotensin ii • antioxidant enzymes • brain • c-jun • c-Jun NH2-terminal kinase
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Introduction |
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Angiotensin II (Ang II), the principle mediator of the renin–angiotensin system, plays a pivotal role in cardiovascular and renal homeostasis. Most of the physiological and pathophysiological effects of Ang II are mediated by the Angiotensin II type 1 receptor (AT1R). The activation of the renin–angiotensin system and the sympathetic nervous system in chronic heart failure (CHF) are critical factors that contribute to the development and progression of CHF in patients.1,2 Evidence also demonstrates that AT1R blockers, when added to conventional treatment for patients with CHF, are associated with a reduction in morbidity and mortality as well as an improvement in quality of life.3 Currently, blockade of the renin–angiotensin system has become the primary treatment for patients with CHF. Therefore, a comprehensive understanding of the regulation of the AT1R is necessary for developing new therapies to target this receptor in the setting of CHF.
The role of reactive oxygen species (ROS) in the cardiovascular system has been investigated extensively over the past decade.4 It has been reported that, in the rostral ventrolateral medulla (RVLM), the levels of ROS are elevated in stroke-prone spontaneously hypertensive rats. Scavenging superoxide anion (O2
–) in the RVLM reduces both sympathetic nerve activity and blood pressure.5 Similar evidence has been provided for rabbits with CHF.6 Ang II induces a rapid and transient increase in ROS in a wide variety of cell types.7 Ang II–induced ROS-formation is attenuated by the NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI), suggesting that it is mediated through NADPH Oxidase.7 In addition, Ang II–induced ROS-formation can also be blocked by losartan,8 suggesting that AT1R and its downstream pathway are involved.
However, the downstream targets of ROS have remained largely unexplored, at least for the regulation of the AT1R. Extracellular administration of nonlethal concentrations of H2O2 has been demonstrated to activate mitogen-activated protein kinase (MAPK) as well as the c-Jun amino-terminal kinase (JNK).9 Endogenous ROS induced by Ang II has also been shown to activate stress activated protein kinase (SAPK), which can be inhibited by treating cells with chemical or enzymatic antioxidants.10–13 This has also been demonstrated for JNK activation, where it has been noted in several cell types that activation of JNK is inhibited by pretreatment with the antioxidant N-acetylcysteine.14
The transcription factor AP1 is stimulated by a host of ligands leading eventually to serine phosphorylation and subsequent AP1 translocation to the nucleus. ROS are important in AP1 activation because most of the activation can be blocked by antioxidant treatment.15
We and other investigators have reported that Ang II infusion into the RVLM can alter sympathetic outflow. The expression of AT1R is upregulated in the RVLM of rabbits with CHF or in normal rabbits subjected to chronic introcerebroventricular (ICV) infusion of Ang II. The upregulation of central AT1R occurs with activation of the renin–angiotensin system and results in increased sympathetic nerve activity in CHF animals.16,17 We observed that losartan attenuated the AT1R upregulation in rabbits with CHF or after ICV Ang II infusion in normal rabbits.18 Central blockade of the AT1R in the CHF state reduced sympathetic nerve activity and increased baroreflex function.19,20 In rat hypertensive models, losartan suppresses the expression of both basal and enhanced cardiac AT1R.21
We hypothesized that Ang II upregulates AT1R through ROS modulation of a downstream signaling pathway that includes AP1. The effects of losartan in CHF on the amelioration of AT1R upregulation may be mediated by blocking the formation of endogenous ROS. To test our hypothesis and provide additional molecular targets for central intervention in the CHF state, we investigated the relationship between oxidative stress, antioxidant treatment, AT1R regulation, and the transcription factor AP1 in a neuronal cell line and in the RVLM of intact CHF rabbits.
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Materials and Methods |
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These experiments were reviewed and approved by the University of Nebraska Medical Center Institutional Animal Care and Use Committee and conformed to the Guidelines for the Care and Use of Experimental Animals of the American Physiological Society and the National Institutes of Health.
Cell Culture
A neuronal cell line (CATH.a) was purchased from ATCC, and the cells were grown in RPMI 1640 containing 8% horse serum, 4% FBS, 100 IU/L penicillin, at 37°C in 95% air and 5% CO2 in a humidified atmosphere. For experiments, cells were plated on polystyrene tissue culture dishes at a density of 1x107 cells per 100-mm plate, or 1.5x106 cells per well in 6-well culture plates. After subculture, cells were allowed to grow for 2 days and then pretreated with either an AT1 receptor blocker (losartan, 10 µmol/L, Merck), an NADPH oxidase inhibitor (Apocynin, 20 µmol/L, Calbiochem), an SOD mimetic (Tempol, 0.2 µmol/L, Calbiochem), or a SAPK inhibitor (Jnk inhibitor I, 1 µmol/L, Calbiochem) for 30 minutes, then treated with Ang II (100 nmol/L, Sigma) for 3 hours. DMSO or water was used a vehicle control.
Induction of CHF and ICV Infusion in Rabbits
Ten male New Zealand White rabbits weighing between 3.5 and 4.3 Kg were induced to develop CHF by chronic ventricular tachycardia, as previously described.20 The 10 CHF rabbits were divided into 2 groups (n=5 per group): one treated with ICV vehicle infusion and one with ICV Tempol infusion (10 µg/µL/h). For chronic ICV infusions, a 19-gauge cannula was implanted into a lateral cerebral ventricle as previously described.22 An osmotic minipump (Model 2001, Durect Corp) filled with Tempol or artificial cerebrospinal fluid (1 µL/h) was implanted subcutaneously in the back of the neck and connected to the ICV cannula. The infusion was continued for 7 days.
Cardiac Function, Arterial Pressure, Heart Rate, LV Pressure, Ejection Fraction
Cardiac function was measured by echocardiography (Acuson Sequoia 512C), with the rabbits hand-held in the conscious state. Arterial pressure was measured with a radiotelemetry unit (Data Sciences International), the catheter of which was inserted into the right femoral artery. LV pressure was measured with a Millar transducer (Model SPR-524, Millar Instruments, Inc.) under anesthesia at the conclusion of the experiment.
Preparation of RVLM Tissue
At the end of the experiment, the rabbits were killed with pentobarbital sodium. The brain was removed and immediately frozen on dry ice, blocked in the coronal plane, and sectioned at 300-µm thickness in a cryostat. The RVLM was punched according to the method of Palkovits and Brownstein23 for analysis of mRNA and protein of the AT1R receptor and other molecular studies.
RNA Extraction, cDNA Synthesis, and Real-Time PCR
Total RNA from 3-mm punches of RVLM or CATH.a cells was isolated using the RNeasy kit (Qiagen). cDNA was generated using the iScript cDNA Sysnthesis Kit (Bio-Rad). Gene Specific primers and probes were synthesized in the UNMC Eppley Cancer Center DNA synthesis Core Facility (please see supplemental Table I, available online at http://circres.ahajournals.org, for the sequences of the gene specific primers and probes). Gene-specific probes were labeled with FAM at the 5' site with Blackhole at the 3' site to add specificity and sensitivity. Beta-actin was used as an internal control for calculation of relative expression levels of target genes in the rabbit studies. GAPDH was used as an internal control for calculation of relative expression levels of target genes in CATHa cells. Both β-actin and GAPDH are commonly used as internal controls in Ang II studies by others.22,24 Real-time PCR was performed by using HotStarTaq DNA polymerase (Qiagen) on CHROMO4 Continuous Fluorescence Detector (Bio-Rad). Analysis of relative gene expression was based on the method described by Livak et al.25
Western Blot Analysis
Homogenates were prepared from RVLM. Protein concentration was measured using a bicinchoninic acid protein assay kit (Pierce). Equal amounts of homogenate were loaded and then separated on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The proteins of these samples were electrophoretically transferred at 100 V for 1.5 hour onto nitrocellulose membranes. The membrane were blocked with 5% nonfat milk in tris-buffered saline-tween 20, and probed at room temperature with primary antibodies against the AT1R, GAPDH (Santa Cruz Biotechnology), p-Jnk, Jnk, c-Jun, Phospho-c-Jun (Cell Signaling Technology), and β-tubulin (Sigma). After washing, the membranes were incubated for 30 minutes with peroxidase-conjugated secondary antibody. The membranes were then washed and incubated for 5 minutes with Supersignal West Pico Chemiluminescent detection reagents (Pierce). The bands on the membrane were digitized and analyzed using UVP BioImaging Systems (UVP).
Activity of NADPH Oxidase by Lucigenin-Enhanced Chemiluminescence
Superoxide anion production by CATHa cells was measured by the lucigenin enhanced chemiluminescence (ECL) method (TD-20/20 luminometer; Turner Designs). After subculture, CATHa cells were allowed to grow for 2 days and then treated with either Ang II (100 nmol/L) alone or with Ang II for 3 hours after pretreatment with losartan (10 µmol/L) for 30 minutes. Cells were collected and total protein concentration was determined using a bicinchoninic acid protein-assay kit (Pierce). NADPH (100 µmol/L, Sigma) and dark-adapted lucigenin (5 µmol/L, Sigma) were added into 0.5-mL microcentrifugal tubes just before reading. Light emission was recorded over 5 minutes, and values were expressed as mean light units per minute per milligram of protein. Using this method, the superoxide anion production also represents NADPH oxidase activity. The NADPH oxidase activity was expressed in relative ratios by comparing the treatment groups to the control groups.
Detection of Superoxide Production by Dihydroethidium Fluorescence
To measure the production of superoxide anion, dihydroethidium (DHE; Molecular Probes) stock (10 mmol/L) was made in DMSO, and dilutions of the stock were used only on the experimental day. Dilutions of DHE were made in reoxygenation solution that resulted in a final concentration of 1 µmol/L DHE in each experimental well. CATH.a cells then were incubated with DHE at 37 °C in a light-protected humidified chamber for 30 minutes. After washing with phosphate-buffered saline, DHE fluorescence was visualized by a Leica fluorescent microscope with appropriate excitation/emission filters. Pictures were captured by a digital camera system.
Electrophoretic Mobility Shift Assay
The AP1-DNA binding activity of rabbit RVLM was measured by EMSA, the protein specificity was tested by adding antibodies of c-fos and c-Jun (Santa Cruz Biotechnology). Detailed methods were described by this laboratory in a previous study.18
Statistics
Data are expressed as mean±SEM. The statistical analyses were performed by nonpaired Student t test when comparing data between the 2 groups of CHF rabbits. A 1-way analysis of variance (ANOVA) for the multiple groups of CATHa cell experiments was performed. The statistical software SigmaStat (SPSS) was used for analysis. If significance was found between groups, post hoc analyses were carried out using the Bonferroni correction. A probability value <0.05 was considered significantly different.
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Results |
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AT1R mRNA Expression in CATHa Cells and Effects of Antioxidants
Ang II (100 nmol/L) significantly increased AT1R mRNA expression by 123±11% compared with the control group (Figure 1). Ang II–induced AT1R mRNA expression was inhibited by the AT1R blocker (Losartan, 10 µmol/L), the NADPH oxidase inhibitor (Apocynin, 20 µmol/L), the SOD mimetic (Tempol, 0.2 µmol/L), and Jnk Inhibitor I (1 µmol/L). These data suggest there is a positive feedback pathway for Ang II to stimulate AT1R expression and that this pathway involves oxidant stress and Jnk.
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Superoxide Production and NADPH Oxidase Activity in CATHa Cells
Ang II induced superoxide production. Both the NADPH oxidase inhibitor Apocynin (20 µmol/L) and the SOD mimetic Tempol (0.2 µmol/L) reversed Ang I–induced superoxide production (Figure 2). This suggests Ang I–induced superoxide is mediated by activation of NAPDH oxidase. The activity of NADPH oxidase in CATHa cells was measured by using lucigenin-enhanced chemiluminescence (Figure 3). Ang II increased NADPH oxidase activity by 126±43%, *P<0.01 compared to control group. The increased activity was normalized by losartan (P<0.01) compared to the Ang II treated group. These further suggest that Ang II can induce superoxide by activation of NADPH oxidase through the AT1R.
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c-Jun and c-fos mRNA Expression in CATHa Cells and the Effects of Antioxidants
Ang II (100 nmol/L) significantly increased c-Jun and c-fos mRNA expressions compared with the control group (Figure 4). Ang I–induced c-Jun and c-fos mRNA expressions were inhibited by the AT1R blocker Losartan (10 µmol/L), the NADPH oxidase inhibitor Apocynin (20 µmol/L), and the SOD mimetic Tempol (0.2 µmol/L). These data suggest that Ang II can induce AP1 expression through ROS by activation of NADPH oxidase.
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Body Weight, Heart Weight, Hemodynamics, and Echocardiographic Data in CHF Rabbits
The Table shows the values for body weight, ratio of organ weight to body weight, hemodynamics, and echocardiographic data of CHF rabbits treated with vehicle or Tempol. After 3 weeks of pacing, rabbits with CHF showed significant decreases in ejection fraction (EF), increases in heart rate (HR), left ventricle systolic diameter (LVSD), and left ventricle end-diastolic diameter (LVEDD). There were no differences between Vehicle and Tempol treatment groups in the prepaced or postpaced states. These data also suggest that 7 days of ICV Tempol treatment does not alter hemodynamic parameters of CHF rabbits.
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The Effect of Tempol on AT1R mRNA, c-Jun mRNA, and c-fos mRNA in the RVLM of Rabbits With CHF
After 7 days of ICV infusion of Tempol the RVLM was punched out and analyzed for AT1R mRNA, c-Jun mRNA, and c-fos mRNA expressions using real-time RT-PCR. Compared with the vehicle-treated group, AT1R mRNA expression was significantly decreased in the Tempol group, *P<0.01(Figure 5A). Compared with the ICV vehicle group, c-Jun mRNA and c-fos expressions in the Tempol treated group were significantly decreased, P<0.05 (Figure 5B and 5C). These data indicate that the change in c-Jun and c-fos occur in a similar fashion as the up regulation of the AT1R, and they are all dependent on superoxide production.
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Effects of Tempol on Protein Expression of AT1R, p-JNK, and p-c-Jun
Rabbit RVLM protein was extracted. The cytosolic protein and nuclear protein were separated by using the kit of NE-PER (Pierce). AT1R, phosphorylated c-Jun, phosphorylated Jnk were detected by Western blotting. Tempol decreased AT1R (1.6±0.29 versus 0.88±0.16, P<0.05) (figure 6A); decreased the ratio of p-c-Jun to total c-Jun in cytosolic compartment (0.56±0.10 versus 0.36±0.05, P<0.05) and in the nuclear compartment (0.67±0.1 versus 0.30±0.08, P<0.01) (Figure 6C and 6D); decreased the ratio of p-Jnk protein to total Jnk (0.40±0.05 versus 0.20±0.04, P<0.05) compared to the Vehicle treated group (Figure 6B). These data suggest that oxidant stress can up regulate AT1R through the Jnk-c-Jun-AP1 pathway in CHF.
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Effects of Tempol on AP1-DNA Binding Activity
In our previous study, we showed the binding activity of the transcription faction AP-1 was increased significantly in CHF compared to sham rabbits.18 Here after giving ICV tempol to the CHF rabbits AP-1 DNA binding activity was significantly decreased compared to ICV vehicle CHF rabbits (Figure 7A and 7B). The DNA/protein complex could be suppressed by adding 200-fold molar excess of unlabeled competitor AP-1 DNA sequence. The DNA/protein complex could be supershifted by adding antibodies of c-fos and c-Jun (Figure 7A). This indicates the DNA/protein components have c-fos and c-Jun proteins that can form a dimer of AP1 transcription factor.
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Discussion |
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We have previously reported that the expression of AT1R was upregulated in the RVLM of rabbits with CHF. We also observed that the upregulation depends on the increase in the transcription factor AP1 which is activated by the SAPK/Jnk pathway but not by p38 or ERK1/2 MAP Kinase pathway.18 AP1 is redox-sensitive through the conserved cysteine residues located in its DNA-binding domain.26 The present study provides further evidence for the role of ROS in up regulation of AT1R expression in CHF.
The NADPH oxidase enzymes are a particularly important source of reactive oxygen species that are implicated in redox signaling.27,28 It is now well accepted that Ang II can stimulate NADPH oxidase dependent superoxide (O2–) generation.29 Substantial clinical and experimental evidence support the important role of oxidant stress in cardiac remodeling and heart failure.30 Markers of oxidant stress are elevated in patients with heart failure and correlate with the severity of contractile dysfunction.31,32 Enhanced ROS generation has been directly documented in the failing heart using highly specific methods such as electron spin resonance spectroscopy.33 However, there are few studies concerning the role of ROS and antioxidants in the central control of sympathetic nerve activity in CHF.
The upregulation of AT1R in CHF contributes to a positive feedback system that evokes a further deterioration of the CHF state. We have also reported that NADPH oxidase activity is increased in the cardiovascular control areas of the rabbit brain with CHF.34 Accordingly, in the present study, we used a neuronal cell line to further explore the relationship between AT1R expression, NADPH oxidase, and ROS. We observed superoxide release after Ang II treatment in CATH.a cells. We also measured NADPH oxidase activity which was increased by Ang II treatment in CATH.a cells. The increased activity was attenuated by losartan. The expression of AT1R was upregulated by treating CATHa cells with Ang II and was inhibited by losartan. Interestingly, the upregulation of AT1R mRNA expression was also blocked by treating the cells with Tempol and Apocynin. These data lead to the speculation that Ang II may induce the upregulation of AT1R expression via a pathway dependent on ROS production. An important aspect of this work is that a similar conclusion can be derived from our experiments in rabbits with CHF after ICV administration of Tempol. These in vivo and in vitro studies suggest that ROS plays a crucial role in the central upregulation of AT1R expression in CHF.
The influence of NADPH oxidase–derived ROS on downstream cellular effects has been extensively studied in the failing heart.35,36 These include alterations in the activity of redox-sensitive protein kinases, such as SAPK and other MAPKs, which may occur indirectly following inactivation of tyrosine phosphatases or through direct activation; these also include alterations in the activity of transcription factors, including NF-
B, AP-1, hypoxia-inducible factor-1, and STATs. We have reported that the phosphorylation of Jnk was increased in the RVLM of CHF rabbits. Exogenous (ICV) Ang II also increased activity of phosphorylated Jnk.18 In the present study, we investigated the effect of Tempol on the SAPK/Jnk pathway in the central sympathetic nervous system of CHF rabbits. We observed that ICV Tempol decreased Jnk phosphorylation in the RVLM of CHF rabbits compared to vehicle treated rabbits, providing evidence that ROS can regulate Jnk phosphorylation in CHF.
The exposure of mammalian cells to extracellular stress induces the expression of immediate early genes such as c-fos and c-jun and activates various transcription factors, among which is AP-1.37 AP-1 is ubiquitously expressed in the brain and has been shown to be associated with a variety of physiological and pathophysiological states.38 For instance, Wang et al reported that Fos protein is required for the reexpression of AT1R in the nucleus tractus solitarii (NTS) after baroreceptor activation in the rat.39 Chan et al showed an augmented upregulation by c-fos of AT1R in the NTS of spontaneously hypertensive rats.40 Fleegal et al demonstrated that Ang II induction of AP-1 in neurons requires stimulation of Jnk.41 We previously showed that phosphorylated c-Jun and c-Jun mRNA increased in the RVLM of CHF rabbits. We also showed that ICV administration of Ang II to normal rabbits could induce c-Jun phosphorylation and its mRNA expression.18 In the present study ICV Tempol attenuated the expression of phosphorylated c-Jun protein, c-Jun mRNA and c-fos mRNA compared to vehicle-treated animals.
Therefore, the findings that Ang II–treated CATHa cells exhibited an increase in c-Jun and c-fos mRNA, which was attenuated by Tempol or Apocynin, strongly suggests that oxidant stress plays a pivotal role in this pathway. In CHF rabbits, Tempol decreased AP1 DNA binding activity. Given that c-Jun and c-fos are the major components of AP-1, these data support the idea that ROS is involved in AP-1 activation. Furthermore, Tempol also suppressed the p-Jnk level of CHF suggesting that that ROS activation of AP-1 may occur through phosphorylation of Jnk.
In order to put these data into clinical perspective, a variety of human diseases have been linked to an overproduction of ROS. The growing realization that oxidants may function in signaling pathways may, in turn, causes a reevaluation of the pathways and sources of oxidant production linked to human disease. In addition, a variety of clinical and epidemiological evidence supports a protective role for antioxidants in cardiovascular disease.42–45 Gao et al34 showed central Ang II activates sympathetic outflow by stimulation of NADPH oxidase and ROS in the CHF state. Chan et al showed NADPH oxidase-derived superoxide anion in the RVLM mediates Ang II–induced pressor responses.46 Likewise, Campese et al demonstrated that oxidant stress mediates Ang II–dependent stimulation of sympathetic nerve activity.47 In previous studies from this laboratory Gao et al also showed that ICV Tempol could lower renal sympathetic nerve activity (RSNA).22,34 Xu et al showed systemic high dose Tempol could decrease blood pressure and RSNA, which was mediated largely by NO-independent sympatho-inhibition in normotensive and hypertensive rats.48,49 The dose of Tempol used in the present study did not lower blood pressure, most likely because arterial pressure was already lower in the CHF state. The fact that ICV Tempol inhibited the upregulation of the AT1R in the RVLM suggests that this intervention would also suppress sympathetic nerve activity as indicated in our previous work22,34. Infanger et al50 showed CuZnSOD transfection to the paraventricular nucleus of myocardiac infarcted mice improved the decreased ejection fraction and increased left ventricle end-volume. Ritchie et al51 showed Tempol treatment reduced morphological and molecular evidence of cardiac hypertrophy in the GLUT4-knockout insulin-resistant mouse in vivo. Curiously, there was no effect of ICV Tempol on cardiac function in our CHF rabbits. It is possible that other factors that modulate sympathetic outflow are dominant in this setting or that a reduction in sympathetic outflow after Tempol is targeted primarily to the periphery. Indeed our previous study clearly showed a reduction in RSNA after ICV Tempol.22,34 In addition, in this model the continuation of cardiac pacing during the experiment may have overrode the beneficial effects of ICV Tempol on the cardiac hemodynamics.
The present study extends these findings and shows that the antioxidant Tempol decreased the expression of AT1R, phosphorylated Jnk, c-Jun, and c-fos in the RVLM. The Jnk pathway may provide an explanation for how AP-1 is activated by oxidant stress. Taken together, these data strongly suggest that antioxidant agents may be beneficial in CHF by decreasing AT1R expression through inhibition of the Jnk and AP1 signaling pathway at the molecular level.
In the future, additional studies are needed to elucidate the effect of hydrogen peroxide, hydroxyl radical, and the specific molecular sites at which ROS act to exert their effects. Except for superoxide dismutase (SOD)-like activity, other possible mechanisms of Tempol action should be kept in mind, including scavenging oxy- and carbon based free radicals, and oxidation of reduced transition metals.
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Acknowledgments |
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The authors thank Phyllis Anding, Pamela Curry, and Johnnie Hackley for their excellent technical assistance.
Sources of Funding
This study was supported by NIH grant PO-1 HL62222. L. Gao was supported by a Scientist Development Grant 0635007N from the American Heart Association.
Disclosures
None.
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Footnotes |
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Original received January 9, 2008; resubmission received May 14, 2008; revised resubmission received June 6, 2008; accepted June 11, 2008.
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