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(Circulation Research. 2006;99:1004.)
© 2006 American Heart Association, Inc.

Integrative Physiology

Neuronal Angiotensin II Type 1 Receptor Upregulation in Heart Failure

Activation of Activator Protein 1 and Jun N-Terminal Kinase

Dongmei Liu, Lie Gao, Shyamal K. Roy, Kurtis G. Cornish, Irving H. Zucker

From the Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, 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

	Abstract

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Chronic heart failure (CHF) is a leading cause of mortality in developed countries. Angiotensin II (Ang II) plays an important role in the development and progression of CHF. Many of the important functions of Ang II are mediated by the Ang II type 1 receptor (AT₁R), including the increase in sympathetic nerve activity in CHF. However, the central regulation of the AT₁R in the setting of CHF is not well understood. This study investigated the AT₁R in the rostral ventrolateral medulla (RVLM) of rabbits with CHF, its downstream pathway, and its gene regulation by the transcription factor activator protein 1 (AP-1). Studies were performed in 5 groups of rabbits: sham (n=5), pacing-induced (3 to 4 weeks) CHF (n=5), CHF with intracerebroventricular (ICV) losartan treatment (n=5), normal with ICV Ang II treatment (n=5), and normal with ICV Ang II plus losartan treatment (n=5). AT₁R mRNA and protein expressions, plasma Ang II, and AP-1–DNA binding activity were significantly higher in RVLM of CHF compared with Sham rabbits (240.4±30.2%, P<0.01; 206.6±25.8%, P<0.01; 280±36.5%, P<0.05; 207±16.4%, P<0.01, respectively). Analysis of the stress-activated protein kinase/Jun N-terminal kinase (SAPK/JNK) pathway showed that phosphorylated c-Jun proteins, phosphorylated JNK proteins, and JNK activity increased significantly in RVLM of CHF compared with sham (262.9±48.1%, 213.8±27.7%, 148.2±10.1% of control, respectively). Importantly, ICV losartan in CHF rabbits attenuated these increases. ICV Ang II in normal rabbits simulated the molecular changes seen in CHF. This effect was blocked by concomitant ICV losartan. In addition, Ang II–induced AT₁R expression was blocked by losartan and a JNK inhibitor, but not by extracellular signal-regulated kinase or p38 MAP kinase inhibitors in a neuronal cell culture. These data suggest that central Ang II activates the AT₁R, SAPK/JNK pathway. AP-1 may further regulate gene expression in RVLM in the CHF state.

Key Words: angiotensin II • sympathetic nerve activity • chronic tachycardia

	Introduction

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Chronic heart failure (CHF) is a leading cause of mortality in developed countries. CHF is characterized, in part, by activation of the renin/angiotensin/aldosterone system and the sympathetic nervous system. Excessive activity of the sympathetic nervous system contributes to the development and progression to CHF in patients.^1,2 In experimental animals, it has been well established that the renin/angiotensin system is markedly activated and angiotensin II (Ang II) is elevated in moderate to severe CHF.^3–5

Ang II is considered to be a prime candidate in the regulation of sympathetic outflow in the CHF state, because Ang II can alter sympathetic function at several sites from the central nervous system to the periphery.^6,7 Our laboratory has clearly demonstrated that rabbits with pacing-induced CHF exhibit elevated plasma Ang II compared with sham rabbits.^8,9 We have also shown that central blockade of the Ang II type 1 receptor (AT₁R) reduces sympathetic nerve activity and increases baroreflex function in the CHF state.^10–12 DiBona and colleagues reported similar results in rats with chronic myocardial infarction induced CHF.^13–15 Suppression of the AT₁R gene in the brain using antisense techniques reduces resting sympathetic nerve activity in rats with CHF but not in sham rats.¹⁶

Complementary to the above evidence, in previous studies, we observed that the AT₁R was upregulated in the rostral ventrolateral medulla (RVLM) of rabbits with CHF.⁹ Intracerebroventricular (ICV) Ang II given to normal rabbits exhibits an upregulation of AT₁R expression and an increase in sympathetic outflow. Losartan, however, normalized these changes.^9,17

It is well known that Ang II activates p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated protein kinase (ERK), and stress-activated protein kinase/Jun N-terminal kinase (SAPK/JNK), which are critical protein kinases for cell growth, cell death, and gene expression.¹⁸ Ang II has been shown to activate both p38 MAPK¹⁹ and JNK²⁰ in cultured vascular smooth muscle cells (VSMCs). p38 MAPK positively regulates VSMC growth induced by Ang II,¹⁹ whereas JNK was activated in a balloon-injured artery and could be inhibited by an AT₁R antagonist.²¹ It has also been well established in other studies that JNK is involved in the activation of the transcription factor AP-1.^22–24 Although these pathways have been relatively well studied in VSMC and cardiac myocytes, there has been little investigation of this important Ang II signaling pathway in neurons that control sympathetic outflow, especially in the CHF state.

We hypothesized that based on data in the peripheral circulation, AT₁R expression in central cardiovascular neurons would be regulated by the JNK pathway and that AP-1 plays a major role in AT₁R gene transcription. The current study examined the relationship between plasma Ang II and the expression of AT₁R in CHF. We further investigated the intracellular mechanism for the upregulation of the AT₁R by Ang II.

	Materials and Methods

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Animals
Experiments were performed on 25 male New Zealand White rabbits weighing between 3.2 and 4.1 kg. 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 NIH. Rabbits were assigned to 5 different groups (n=5 per group). These groups were sham, pacing-induced CHF, CHF plus intracerebroventricular (ICV) infusion of losartan, sham plus ICV Ang II, and sham plus ICV Ang II and losartan.

Cell Culture
A neuronal cell line (CATH.a) was purchased from American Type Culture Collection and 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/100-mm plate, or 1.5x10⁶ cells/well in 6-well culture plates. After subculture, cells were allowed to grow for 2 days and then treated with Ang II (100 nmol/L), an AT₁ receptor blocker (losartan, 10 µmol/L), a JNK inhibitor (SP600125, 10 µmol/L), a p38 inhibitor (SB202190, 10 µmol/L), or an ERK inhibitor (PD98059, 10 µmol/L).

Induction of CHF
CHF was induced by chronic ventricular tachycardia, as previously described.²⁵ Sham animals were prepared identically to CHF animals but were not paced.

Chronic ICV Infusion
In the ICV infusion groups, a 19-gauge cannula was implanted into a lateral cerebral ventricle as previously described.¹⁷ An osmotic minipump (Model 2001, Durect Corp) filled with Ang II 100 ng/µL per hour or losartan 30 µg/µL per hour in artificial cerebrospinal fluid was implanted subcutaneously in the back of the neck and connected to the ICV cannula. The infusion was continued for 6 days.

Cardiac Function, Arterial Pressure, Heart Rate, Left Ventricular Pressure, and 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). Left ventricular (LV) pressure was measured with a Millar transducer (Model SPR-524, Millar Instruments Inc). Details of the procedure can be found in the online data supplement at http://circres.ahajournals.org.

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 Brownstein²⁶ for analysis of mRNA and protein of the AT₁R receptor and other molecular studies.

Plasma Ang II Radioimmunoassay
Ang II peptide in rabbit plasma was measured using a radioimmunoassay (RIA). The protocol for Ang II RIA was modified from that described by Raff et al²⁷ to increase the recovery rate and sensitivity. Details of the procedure can be found in the online data supplement.

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 Synthesis Kit (Bio-Rad). Gene Specific primers and probes are listed in the Table 1 and were synthesized in the University of Nebraska Medical Center Eppley DNA Synthesis Core Facility. Gene-specific probes were labeled with carboxyfluorescein (FAM) at the 5' site with Black Hole Quenches at the 3' site to add specificity and sensitivity (Glen Research). ß-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 CATH.a cells. Both ß-actin and GAPDH are commonly used as internal controls in Ang II studies by others.^17,28 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 and Schmittgen.²⁹

View this table: [in this window] [in a new window]   Table 1. Gene-Specific Primers and Probes

Western Blot Analysis
Homogenates were prepared from RVLM. Protein concentration was measured using a bicinchoninic acid protein assay kit (Pierce, Rockford, Ill). Details of the procedure can be found in the online data supplement.

SAPK/JNK Activity Assays
SAPK/JNK activity were measured by using the ASPK/JNK assay kit from Cell Signaling Technology. Details of the procedure can be found in the online data supplement.

Electrophoretic Mobility-Shift Assay
Nuclear extracts of rabbit brain tissues were prepared with the NE-PER nuclear extraction reagent (Pierce). Details of the procedure can be found in the online data supplement.

Statistics
Data are expressed as mean±SEM. All statistical analyses were performed by a 1-way analysis of variance (ANOVA) for repeated measurements using SigmaStat (SPSS, Chicago, Ill). If significance was found between groups post hoc analyses were performed using the Bonferroni correction. P<0.05 was considered significantly different.

	Results

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Body Weight, Heart Weight, Hemodynamics, and Echocardiographic Data
Table 2 shows the values for body weight, ratio of organ weight to body weight, hemodynamics, and echocardiographic data in the rabbits from the 5 groups studied. The CHF group exhibited a significantly higher ratio of wet lung weight to body weight, heart rate, LV systolic diameter, LV end-diastolic diameter, plasma Ang II, and a significantly lower ejection fraction and cardiac output compared with the normal group. No significant differences in these parameters were found between CHF and CHF plus ICV losartan. There was a trend for MAP to increase in response to ICV Ang II infusion in sham animals, but this did not reach statistical significance.

View this table: [in this window] [in a new window]   Table 2. Baseline Hemodynamic Parameters in CHF and Sham Rabbits

AT₁R Expression
AT₁R protein and mRNA expression in the RVLM of the CHF group were significantly higher compared with sham (Figure 1). Animals with CHF exhibited a 240.4±30.2% increase in AT₁R mRNA (P<0.01) and a 206.6±25.8% (P<0.01) in AT₁R protein. Following 6 days of ICV losartan infusion to CHF rabbits, AT₁R protein and mRNA expressions were normalized. A similar increase in AT₁R protein and mRNA expressions was observed when normal rabbits were infused with ICV Ang II for 6 days. In Ang II–infused normal rabbits that were given losartan the increase in AT₁R expression was inhibited (Figure 1). These data strongly suggest that Ang II plays an important positive feedback role in AT₁R upregulation in CHF.

View larger version (16K): [in this window] [in a new window]   Figure 1. The expression of AT1R. A, Western blots of AT1R protein (top) and densitometric analysis of AT1R protein (bottom) from the RVLM in the 5 groups of rabbits. The data are the mean ratio of AT1R to GAPDH protein. B, Relative AT1R mRNA levels determined with real-time PCR. The data are the mean of AT1R mRNA levels. Error bars represent the SEM. *P<0.01 vs sham group, P<0.01 vs CHF group, #P<0.01 vs Ang II group.

Increases of AP-1–Binding Activity in CHF and in Response to ICV Ang II Infusion
Binding activity of the transcription factor AP-1 was increased significantly in CHF rabbits and in sham rabbits subjected to ICV infusion of Ang II compared with sham rabbits (Figure 2A). Administration of ICV losartan to CHF and Ang II–infused sham rabbits inhibited AP-1 activity compared with their respective controls but were not different compared with sham rabbits (Figure 2A).

View larger version (20K): [in this window] [in a new window]   Figure 2. AP-1–DNA binding. A, Electrophoretic mobility-shift assay (EMSA) (top) and densitometric analysis (bottom) of AP-1–DNA binding activities in the RVLM from each group of rabbits. The data are the mean densities. Error bars represent the SEM. B, Competitive and supershift analyses of AP-1–DNA binding activities. *P<0.01 vs sham group, P<0.01 vs CHF group, #P<0.01 vs Ang II group.

The specificity of the DNA/protein complex was characterized by competitive and supershift assays. The binding to the AP-1–specific oligonucleotide was inhibited in the presence of unlabeled oligonucleotides with the same sequence. The addition of anti–c-Jun antibody to the binding reaction produced a supershift complex as shown in Figure 2B. These data suggest that more active c-Jun transcription factors are available in CHF and Ang II–infused rabbits to bind to the AP-1 sequence.

Increases of c-Jun mRNA and Phosphorylated c-Jun in CHF and ICV Ang II infusion
To determine the role of c-Jun in transcriptional regulation of the AT₁R, we measured c-Jun activation in punches of RVLM. The AP-1 transcription factor is a dimer of Fos and Jun or a dimer of the members of the Jun family. To activate AP-1 transcription, both components of AP-1 have to be phosphorylated. We measured phosphorylated c-Jun as an indicator of activation of AP-1. As shown in Figure 3, c-Jun mRNA expressions and phosphorylated c-Jun level increased significantly in CHF and Ang II–infused groups compared with sham. Losartan inhibited c-Jun activation in both CHF and Ang II–infused groups (Figure 3). These data strongly suggest that c-Jun as a monomer of the AP-1 dimer is activated in CHF and that Ang II and its binding to the AT₁R may be responsible for AP-1 activation.

View larger version (16K): [in this window] [in a new window]   Figure 3. The expression of c-Jun. A, Relative c-Jun mRNA expression detected by real-time PCR. The data are the mean of relative c-Jun mRNA levels. Error bars represent the SEM. B (top), Western blots of phosphorylated (top row) and total (bottom row) c-Jun. B (bottom), densitometric analysis of phosphorylated protein levels. The data are the mean ratio of phosphorylated c-Jun to total c-Jun. Error bars represent the SEM. *P<0.01 vs sham group, P<0.01 vs CHF group, #P<0.01 vs Ang II group.

Activation of JNK in CHF and in Response to ICV Ang II Infusion
We measured phosphorylated JNK to determine whether the JNK pathway was necessary for AP-1 activation and AT₁R upregulation. As shown in Figure 4, phosphorylated JNK and JNK activities increased significantly in CHF and Ang II–infused groups compared with sham rabbits. Again, losartan reduced this response. These data suggest that increased phospho-JNK may be responsible for increased levels of phospho-c-Jun and thereby increased AP-1 activity in CHF.

View larger version (18K): [in this window] [in a new window]   Figure 4. The phosphorylation and activity of JNK. A (top), Western blots of phosphorylated (top row) and total (bottom row) JNK. A (bottom), Densitometric analysis of phosphorylated protein levels. The data are the mean ratio of phosphorylated JNK to total JNK. Error bars represent the SEM, where the bars with different lower case letters are significantly different from one another at P<0.01 among the 5 groups. B, Western blots (top) of phosphorylated c-Jun fusion protein in the assay of JNK1/2 activity and densitometric analysis (bottom) of phosphorylated c-Jun fusion protein. The data are the mean of densities. Error bars represent the SEM. *P<0.01 vs sham group, P<0.01 vs CHF group, #P<0.01 vs Ang II group.

Ang II–Induced Stimulation of AT₁R mRNA Through JNK but Not p38 or ERK
To further explore the mechanism of AT₁R upregulation in CHF, we used a neuronal cell line to investigate the specific pathway for AT₁R mRNA transcriptional regulation. Treatment of CATH.a cells with Ang II (100 nmol/L) for 0 to 24 hours at 37°C produced a time-dependent increase in AT₁R mRNA expression (Figure 5A), which peaked at 6 hours. The effect of various agents on AT₁R mRNA expression in response to Ang II (100 nmol/L) was examined after a 3-hour incubation (Figure 5B). Pretreatment with the AT₁R antagonist losartan (10 µmol/L, 1 hour) inhibited the response to Ang II. Pretreatment with the JNK inhibitor, SP 600125 (10 µmol/L, 1 hour) also inhibited AT₁R expression. The increase in AT₁R mRNA expression in Ang II–treated cells was not blocked by pretreatment with the p38 inhibitor (SB 202190, 10 µmol/L), or the ERK inhibitor (PD 98059, 10 µmol/L). These data suggest that AT₁R transcription in CATH.a cells occurs downstream of JNK activation but not downstream of p38 or ERK activation.

View larger version (11K): [in this window] [in a new window]   Figure 5. The expression of AT1R mRNA in CATH.a cells. A, AT1R mRNA levels were determined by real-time RT-PCR analysis at different time points. The data are the mean of the AT1R mRNA levels. Error bars represent the SEM; *P<0.01 compared with the control group. B, Relative AT1R mRNA levels were determined with real-time RT-PCR among different treatments. The data are the mean of relative AT1R mRNA levels. Error bars represent the SEM. *P<0.01 compared with the control group, P<0.01 compared with the Ang II–treated group.

	Discussion

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The main finding in this study is that the AT₁R is up regulated in the RVLM of rabbits with CHF and that the upregulation depends on the increase in the transcription factor AP-1, which is activated by the SAPK/JNK pathway but not by other MAPK pathways. This conclusion is drawn from the following observations. (1) AT₁R protein and mRNA expressions were increased in the RVLM of CHF rabbits. ICV losartan normalized AT₁R up regulation. ICV infusion of Ang II in normal rabbits mimicked the change in AT₁R expression in the CHF state, even though there were no changes in hemodynamic parameters. (2) An increase in AP-1–DNA binding indicated that AP-1 activity increases in the regulation of AT₁R gene transcription in CHF. (3) The AP-1 upstream signaling pathway consisting of phosphorylated JNK and phosphorylated c-Jun expression which were also increased in CHF and ICV Ang II–infused rabbits. (4) In a neuronal cell culture, Ang II–stimulated AT₁R mRNA expression. Losartan and a JNK inhibitor blocked Ang II–induced AT₁R expression; however, inhibition of ERK or p38 had no effect.

Increasing evidence indicates that the activation of the AT₁R occurs in diverse tissues and mediates the pathogenesis of various diseases.³⁰ Ang II has been reported to rapidly cause downregulation of its receptor.³¹ Binding studies indicate that Ang II receptor density in the glomerulus and peripheral vasculature is decreased under conditions of elevated circulating Ang II and is increased when plasma Ang II is low.^32,33 However, chronic increases in peripheral or central Ang II are associated with a gradually developing hypertension, which would require continued expression of the AT₁R.³⁴ Porter³⁵ found that 1 week of ICV Ang II infusion produced a significant increase in brain AT₁R in young rats, which was thought to play a role in the development of cardiovascular control mechanisms. Moellenhoff et al³⁶ showed an increase in AT₁R receptor number in the hypothalamus of rats after repetitive stimulation with Ang II. Cheng et al³⁷ found that Ang II upregulated AT₁R in the renal proximal tubule which had a significant impact on sodium reabsorption.

In this study, we observed that AT₁R expression in the RVLM was significantly higher in CHF than in sham rabbits. Zhu et al¹⁶ observed AT₁R expression increased in the paraventricular nucleus (PVN) of CHF rats. Previous data from this laboratory⁹ showed that an increase in AT₁R expression in RVLM correlated with an increase in renal sympathetic activity and that chronic ICV losartan reduced sympathetic nerve activity and normalized the AT₁R increase in CHF. In the present study, ICV losartan reversed the changes in AT₁R expression and its signaling pathways in CHF. The changes in AT₁R expression along with c-Jun did not reflect normalization in hemodynamic parameters following losartan treatment. There may be several reasons for this discrepancy. First, it may suggest that early molecular changes may not be translated into functional changes for a longer period of time. Second, abnormalities in cardiac and peripheral function are multifactorial. Central sympathetic excitation is just one of many factors responsible for the progression of heart failure. Ribeiro reviewed several losartan clinical trials over the past 15 years and concluded that losartan confers its cardiovascular and renal protective effects beyond its ability to lower blood pressure.³⁸

In a previous study from our laboratory, we observed an increase in renal sympathetic nerve activity in rabbits subjected to chronic ICV infusion of Ang II.¹⁷ This study also showed a central upregulation of AT₁R similar to that observed here. Furthermore, chronic central stimulation with Ang II evoked a profound increase in reactive oxidant stress in the RVLM. These data, along with that reported here, suggest a novel pathway by which Ang II exerts its deleterious effects on the central regulation of sympathetic outflow. Since Shibanuma et al³⁹ reported that the treatment of cells with H₂O₂ induced the transcription of c-fos and c-jun, redox regulation of transcription factor function has emerged as a potentially important and widespread mechanism of gene regulation. The growing list of redox-regulated transcription factors currently includes such well-known factors as AP-1, Egr-1, nuclear factor B, and p53.^40,41 It has also been reported that Ang-II stimulates the binding activity of AP-1 via a reactive oxygen species (ROS)-signaling pathway in cultured neonatal rat ventricular myocytes.²³ A role for ROS in AP-1 activation in the central nervous system following Ang II infusion or in CHF remains to be determined.

This study provides evidence that AP-1–DNA binding activity is significantly increased in the RVLM of CHF rabbits and of rabbits exposed to ICV Ang II. AP-1 is redox sensitive through the conserved cysteine residues located in the DNA-binding domain.⁴² AP-1 consists of a dimer of Jun (c-Jun, JunB, and JunD) and Fos (c-Fos, FosB, Fra1, and Fra2) family members. Jun family members form homo- and heterodimers that recognize a TGAGTCA consensus DNA sequence. Fos family members, which are unable to dimerize with each other, augment transcriptional activation by the association with Jun family members.^43,44 ROS generation in response to various external stimuli has been shown to be related to changes in AP-1.^45,46 AP-1 is involved in the expression of numerous genes responsible for cell proliferation and tissue remodeling by binding to the AP-1 consensus sequence present in their promoter regions. The promoter region of the AT₁R gene contains an AP-1 consensus sequence. We analyzed the promoter regions of human, rat, and mouse AT₁R genes for the putative transcription factor binding sites by using Transcription Element Search Software (TESS) (http://www.cbil.upenn.edu/cgi-bin/tess/tess). Human, mouse, and rat AT₁R genes harbor the consensus transcription factor binding sites for AP-1. The increased binding properties of these AP-1 complexes can result in persistent gene transcription. Thus, an increased expression of the AT₁R may reflect the fact that the expression of the AT₁R gene is under positive control by AP-1 activity.

We next determined the signal transduction pathway for the Ang II–induced AP-1 transactivation for AT₁R upregulation. Increasing evidence suggests that JNK plays an important role in mediating neuronal injury and apoptosis by oxidative stress.⁴⁷ Fleegal and Sumners⁴⁸ have reported that Ang II acts via AT₁R-stimulated AP-1–DNA binding in neurons of newborn Sprague–Dawley rats and the stimulatory effects of Ang II on AP-1–DNA binding require activation of JNK. To this end, we measured phosphorylated JNK and its activity in CHF and Ang II–infused normal rabbits. Phosphorylated JNK and its activity in the RVLM were increased in both groups. Losartan blocked the activation of JNK. The effects of Ang II on neuronal c-Jun mRNA and phosphorylated c-Jun protein have also been evaluated. The increased level of phospho–c-Jun in CHF rabbits further suggests the activation of AP-1.

Because ICV infusion of Ang II stimulates a variety of tissue types in the intact animal, we examined the effect of Ang II in a neuronal cell line (CATH.a). These results confirmed an Ang II–mediated upregulation of AT₁R expression, which was totally blocked by the AT₁R blocker losartan. In CATH.a cells, we also tested the effects of specific blockers for JNK, ERK, and p38 on AT₁R expression. Ang II–induced AT₁R expression was blocked by the JNK inhibitor but not by ERK or p38 blockers. These results suggest that AT₁R transcription occurs downstream of JNK activation and does not involve p38 or ERK activation.

Overall, this study provides insight into the mechanisms that may contribute to AT₁R upregulation in RVLM neurons of CHF rabbits. We recognize that other areas in the mid- and hindbrain may be important Ang II–dependent regulators of sympathetic nerve activity; however, because the RVLM is the final common pathway for premotor sympathetic neurons projecting to the spinal cord, this mechanism may be important in setting the level of sympathetic outflow in CHF and other hyperadrenergic states. Figure 6 illustrates a possible mechanism for AP-1 activation and AT₁R upregulation, consistent with the data presented here. Further investigations will be necessary to elucidate additional complex mechanisms such as posttranscriptional modification of the AT₁R (eg, mRNA stability and/or alternative splicing).

View larger version (49K): [in this window] [in a new window]   Figure 6. Schematic diagram for AT1R upregulation. Ang II binds to the neuronal AT1R and activates JNK, then phosphorylates a c-Jun+c-fos dimer to form AP-1, which translocates to the nucleus and binds to the promoter region of the AT1R gene to initiate transcription.

	Acknowledgments

 
We thank Phyllis Anding, Pamela Curry, and Johnnie Hackley for excellent technical assistance.

Sources of Funding

This study was supported by NIH grant PO-1 HL62222. L.G. was supported by a Postdoctoral Fellowship from the American Heart Association.

Disclosures

I.H.Z. is a member of the scientific advisory board of CVRx Inc (Maple Grove, Minn).

	Footnotes

 
Original received June 12, 2006; resubmission received July 20, 2006; revised resubmission received August 21, 2006; accepted September 14, 2006.

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