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Am J Physiol Heart Circ Physiol 288: H2271-H2279, 2005. First published January 6, 2005; doi:10.1152/ajpheart.00949.2004
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Sympathoexcitation by central ANG II: Roles for AT1 receptor upregulation and NAD(P)H oxidase in RVLM

Lie Gao, Wei Wang, Yu-Long Li, Harold D. Schultz, Dongmei Liu, Kurtis G. Cornish, and Irving H. Zucker

Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, Nebraska

Submitted 10 September 2004 ; accepted in final form 5 January 2005


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Chronic heart failure is often associated with sympathoexcitation and blunted arterial baroreflex function. These phenomena have been causally linked to elevated central ANG II mechanisms. Recent studies have shown that NAD(P)H oxidase-derived reactive oxygen species (ROS) are important mediators of ANG II signaling and therefore might play an essential role in these interactions. The aims of this study were to determine whether central subchronic infusion of ANG II in normal animals has effects on O2– production and expression of NAD(P)H oxidase subunits as well as ANG II type 1 (AT1) receptors in the rostral ventrolateral medulla (RVLM). Twenty-four male New Zealand White rabbits were divided into four groups and separately received a subchronic intracerebroventricular infusion of saline alone, ANG II alone, ANG II with losartan, and losartan alone for 1 wk. On day 7 of intracerebroventricular infusion, mean arterial pressure (MAP), heart rate (HR), and renal sympathetic nerve activity (RSNA) values were recorded, and arterial baroreflex sensitivity was evaluated while animals were in the conscious state. We found that ANG II significantly increased baseline RSNA (161.9%; P < 0.05), mRNA and protein expression of AT1 receptors (mRNA, 66.7%; P < 0.05; protein, 85.1%; P < 0.05), NAD(P)H oxidase subunits (mRNA, 120.0–200.0%; P < 0.05; protein, 90.9–197.0%; P < 0.05), and O2– production (83.2%; P < 0.05) in the RVLM. In addition, impaired baroreflex control of HR (Gainmax reduced by 48.2%; P < 0.05) and RSNA (Gainmax reduced by 53.6%; P < 0.05) by ANG II was completely abolished by losartan. Losartan significantly decreased baseline RSNA (–49.5%; P < 0.05) and increased baroreflex control of HR (Gainmax increased by 64.8%; P < 0.05) and RSNA (Gainmax increased by 67.9%; P < 0.05), but had no significant effects on mRNA and protein expression of AT1 receptor and NAD(P)H oxidase subunits and O2– production in the RVLM. These data suggest that in normal rabbits, NAD(P)H oxidase-derived ROS play an important role in the modulation of sympathetic activity and arterial baroreflex function by subchronic central treatment of exogenous ANG II via AT1 receptors.

baroreflex; renal sympathetic nerve activity; free radicals; angiotensin II type 1 receptor; rostral ventrolateral medulla; reactive oxygen species

CHRONIC HEART FAILURE IS often associated with sympathoexcitation (24, 40, 41, 63) and impaired arterial baroreflex function (15, 50, 54, 55). These phenomena have been causally linked to elevated central ANG II (14, 16, 39, 56). Indeed, increases in central ANG II have been shown to increase sympathetic outflow and blunt arterial baroreflex responses in normal animals (4, 57). Conversely, central administration of losartan has been shown to reduce sympathetic outflow and enhance baroreflex sensitivity in rats with chronic heart failure (14, 34). Low-dose, chronic subpressor infusion of ANG II into conscious rabbits is capable of resetting the arterial baroreflex (3, 4). However, high-dose, chronic central ANG II infusions in conscious rabbits did reduce the baroreflex control of heart rate (HR) (17).

Recent evidence indicates that NAD(P)H oxidase-derived reactive oxygen species (ROS) are important mediators of ANG II signaling (22, 61). ANG II not only augments ROS formation and increases oxidase activity but also upregulates mRNA and protein expression of most NAD(P)H oxidase subunits both in vitro (44) and in vivo (38). In addition, ROS have been shown to play an important role in various physiological and pathophysiological processes (25, 47) in the central nervous system. ROS have been linked to regulation of sympathetic nerve activity. For example, treatment with the cell membrane-permeable superoxide dismutase (SOD) mimetic tempol decreased renal sympathetic nerve activity (RSNA) in both normotensive and hypertensive rats (58, 59). Conversely, the SOD inhibitor diethyldithiocarbamate has the opposite effect (48).

AT1 receptors are widely distributed in the central nervous system from the forebrain to the brain stem (28). The rostral ventrolateral medulla (RVLM) contains a high density of these receptors (1) and is a major site of sympathoexcitation in response to ANG II administration into the cerebrospinal fluid (23). The RVLM not only plays a critical role in the generation and maintenance of sympathetic nerve activity (11, 12) but also is an essential part of the central baroreflex pathway (10, 18).

Therefore, we hypothesized that centrally administered ANG II may act via an AT1 receptor mechanism to activate sympathetic outflow and impair arterial baroreflex function by stimulation of NAD(P)H oxidase and ROS in the RVLM. The purpose of this study was to determine whether central subchronic infusion of ANG II stimulates O2– production and expression of NAD(P)H oxidase subunits and AT1 receptors in the RVLM. Second, we identified the effects on sympathetic nerve activity and arterial baroreflex function and determined whether these central effects of ANG II are mediated by AT1 receptors.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Experiments were carried out on 24 male New Zealand White rabbits that weighed 3.5–4.2 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 National Institutes of Health. Rabbits were housed in individual cages under controlled temperature and humidity with a 12:12-h dark-light cycle and were fed standard rabbit diet (sodium content, 0.31%; Harlan Techlad) with water available ad libitum.

Subchronic lateral cerebral ventricle infusion. The skull was exposed through an incision on the midline of the scalp. After the bregma was identified, a 19-gauge stainless steel cerebroventricular cannula was implanted into the right lateral cerebral ventricle, 4 mm lateral to the bregma and 6 mm below the cerebral surface, and fixed tightly to the skull with super-glue adhesive and dental cement. The position of the cannula in the lateral cerebral ventricle was confirmed by the staining of all four ventricles after injection of 0.1 ml of Evans blue dye at the end of the experiments. An osmotic minipump (model 2001; Durect; Cupertino, CA) filled with isotonic saline (1 µl/h), ANG II (80 ng·µl–1·h–1), ANG II (80 ng·µl–1·h–1) plus losartan (20 µg·µl–1·h–1), or losartan alone (20 µg·µl–1·h–1) was implanted subcutaneously in the back of neck and connected to the cerebroventricular cannula. The infusion was continued for 7 days.

Arterial pressure and HR recording. While animals were under anesthesia and aseptic conditions, a catheter connected to a radio-telemetry unit (Data Sciences International; St. Paul, MN) was inserted into the descending aorta via a branch of the right femoral artery for direct measurement of arterial pressure (AP) with rabbits in the conscious state. HR was derived from the AP pulse using a PowerLab (model 8S; ADInstruments; Colorado Springs, CO) data-acquisition system.

RSNA recording. RSNA-recording electrodes were implanted as described previously (32). In brief, while animals were under anesthesia and under sterile conditions, the left kidney was exposed retroperitoneally, and a branch of renal nerve was separated from the renal plexus and the surrounding connective tissues. A pair of stainless steel, stranded Teflon-coated recording electrodes were placed around the nerve branch. The nerve-electrode junction was insulated electrically from the surrounding tissues and covered with fast-setting silicone (Kwik-Sil; World Precision Instruments; Sarasota, FL). A ground wire was secured to a nearby muscle. The recording electrodes and ground wire were tunneled under the skin and exteriorized in the midscapular area. During the experiment, the electrical signal from the electrode was amplified with a Grass P55 preamplifier (Grass Instruments; West Warwick, RI) with high- and low-frequency cutoffs of 1,000 and 100 Hz, respectively. The output from the Grass amplifier was directed to the PowerLab system, which sampled at 1,000 samples/s. The signal was also full-wave rectified and integrated. The average rectified signal (RC filtered; time constant, 0.5 s) was then recorded and stored for later analysis. The frequency of nerve discharge was counted by using a window discriminator and rate meter. The cursor of the window discriminator was set just above the electrical noise. Both frequency and integrated nerve activity were recorded continuously along with the raw nerve activity. The units of RSNA used in this experiment are percent of maximum (% max) and represent the maximum RSNA induced by nasopharyngeal stimulation with cigarette smoke, which has been shown to be a suitable and reliable method of comparing RSNA baroreflex curves under a variety of different conditions (5).

Evaluation of arterial baroreflex function. Evaluation of the arterial baroreflex was carried out as previously described (31). In brief, rabbits were studied in a quiet, dimly lit room while they stood in a Plexiglas box. AP, HR, and RSNA values were recorded using a PowerLab system. Intravenous infusions were administered via a lateral ear vein. After the animal had adjusted to the environment and all hemodynamics were stable, an intravenous infusion of sodium nitroprusside (SNP) was started at a rate of 100 µg·kg–1·min–1 at 0.5 ml/min. When AP reached its nadir (usually 40–50 mmHg), the SNP infusion was stopped, and a phenylephrine (PE) infusion was started at a rate of 80 µg·kg–1·min–1 at 0.5 ml/min. The PE infusion continued until AP reached ~110 mmHg. The baroreflex was analyzed over the pressure range from lowest to highest pressure. This infusion rate increased AP at a rate of ~1 mmHg/s.

The HR, MAP, and RSNA data were acquired every 2 s from the thresholds to the saturation points. A sigmoid logistic function was fit to the data using a nonlinear regression program (SigmaPlot 8.0; Jandel). Four parameters can be derived from the following equation: HR or RSNA = A/{1 + exp[B(MAP – C)]} + D, where A is the HR or RSNA range, B is the slope coefficient, C is the pressure at the midpoint of the range (BP50), and D is the minimum HR or RSNA. The peak slope (or maximum gain) was determined by taking the first derivative of the baroreflex curve and was calculated using the equation Gainmax = A1 x A2 x, where A1 is the range and A2 is the average slope.

O2– production in RVLM. O2– production was measured by the lucigenin enhanced chemiluminescence (ECL) method (TD-20/20 luminometer; Turner Designs; Sunnyvale, CA). Total protein concentration was determined using a bicinchoninic acid protein-assay kit (Pierce; Rockford, IL). NADPH (100 µM) and dark-adapted lucigenin (5 µM) were added into 0.5-ml microcentrifugal tubes just before reading. Light emission was recorded over 10 min, and values are expressed as mean light units per minute per milligram of protein.

Preparation of RVLM tissue. At the end of the experiment, each rabbit was euthanized with pentobarbital sodium. The brain was removed and immediately frozen on dry ice, blocked in the coronal plane, and sectioned into 300-µm-thick slices using a cryostat. The RVLM was punched according to the method of Palkovits and Brownstein (42) for the analysis of O2– production and mRNA and protein of AT1 receptor and NAD(P)H subunits.

RT-PCR analysis of AT1 receptor and NAD(P)H subunit mRNA in RVLM. Total RNA of the RVLM was isolated using the RNeasy Mini Kit Total RNA Isolation System (Qiagen; Valencia, CA), after which cDNA was synthesized by means of Moloney murine leukemia virus (MMLV) reverse transcriptase (Invitrogen Life Technologies; Carlsbad, CA) according to the manufacturers' instructions. RNA was treated in parallel in the presence or absence of reverse transcriptase. PCR amplification was performed by means of a PTC-100 programmable thermal controller (MJ Research; Watertown, MA) as follows: 1 cycle at 95°C for 15 min, followed by 35 cycles of 94°C for 45 s, 55°C for 45 s, and 72°C for 1 min. The primer pairs were based on the cDNA sequences of rabbit AT1 receptor (GenBank accession no. S59041), gp91phox (GenBank accession no. AF323788), p67phox (GenBank accession no. AF323789), p47phox (GenBank accession no. AF324409), p40phox (GenBank accession no. AF323790), and p22phox (GenBank accession no. AF323787) with {beta}-actin (GenBank accession no. AF309819) as an internal control. The primer pairs were as follows: AT1 receptor, 5'-TTTGGGAACAGCTTGGCGGT-3' and 5'-GCCAGCCAGCAGCCAAATAA-3'; gp91phox, 5'-gcttgtggctgtgataagca-3' and 5'-ctcctgcatctgtgtctcca-3'; p67phox, 5'-aactcagtgggtgaccaagg-3' and 5'-gttcttccacgaaggctctg-3'; p47phox, 5'-acgagagtggttggtggttc-3' and 5'-tagccagtgacgtcctcctt-3'; p40phox, 5'-tcactgggaacagcaaactg-3' and 5'-ctgctgaggtcttcctccac-3'; p22phox, 5'-cgcttcacccagtggtactt-3' and 5'-gcagccagcaggtagatgat-3'; and {beta}-actin, 5'-GATCGCTGACCGTATGCAG-3' and 5'-GTCGTACTCCTGCTTGGTG-3'. The amplification products were visualized on 2% agarose gels by the use of ethidium bromide and were sequenced so that their identities could be confirmed. The bands were analyzed using UVP BioImaging Systems.

Western Blot analysis of AT1 receptor and NAD(P)H subunit protein in RVLM. The RVLM was homogenized with the homogenizer in RIPA buffer. Protein extraction from homogenates was used for Western blot analysis for rabbit AT1 receptor, gp91phox, p67phox, p47phox, and p40phox. Protein concentration was measured using a protein assay kit (Pierce). Samples were adjusted to the same concentrations of protein, mixed with equal volumes of 2x 4% SDS sample buffer, boiled for 5 min, and then loaded onto a 7.5% SDS-PAGE gel (5 µg protein/30 µl per well) for electrophoresis using a Bio-Rad minigel apparatus at 40 mA (for each gel) for 45 min. The fractionized proteins on the gel were electrophoretically transferred onto the polyvinylidene difluoride membrane (Millipore) at 300 mA for 90 min. The membrane was probed with primary antibody (1:1,000 dilutions of rabbit anti-human AT1 receptor polyclonal antibody and goat anti-human gp91phox, p67phox, p47phox, and p40phox polyclonal antibodies; Santa Cruz) and secondary antibody (1:2,500 dilutions of goat anti-rabbit IgG-horseradish peroxidase and rabbit anti-goat IgG-horseradish peroxidase; Santa Cruz) and then treated with enhanced chemiluminescence substrate (Pierce) for 5 min at room temperature. The bands in the membrane were visualized and analyzed using UVP BioImaging Systems.

Data and statistical analysis. Data are expressed as means ± SE. Differences between groups were determined with a two-way ANOVA followed by a Newman-Keuls test for post hoc analysis of significance. The differences before and after intracerebroventricular infusion treatment in each group were analyzed with a paired t-test. A P value of <0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Cardiovascular and sympathetic effects of ANG II and losartan. Basal MAP, HR, and RSNA values obtained before any treatments were similar among the four experimental groups (Table 1). The intracerebroventricular infusion of ANG II significantly increased RSNA (115.3%; P < 0.05), and losartan completely abolished the effects of ANG II on RSNA (P < 0.01). Although there was a tendency for the intracerebroventricular infusion of ANG II to increase MAP and HR, this did not reach statistical significance. The intracerebroventricular infusion of losartan significantly decreased baseline RSNA (–49.5%; P < 0.05) but had no effects on MAP and HR (Table 1). Original 7-day recordings of RSNA, MAP, and HR after intracerebroventricular infusion of saline (Fig. 1A) or ANG II (Fig. 1B) show that the frequency and integrated RSNA values appear to be greater after ANG II treatment than after saline treatment. Mean data for baseline RSNA (expressed as a percent of maximum nerve activity) after infusion are shown in Table 2. As can be seen, RSNA was significantly increased in the ANG II group compared with the saline group (control), RSNA in the ANG II plus losartan group was significantly lower than that from the ANG II group, and RSNA from the losartan group was significantly lower than that from the saline group (control).


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  		Table 1. Baseline MAP, HR, and RSNA values before and after intracerebroventricular infusion in conscious rabbits

 


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  		Fig. 1. Effects of subchronic intracerebroventricular infusion of ANG II on renal sympathetic nerve activity (RSNA). Original recordings of RSNA, arterial blood pressure (AP), mean arterial pressure (MAP), and heart rate (HR) after intracerebroventricular infusion of saline (A) or ANG II (B) for 7 days in conscious rabbits. Note the increased RSNA without a significant change in AP and HR by ANG II infusion compared with saline infusion. Freq, frequency; bpm, beats per minute.

 

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  		Table 2. Arterial baroreflex curve parameters after central administration of ANG II and losartan

 
Baroreflex effects of infusion of ANG II and losartan. Original 7-day recordings of AP show the changes induced by PE infusion after SNP infusion and the attendant HR and RSNA responses after intracerebroventricular infusion of saline (Fig. 2A) or ANG II (Fig. 2B). It is evident that the reflex bradycardia and RSNA response to PE after ANG II treatment were depressed compared with saline treatment.



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  		Fig. 2. Original recordings of AP changes induced by intravenous infusion of phenylephrine and attendant RSNA and HR reflex responses after 7 days of intracerebroventricular infusion of saline or ANG II. Note the suppressed reflex RSNA response and bradycardia responses to phenylephrine-induced pressor effect in ANG II-treated (B) compared with saline-treated (A) rabbits.

 
Composite arterial baroreflex curves for the control of HR and RSNA in the four groups of rabbits are shown in Fig. 3, and the other parameters that describe MAP-HR and RSNA-HR baroreflex curves are shown in Table 2. As can be seen, rabbits that received intracerebroventricular infusion of ANG II exhibited depressed baroreflex control of HR. This depression was due primarily to a reduction in the range of HR induced by altering AP and the minimum HR achieved during increases in AP (Table 2). The rabbits that received intracerebroventricular infusion of ANG II plus losartan had restored baroreflex function compared with the ANG II group. However, the rabbits that received intracerebroventricular infusion of losartan alone exhibited enhanced baroreflex control of HR that was due primarily to an increase in the average slope of the baroreflex function (Table 2).



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  		Fig. 3. Composite arterial baroreflex curves and mean data of maximum gain (Gainmax) of arterial baroreflex curves for HR (A and B) and RSNA (C and D) after 7 days of intracerebroventricular infusion of saline, ANG II, ANG II plus losartan (Los), or losartan alone. *P < 0.05; n = 6 rabbits/group. Gain curves of mean baroreflex curves are shown (A and C, insets).

 
Regarding the baroreflex control of RSNA, intracerebroventricular infusion of ANG II blunted the sensitivity with a significant decrease in the average slope and increase in the minimum RSNA achieved during increases in AP (Table 2). The BP50 value also was significantly elevated in this case and was completely abolished by losartan. On the other hand, rabbits that received intracerebroventricular infusion of losartan alone exhibited enhanced baroreflex control of RSNA that was due primarily to the increase in range of the baroreflex curve (Table 2).

mRNA expression of AT1 receptor and NAD(P)H oxidase subunits in RVLM after ANG II and losartan infusions. The mRNA expression of AT1 receptor and NAD(P)H oxidase subunits in the RVLM was assessed by RT-PCR. As shown in Fig. 4B, intracerebroventricular infusion of ANG II for 7 days significantly increased AT1 receptor expression by 66.7%, p40phox expression by 120.0%, p47phox and p67phox expression by 200.0%, and gp91phox expression by 175.0%. We failed to detect mRNA expression for p22phox in the RVLM of either the saline- or ANG II-treated group. However, as a positive control, we observed mRNA expression of p22phox in liver and kidney of these rabbits (data not shown). In the rabbits treated with ANG II plus losartan, mRNA expression of AT1 receptor and NAD(P)H oxidase subunits in the RVLM was significantly decreased compared with the ANG II group and almost the same as the saline group (Fig. 4B). In addition, mRNA expression of AT1 receptor and NAD(P)H oxidase subunits in the RVLM of losartan-treated rabbits was not significantly different from saline-treated rabbits (Fig. 4B). Figure 4A is a representative RT-PCR image that shows the upregulation of AT1 receptor, p40phox, p47phox, p67phox, and gp91phox mRNA expression in RVLM of ANG II-treated rabbits (Fig. 4A) compared with saline-treated animals (controls, Fig. 4C).



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  		Fig. 4. RT-PCR analysis for mRNA expression of ANG II type 1 receptor (AT1R) and NAD(P)H subunits in rostral ventrolateral medulla (RVLM) after 7 days of intracerebroventricular infusion of saline, ANG II, ANG II plus losartan, or losartan. A representative RT-PCR image (A) shows the upregulation of AT1 receptor, p40phox, p47phox, p67phox, and gp91phox mRNA expression in RVLM of ANG II-treated rabbits compared with saline-treated group [A and C (control), respectively]. {beta}-Actin was used as internal control. Results of densitometric analysis (B) include the means ± SE. *P < 0.05 and **P < 0.01 compared with control group; #P < 0.05 compared with ANG II-treated group; n = 6 rabbits/group.

 
Protein expression of AT1 receptor and NAD(P)H oxidase subunits in RVLM after infusion of ANG II and losartan. As shown in Fig. 5B, protein expression for AT1 receptor, p40phox, p47phox, p67phox, and gp91phox was significantly increased in rabbits treated with ANG II compared with rabbits treated with saline. In the rabbits treated with ANG II plus losartan, the protein expression of AT1 receptor and NAD(P)H oxidase subunits in RVLM was significantly decreased compared with the ANG II-treated group and almost the same as the saline-treated group (Fig. 5B). In addition, the protein expression of AT1 receptor and NAD(P)H oxidase subunits in the RVLM of losartan-treated rabbits was not significantly different from the saline-treated rabbits (Fig. 5B). Representative Western blots show the upregulation of AT1 receptor, p40phox, p47phox, p67phox, and gp91phox protein expression in the RVLM of ANG II-treated rabbits compared with the saline-treated group (controls).



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  		Fig. 5. Western blot analysis for protein expression of AT1 receptor and NAD(P)H subunits in RVLM after 7 days of intracerebroventricular infusion of saline, ANG II, ANG II plus losartan, or losartan. Representative Western blots (A) show the upregulation of AT1 receptor, p40phox, p47phox, p67phox, and gp91phox protein expression in RVLM of ANG II-treated rabbits compared with the saline-treated group. Results of densitometric analysis (B) include means ± SE. *P < 0.05 and **P < 0.01 compared with control group; #P < 0.05 compared with ANG II-treated group; n = 6 rabbits/group.

 
Measurements of O2– production in RVLM after infusion of ANG II and losartan. Using the lucigenin assay, we detected superoxide anion in punches of RVLM. Superoxide anion was significantly greater in the RVLM of ANG II-treated rabbits compared with saline-treated rabbits (1.74 ± 0.18 vs. 0.95 ± 0.13; P < 0.05), whereas in rabbits treated with losartan or ANG II plus losartan, the superoxide anion in the RVLM was not different from saline-treated rabbits (Fig. 6).



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  		Fig. 6. Mean data of NAD(P)H-dependent O2– production in RVLM after 7 days of intracerebroventricular infusion of saline, ANG II, ANG II plus losartan, or losartan alone measured by lucigenin chemiluminescence. MLU, mean light units. *P < 0.01; n = 6 rabbits/group.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The major findings of this study are that subchronic intracerebroventricular infusion of ANG II increases mRNA and protein levels of NAD(P)H oxidase components concomitant with increased local O2– production in the RVLM, elevations in basal RSNA, and impaired arterial baroreflex function. These results strongly suggest that NAD(P)H oxidase-derived ROS play an important role in the modulation of sympathetic activity and arterial baroreflex function via AT1 receptors. Moreover, in the present study we also found that subchronic intracerebroventricular infusion of the specific AT1 receptor antagonist losartan alone significantly decreased basal RSNA and improved arterial baroreflex control of HR and RSNA without any change in RVLM mRNA and protein levels of NAD(P)H oxidase components and local O2– production, which suggests that a non-ROS pathway may play a role in the modulation of sympathetic activity and arterial baroreflex function by endogenous ANG II in conscious normal rabbits.

Strong evidence is accumulating to suggest that central ROS are involved in the action of ANG II in the regulation of cardiovascular activity and function of central autonomic networks. For example, the adenoviral vector-mediated overexpression of SOD abolishes the cardiovascular effects of intracerebroventricular-injected ANG II in mice (61), and chronic systemic infusion of ANG II in mice causes a gradually developing hypertension that is correlated with marked elevations in O2– production specifically in the subfornical organ (62). Moreover, in the nucleus of the solitary tract, the essential NAD(P)H oxidase subunit gp91phox is present in somatodendritic and axonal profiles that contain AT1 receptors, and both the SOD scavenger Mn(III)tetrakis(4-benzoic acid)porphyrin chloride and NAD(P)H oxidase assembly inhibitor apocynin blocked the potentiation of ANG II on L-type Ca2+ currents (53). In the present study, we found that subchronic intracerebroventricular infusion of ANG II increases O2– production in the RVLM concomitant with elevation in basal RSNA and impaired arterial baroreflex function, which suggests that O2– is involved in the modulation of RSNA and arterial baroreflex function by long-term central ANG II treatment. In this regard, in a recent study by Mayorov et al. (35), it was nicely demonstrated that bilateral microinjections of tempol or tiron into the RVLM attenuated the pressor, sympathetic, and tachycardic responses to stress or microinjection of ANG II into the RVLM. The studies by Zanzinger and Czachurski (60) showed that SOD injected into the RVLM of pigs caused sympathoinhibition to a greater extent when the animals were under conditions of chronic overproduction of ROS, which further supports our hypothesis that ROS in the RVLM plays an important role in modulation of central autonomic nervous system activity and cardiovascular function by central ANG II. However, the mechanisms by which ROS excites the RVLM neurons are still unknown. Some studies (2, 29) indicate that ROS may directly activate Ca2+ channels to enhance Ca2+ currents and therefore might depolarize neurons and increase excitability and spontaneous activity.

The main source of ANG II-derived ROS is NAD(P)H oxidase (20, 37), an enzyme first described in phagocytes. NAD(P)H oxidase is composed of two membrane-bound subunits (gp91phox and p22phox), several cytoplasmic subunits (p40phox, p47phox, and p67phox), and the small G protein Rac 1a (26). After stimulation of AT1 receptors by ANG II, the cytoplasmic subunits bind to the membrane subunits and activate the enzyme, which results in production of O2– (21, 30, 49). In the present study, RT-PCR and Western blots showed a significant enhancement of most NAD(P)H oxidase subunit mRNA and protein in the RVLM from ANG II-infused vs. vehicle-infused and ANG II plus losartan-treated rabbits that correlated positively with changes in O2– production. This would be expected to contribute to higher NAD(P)H oxidase O2–-generating activity, because increases in even individual components have been shown to enhance NAD(P)H oxidase activity in cell-free assays (13). Previous reports by other groups have shown that prolonged subcutaneous or intraperitoneal infusion of ANG II significantly increased renal cortical (6) or aortic (7, 38) NAD(P)H oxidase subunit expression. The data reported here extend those findings to rabbit RVLM, which is a specific central region that not only maintains sympathetic vasomotor outflow and plays a key role in controlling the baroreceptor reflex (9) but also has a high density of AT1 receptors (1) and is a major site of sympathoexcitation in response to ANG II administered into the cerebrospinal fluid (23). This suggests close relationships between the brain renin-ANG II system, central redox signaling, and the regulation of cardiovascular function and autonomic nerve activity.

In the present study, we failed to detect mRNA expression for p22phox in the RVLM of rabbits. This does not appear to be due to a technical problem, because as a positive control, we did observe mRNA expression of p22phox in liver and kidney of these rabbits. However, this is not to say that NAD(P)H oxidase works well even without p22phox. One possibility, we believe, for the lack of p22phox in the RVLM of rabbit is the existence of some yet-unknown p22phox homolog, similar to what is observed in smooth muscle cells that lack gp91phox; recent studies have identified the existence of several gp91phox homologs such as Nox1 and Nox4 (27).

The ANG II receptor is a central component of the renin-angiotensin system. Activation of ANG II receptors mediates a variety of effects from contraction of vascular smooth muscle to secretion of hormones including the effects on NAD(P)H oxidase and ROS. Thus regulation of their expression is important in cardiovascular and central responsiveness to ANG II. The existence of two types of ANG II receptors, AT1 and AT2, has been demonstrated. Although little is known about the function of AT2 receptors, AT1 receptors mediate many of the functions described. The other finding of the present study is that subchronic intracerebroventricular infusion of ANG II significantly increased the mRNA and protein expression of AT1 receptors in the RVLM of rabbits, which might give an explanation for our previous finding that in the chronic heart-failure animal model, elevated ANG II levels were often concomitant with overexpression of AT1 receptors. Porter (43) also found that 1 wk of intracerebroventricular ANG II infusion produced a significant increase in brain AT1 receptor protein (via Western blot) and mRNA (via relative RT-PCR) expression. Moellehoff et al. (36) showed an increase in AT1 receptor number in some brain regions in rats after repetitive intracerebroventricular injections of ANG II (by immunohistochemical staining). The exact molecular mechanisms by which the expression of AT1 receptors in brain was upregulated by its ligand are still unclear. On the one hand, a number of factors including aldosterone are suggested to modulate the expression of AT1 receptor protein and mRNA (46). Chronic ANG II infusion increases plasma aldosterone and then induces transcription factors through mineralocorticoid receptors, and this is followed by an increase in AT1 receptor expression (51). ANG II also activates various nuclear transcription factors including activator protein-1, the signal transducers and activators of transcription family of transcription factors, cAMP response-element binding protein, and nuclear factor-{kappa}B (45), some of which are involved in the transcription of AT1 receptor genes (8).

In the present study, we also found that subchronic intracerebroventricular infusion of ANG II significantly increased basal RSNA, which may be due to the stimulation of ANG II on NAD(P)H oxidase and ROS in RVLM as described above, and contributed to the impaired baroreflex control of HR and RSNA observed in the same experiments. Many studies have solidly supported the idea that sympathetic activity and/or renin-angiotensin system activity can antagonize arterial baroreflex function in both humans and experimental animals (19, 33, 52). This was in agreement with the work of Gaudet et al. (17), who described diminished sensitivity of the cardiac baroreflex in conscious normotensive rabbits after long-term central administration of ANG II at subpressor doses. Considering these data, the increased NAD(P)H oxidase expression and ROS production in RVLM is critical for the effects of intracerebroventricular ANG II infusion on basal sympathetic activity and arterial baroreflex function. In addition, we still found that subchronic intracerebroventricular infusion of the AT1 receptor antagonist losartan produced enhanced baroreflex control of both HR and RSNA concomitant with suppressed baseline sympathetic activity but without any change of AT1 receptor protein and NAD(P)H oxidase subunit expression or O2– production in RVLM, which indicates that in conscious normal rabbits, there was tonic activation of these receptors from endogenous ANG II on arterial baroreflex function and sympathetic activity but no effect on AT1 receptor and NAD(P)H oxidase subunit expression.

In summary, our results demonstrate that in normal rabbits, subchronic intracerebroventricular infusion of ANG II upregulated AT1 receptor and NAD(P)H oxidase subunit expression and increased O2– production in RVLM mediated by central AT1 receptors and contributed to the enhanced sympathetic outflow and impaired arterial baroreceptor reflex control of HR and RSNA induced by this ANG II treatment. Endogenous ANG II, however, regulates the sympathetic activity and artery baroreflex function independent of the NAD(P)H-ROS pathway in RVLM.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by National Heart, Lung, and Blood Institutes Grant PO1 HL-62222. L. Gao was supported by a postdoctoral fellowship (Award 0425680Z) from the American Heart Association, Heartland Affiliate.


   ACKNOWLEDGMENTS
 
The authors thank Johnnie F. Hackley, Pamela Curry, and Jody Hallgren-Golka for expert technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: I. H. Zucker, Dept. of Cellular and Integrative Physiology,Univ. of Nebraska Medical Center, 985850 Nebraska Medical Center, Omaha, NE 68198-5850 (E-mail: izucker{at}unmc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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  1. Allen AM, Moeller I, Jenkins TA, Zhuo J, Aldred GP, Chai SY, and Mendelsohn FA. Angiotensin receptors in the nervous system. Brain Res Bull 47: 17–28, 1998.[CrossRef][ISI][Medline]
  2. Annunziato L, Pannaccione A, Cataldi M, Secondo A, Castaldo P, Di Renzo G, and Taglialatela M. Modulation of ion channels by reactive oxygen and nitrogen species: a pathophysiological role in brain aging? Neurobiol Aging 23: 819–834, 2002.[CrossRef][ISI][Medline]
  3. Brooks VL. Chronic infusion of angiotensin II resets baroreflex control of heart rate by an arterial pressure-independent mechanism. Hypertension 26: 420–424, 1995.[Abstract/Free Full Text]
  4. Brooks VL, Ell KR, and Wright RM. Pressure-independent baroreflex resetting produced by chronic infusion of angiotensin II in rabbits. Am J Physiol Heart Circ Physiol 265: H1275–H1282, 1993.[Abstract/Free Full Text]
  5. Burke SL and Head GA. Method for in vivo calibration of renal sympathetic nerve activity in rabbits. J Neurosci Methods 127: 63–74, 2003.[CrossRef][ISI][Medline]
  6. Chabrashvili T, Kitiyakara C, Blau J, Karber A, Aslam S, Welch WJ, and Wilcox CS. Effects of ANG II type 1 and 2 receptors on oxidative stress, renal NADPH oxidase, and SOD expression. Am J Physiol Regul Integr Comp Physiol 285: R117–R124, 2003.[Abstract/Free Full Text]
  7. Cifuentes ME, Rey FE, Carretero OA, and Pagano PJ. Upregulation of p67phox and gp91phox in aortas from angiotensin II-infused mice. Am J Physiol Heart Circ Physiol 279: H2234–H2240, 2000.[Abstract/Free Full Text]
  8. Clauser E, Curnow KM, Davies E, Conchon S, Teutsch B, Vianello B, Monnot C, and Corvol P. Angiotensin II receptors: protein and gene structures, expression and potential pathological involvements. Eur J Endocrinol 134: 403–411, 1996.[Abstract]
  9. Dampney RA. Functional organization of central pathways regulating the cardiovascular system. Physiol Rev 74: 323–364, 1994.[Free Full Text]
  10. Dampney RA, Blessing WW, and Tan E. Origin of tonic GABAergic inputs to vasopressor neurons in the subretrofacial nucleus of the rabbit. J Auton Nerv Syst 24: 227–239, 1988.[CrossRef][ISI][Medline]
  11. Dampney RA, Goodchild AK, Robertson LG, and Montgomery W. Role of ventrolateral medulla in vasomotor regulation: a correlative anatomical and physiological study. Brain Res 249: 223–235, 1982.[CrossRef][ISI][Medline]
  12. Dampney RA and Moon EA. Role of ventrolateral medulla in vasomotor response to cerebral ischemia. Am J Physiol Heart Circ Physiol 239: H349–H358, 1980.[Abstract/Free Full Text]
  13. DeLeo FR and Quinn MT. Assembly of the phagocyte NADPH oxidase: molecular interaction of oxidase proteins. J Leukoc Biol 60: 677–691, 1996.[Abstract]
  14. DiBona GF, Jones SY, and Brooks VL. ANG II receptor blockade and arterial baroreflex regulation of renal nerve activity in cardiac failure. Am J Physiol Regul Integr Comp Physiol 269: R1189–R1196, 1995.[Abstract/Free Full Text]
  15. DiBona GF and Sawin LL. Increased renal nerve activity in cardiac failure: arterial vs. cardiac baroreflex impairment. Am J Physiol Regul Integr Comp Physiol 268: R112–R116, 1995.[Abstract/Free Full Text]
  16. Francis GS. The relationship of the sympathetic nervous system and the renin-angiotensin system in congestive heart failure. Am Heart J 118: 642–648, 1989.[CrossRef][ISI][Medline]
  17. Gaudet E, Godwin SJ, and Head GA. Effects of central infusion of ANG II and losartan on the cardiac baroreflex in rabbits. Am J Physiol Heart Circ Physiol 278: H558–H566, 2000.[Abstract/Free Full Text]
  18. Granata AR, Ruggiero DA, Park DH, Joh TH, and Reis DJ. Lesions of epinephrine neurons in the rostral ventrolateral medulla abolish the vasodepressor components of baroreflex and cardiopulmonary reflex. Hypertension 5: V80–V84, 1983.[ISI][Medline]
  19. Grassi G, Cattaneo BM, Seravalle G, Lanfranchi A, Pozzi M, Morganti A, Carugo S, and Mancia G. Effects of chronic ACE inhibition on sympathetic nerve traffic and baroreflex control of circulation in heart failure. Circulation 96: 1173–1179, 1997.[Abstract/Free Full Text]
  20. Griendling KK, Minieri CA, Ollerenshaw JD, and Alexander RW. Angiotensin II stimulates NADH and NADPH oxidase activity in cultured vascular smooth muscle cells. Circ Res 74: 1141–1148, 1994.[Abstract/Free Full Text]
  21. Groemping Y, Lapouge K, Smerdon SJ, and Rittinger K. Molecular basis of phosphorylation-induced activation of the NADPH oxidase. Cell 113: 343–355, 2003.[CrossRef][ISI][Medline]
  22. Hanna IR, Taniyama Y, Szocs K, Rocic P, and Griendling KK. NAD(P)H oxidase-derived reactive oxygen species as mediators of angiotensin II signaling. Antioxid Redox Signal 4: 899–914, 2002.[CrossRef][ISI][Medline]
  23. Head GA, Saigusa T, and Mayorov DN. Angiotensin and baroreflex control of the circulation. Braz J Med Biol Res 35: 1047–1059, 2002.[ISI][Medline]
  24. Kaye DM, Lefkovits J, Cox H, Lambert G, Jennings G, Turner A, and Esler MD. Regional epinephrine kinetics in human heart failure: evidence for extra-adrenal, nonneural release. Am J Physiol Heart Circ Physiol 269: H182–H188, 1995.[Abstract/Free Full Text]
  25. Knapp LT and Klann E. Role of reactive oxygen species in hippocampal long-term potentiation: contributory or inhibitory? J Neurosci Res 70: 1–7, 2002.[CrossRef][ISI][Medline]
  26. Lassegue B and Clempus RE. Vascular NAD(P)H oxidases: specific features, expression, and regulation. Am J Physiol Regul Integr Comp Physiol 285: R277–R297, 2003.[Abstract/Free Full Text]
  27. Lassegue B, Sorescu D, Szocs K, Yin Q, Akers M, Zhang Y, Grant SL, Lambeth JD, and Griendling KK. Novel gp91phox homologues in vascular smooth muscle cells: nox1 mediates angiotensin II-induced superoxide formation and redox-sensitive signaling pathways. Circ Res 88: 888–894, 2001.[Abstract/Free Full Text]
  28. Lenkei Z, Palkovits M, Corvol P, and Llorens-Cortes C. Distribution of angiotensin type-1 receptor messenger RNA expression in the adult rat brain. Neuroscience 82: 827–841, 1998.[CrossRef][ISI][Medline]
  29. Li A, Segui J, Heinemann SH, and Hoshi T. Oxidation regulates cloned neuronal voltage-dependent Ca2+ channels expressed in Xenopus oocytes. J Neurosci 18: 6740–6747, 1998.[Abstract/Free Full Text]
  30. Li JM and Shah AM. Intracellular localization and preassembly of the NADPH oxidase complex in cultured endothelial cells. J Biol Chem 277: 19952–19960, 2002.[Abstract/Free Full Text]
  31. Liu JL, Pliquett RU, Brewer E, Cornish KG, Shen YT, and Zucker IH. Chronic endothelin-1 blockade reduces sympathetic nerve activity in rabbits with heart failure. Am J Physiol Regul Integr Comp Physiol 280: R1906–R1913, 2001.[Abstract/Free Full Text]
  32. Liu JL and Zucker IH. Regulation of sympathetic nerve activity in heart failure: a role for nitric oxide and angiotensin II. Circ Res 84: 417–423, 1999.[Abstract/Free Full Text]
  33. Lucini D, Bertocchi F, Malliani A, and Pagani M. A controlled study of the autonomic changes produced by habitual cigarette smoking in healthy subjects. Cardiovasc Res 31: 633–639, 1996.[CrossRef][ISI][Medline]
  34. Ma R, Zucker IH, and Wang W. Central gain of the cardiac sympathetic afferent reflex in dogs with heart failure. Am J Physiol Heart Circ Physiol 273: H2664–H2671, 1997.[Abstract/Free Full Text]
  35. Mayorov DN, Head GA, and De Matteo R. Tempol attenuates excitatory actions of angiotensin II in the rostral ventrolateral medulla during emotional stress. Hypertension 44: 101–106, 2004.[Abstract/Free Full Text]
  36. Moellenhoff E, Blume A, Culman J, Chatterjee B, Herdegen T, Lebrun CJ, and Unger T. Effect of repetitive icv injections of ANG II on c-Fos and AT1-receptor expression in the rat brain. Am J Physiol Regul Integr Comp Physiol 280: R1095–R1104, 2001.[Abstract/Free Full Text]
  37. Mohazzab KM, Kaminski PM, and Wolin MS. NADH oxidoreductase is a major source of superoxide anion in bovine coronary artery endothelium. Am J Physiol Heart Circ Physiol 266: H2568–H2572, 1994.[Abstract/Free Full Text]
  38. Mollnau H, Wendt M, Szocs K, Lassegue B, Schulz E, Oelze M, Li H, Bodenschatz M, August M, Kleschyov AL, Tsilimingas N, Walter U, Forstermann U, Meinertz T, Griendling K, and Munzel T. Effects of angiotensin II infusion on the expression and function of NAD(P)H oxidase and components of nitric oxide/cGMP signaling. Circ Res 90: E58–E65, 2002.[Abstract/Free Full Text]
  39. Murakami H, Liu JL, and Zucker IH. Angiotensin II blockade [corrected] enhances baroreflex control of sympathetic outflow in heart failure. Hypertension 29: 564–569, 1997.[Abstract/Free Full Text]
  40. Noshiro T, Way D, Miura Y, and McGrath BP. Enalaprilat restores sensitivity of baroreflex control of renal and total noradrenaline spillover in heart failure rabbit. Clin Exp Pharmacol Physiol 20: 373–376, 1993.[ISI][Medline]
  41. Packer M. Pathophysiology of chronic heart failure. Lancet 340: 88–92, 1992.[CrossRef][ISI][Medline]
  42. Palkovits M and Brownstein M. Brain Microdissection Techniques. West Sussex, UK: Wiley, 1983.
  43. Porter JP. Chronic intracerebroventricular infusion of angiotensin II increases brain AT1 receptor expression in young rats. Brain Res Dev Brain Res 112: 293–295, 1999.[Medline]
  44. Rueckschloss U, Quinn MT, Holtz J, and Morawietz H. Dose-dependent regulation of NAD(P)H oxidase expression by angiotensin II in human endothelial cells: protective effect of angiotensin II type 1 receptor blockade in patients with coronary artery disease. Arterioscler Thromb Vasc Biol 22: 1845–1851, 2002.[Abstract/Free Full Text]
  45. Ruiz-Ortega M, Lorenzo O, Ruperez M, Blanco J, and Egido J. Systemic infusion of angiotensin II into normal rats activates nuclear factor-kappaB and AP-1 in the kidney: role of AT1 and AT2 receptors. Am J Pathol 158: 1743–1756, 2001.[Abstract/Free Full Text]
  46. Schiffrin EL, Franks DJ, and Gutkowska J. Effect of aldosterone on vascular angiotensin II receptors in the rat. Can J Physiol Pharmacol 63: 1522–1527, 1985.[ISI][Medline]
  47. Shimohama S, Tanino H, Kawakami N, Okamura N, Kodama H, Yamaguchi T, Hayakawa T, Nunomura A, Chiba S, Perry G, Smith MA, and Fujimoto S. Activation of NADPH oxidase in Alzheimer's disease brains. Biochem Biophys Res Commun 273: 5–9, 2000.[CrossRef][ISI][Medline]
  48. Shokoji T, Nishiyama A, Fujisawa Y, Hitomi H, Kiyomoto H, Takahashi N, Kimura S, Kohno M, and Abe Y. Renal sympathetic nerve responses to tempol in spontaneously hypertensive rats. Hypertension 41: 266–273, 2003.[Abstract/Free Full Text]
  49. Taniyama Y and Griendling KK. Reactive oxygen species in the vasculature: molecular and cellular mechanisms. Hypertension 42: 1075–1081, 2003.[Abstract/Free Full Text]
  50. Thames MD, Kinugawa T, Smith ML, and Dibner-Dunlap ME. Abnormalities of baroreflex control in heart failure. J Am Coll Cardiol 22: 56A–60A, 1993.[Medline]
  51. Ullian ME, Schelling JR, and Linas SL. Aldosterone enhances angiotensin II receptor binding and inositol phosphate responses. Hypertension 20: 67–73, 1992.[Abstract/Free Full Text]
  52. Ulman LG, Potter EK, McCloskey DI, and Morris MJ. Post-exercise depression of baroreflex slowing of the heart in humans. Clin Physiol 17: 299–309, 1997.[ISI][Medline]
  53. Wang G, Anrather J, Huang J, Speth RC, Pickel VM, and Iadecola C. NADPH oxidase contributes to angiotensin II signaling in the nucleus tractus solitarius. J Neurosci 24: 5516–5524, 2004.[Abstract/Free Full Text]
  54. Wang W, Chen JS, and Zucker IH. Carotid sinus baroreceptor sensitivity in experimental heart failure. Circulation 81: 1959–1966, 1990.[Abstract/Free Full Text]
  55. Wang W, Chen JS, and Zucker IH. Carotid sinus baroreceptor reflex in dogs with experimental heart failure. Circ Res 68: 1294–1301, 1991.[Abstract/Free Full Text]
  56. Wang W and Ma R. Cardiac sympathetic afferent reflexes in heart failure. Heart Failure Rev 5: 57–71, 2002.
  57. Wong J, Chou L, and Reid IA. Role of AT1 receptors in the resetting of the baroreflex control of heart rate by angiotensin II in the rabbit. J Clin Invest 91: 1516–1520, 1993.[ISI][Medline]
  58. Xu H, Fink GD, Chen A, Watts S, and Galligan JJ. Nitric oxide-independent effects of tempol on sympathetic nerve activity and blood pressure in normotensive rats. Am J Physiol Heart Circ Physiol 281: H975–H980, 2001.[Abstract/Free Full Text]
  59. Xu H, Fink GD, and Galligan JJ. Nitric oxide-independent effects of tempol on sympathetic nerve activity and blood pressure in DOCA-salt rats. Am J Physiol Heart Circ Physiol 283: H885–H892, 2002.[Abstract/Free Full Text]
  60. Zanzinger J and Czachurski J. Chronic oxidative stress in the RVLM modulates sympathetic control of circulation in pigs. Pflügers Arch 439: 489–494, 2000.[CrossRef][ISI][Medline]
  61. Zimmerman MC, Lazartigues E, Lang JA, Sinnayah P, Ahmad IM, Spitz DR, and Davisson RL. Superoxide mediates the actions of angiotensin II in the central nervous system. Circ Res 91: 1038–1045, 2002.[Abstract/Free Full Text]
  62. Zimmerman MC, Lazartigues E, Sharma RV, and Davisson RL. Hypertension caused by angiotensin II infusion involves increased superoxide production in the central nervous system. Circ Res 95: 210–216, 2004.[Abstract/Free Full Text]
  63. Zucker IH and Wang W. Reflex control of renal sympathetic nervous activity in heart failure. Herz 16: 82–91, 1991.[ISI][Medline]



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