Oxidative Stress Mediates Angiotensin II–Dependent
Stimulation of Sympathetic Nerve Activity
Vito M. Campese, Ye Shaohua, Zhong Huiquin
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Abstract—Evidence indicates that angiotensin II (Ang II) enhances sympathetic nervous system (SNS) activity centrally
and peripherally, but the exact mechanisms of this activation are not well established. We have previously shown that
infusion of Ang II in the lateral cerebral ventricle raises blood pressure (BP), renal sympathetic nervous system activity
(RSNA), and norepinephrine (NE) secretion from the posterior hypothalamic nuclei (PH), and reduces the abundance
of interleukin-1␤ (IL-1␤) and neuronal NO synthase (nNOS) mRNA in the PH. Pretreatment with an Ang II type 1 (AT1)
receptor antagonist abolished these effects of Ang II. The data support the hypothesis that Ang II stimulates SNS through
activation of AT1 receptors and downregulation of nNOS. In the current studies, we tested the hypothesis that the effects
of Ang II on central SNS are mediated by reactive oxygen species. To this end, we evaluated the effects of Ang II alone
or in combination with 2 superoxide dismutase (SOD) mimetics, tempol (4-hydroxy-2,2,6,6-tetramethyl piperidinoxyl)
and polyethylene glycol–SOD (PEG-SOD) on BP, NE secretion from the PH, RSNA, and abundance of IL-1␤ and
nNOS mRNA in the PH Ang II raised BP, NE secretion from the PH, and RSNA and reduced the abundance of IL-1␤
and nNOS mRNA in the PH. Tempol and PEG-SOD completely abolished these actions of Ang II. In conclusion, these
studies support the hypothesis that the effects of centrally administered Ang II on the SNS are mediated by increased
oxidative stress in brain regions involved in the noradrenergic control of BP. (Hypertension. 2005;46:533-539.)
Key Words: angiotensin II 䡲 sympathetic nerve activity 䡲 nitric oxide 䡲 interleukins
S
species (ROS). Ang II activates NADH/NADPH-oxidase and
increases superoxide production in vascular tissue.10,11 Few
studies suggest that ROS could also mediate the effects of
Ang II on SNS activity. Pretreatment of mice with adenoviral
vector–mediated superoxide dismutase (AsSOD) abolished
the effects of Ang II on BP and heart rate.12 In addition, Ang
II increased superoxide generation in primary central nervous
system cell cultures, whereas losartan and AsSOD abolished
these effects. NO reacts with superoxide (O2⫺) and other ROS
to produce peroxynitrate, a highly cytotoxic reactive nitrogen
species, suggesting that increased production of ROS may
activate SNS through enhanced oxidation/inactivation of NO.
Peroxynitrate reacts with other proteins, such as tyrosine, to
produce nitrotyrosine, the footprint of the NO–ROS interaction.13 In the current studies, we tested the hypothesis that the
effects of Ang II on SNS activity are attributable to increased
ROS production. To this end, we evaluated the effects of 2
SOD mimetics. The first is tempol (4-hydroxy-2,2,6,6tetramethyl piperidinoxyl), a membrane-permeable and
metal-independent SOD mimetic; the second SOD mimetic
we used is polyethylene glycol–SOD (PEG-SOD). Both
compounds were tested on Ang II–induced effects on central
and peripheral SNS activation and on the abundance of IL-1␤
and nNOS in brain nuclei involved in the noradrenergic
control of BP, including the PH, PVN, and LC.
ubstantial evidence indicates that angiotensin II (Ang II)
enhances sympathetic nerve (SNS) activity centrally and
peripherally.1–3 Intracerebroventricular administration of Ang
II causes a dose-dependent increase in blood pressure (BP),4
probably through activation of Ang II type 1 (AT1) receptors
localized in the median preoptic nucleus, juxtaventricular
neurons of the subfornical organ, organum vasculosum laminae terminalis,5,6 brain stem, or in preganglionic neurons in
the rostral ventrolateral medulla and the intermediolateral
column.7
In a previous study, we showed that intracerebroventricular
infusion of Ang II raises BP, renal sympathetic nervous
system activity (RSNA), and norepinephrine (NE) secretion
from the posterior hypothalamic nuclei (PH).8 Ang II also
reduced the abundance of interleukin-1␤ (IL-1␤) and the
neuronal isoform of NO synthase (nNOS) mRNA in the PH,
paraventricular nuclei (PVN), and locus coeruleus (LC) and
the secretion of NO from the PH. Losartan, an AT1 receptor
blocker, abolished all these effects of Ang II. In all, these
studies suggest that Ang II binds to specific AT1 receptors in
the brain, resulting in inhibition of IL-1␤ and nNOS. Because
NO exerts a tonic inhibition of SNS activity,9 a decrease in
NO caused by Ang II could mediate the increase in SNS
activity.
A large body of evidence suggests that the hypertensive
action of Ang II is in part mediated by reactive oxygen
Received December 14, 2004; first decision January 6, 2005; revision accepted July 13, 2005.
From the Division of Nephrology, and the Hypertension Center, Keck School of Medicine, University of Southern California, Los Angeles.
Correspondence to Vito M. Campese, MD, Division of Nephrology and Hypertension Center, Keck School of Medicine, USC, 1200 N State St, Los
Angeles, CA 90033. E-mail [email protected]
© 2005 American Heart Association, Inc.
Hypertension is available at http://www.hypertensionaha.org
DOI: 10.1161/01.HYP.0000179088.57586.26
533
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Methods
Animal Preparation
For these studies, we used male Sprague-Dawley rats weighing 300
to 350 g fed with normal rat chow (ICN Nutritional Biochemical)
and tap water. For measurements of arterial pressure and administration of drugs, we anesthetized animals with sodium pentobarbital
(a loading dose of 35 mg/kg IP followed by an infusion of 5 mg/kg
per hour) and implanted catheters (PE-10) in a femoral artery and
vein.
Preparation for Intracerebroventricular Infusion
For intracerebroventricular infusion of Ang II, tempol, and PEGSOD, we placed a cannula (23 gauge) in the right lateral ventricle
(coordinates 1.4 mm lateral, 0.8 mm posterior, and 3.8 mm deep
from the bregma). When 2 of these drugs were administered at the
same time, we used a homemade 22-gauge Y-shaped cannula.
NE Secretion From the PH by the
Microdialysis Technique
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To measure NE secretion from the PH, we placed rats in a stereotaxic
apparatus, implanted a 2-mm-long Teflon 22-gauge guide cannula
(IV Catheter Placement Unit; Critikon, Inc.) using coordinates
anterior-posterior (A-P) ⫺4.0 mm and lateral ⫾0.4 mm; vertical
(V)⫽8 mm, and secured the guide in place with dental cement. A
28-gauge stainless steel stylus was lowered through the guide
cannula to a depth 1.5 mm dorsal to the Dorss ventral coordinate for
PH, namely ⫺8.5 mm from the skull surface. The stylus was
removed from the guide cannula and replaced with a microdialysis
probe (CMA Microdialysis AB), which was secured to the guide
with sticky wax. The inlet tubing of the dialysis probe was connected
by PE-20 tubing to a 1-mL disposable syringe driven by a microinfusion pump (model A-99; Razel Scientific Instruments, Inc.), and an
infusion of artificial cerebrospinal fluid (aCSF; in mmol/L: 150 Na⫹,
3.0 K⫹, 1.4 Ca2⫹, 0.8 Mg2⫹, 1.0 phosphorus, and 155 Cl-, pH 7.2) was
initiated at a rate of 1.7 ␮L per minute. PE-10 tubing was attached
to the outlet side of the probe, and the free end led to a 0.5-mL vial
set in a small box of ice. The vial contained 2 ␮L of 0.1 N HCl for
preservation of NE. All samples were immediately frozen and stored
at ⫺70°C until the time of assay. After 90 minutes of dialysis
equilibration, dialysate samples were collected every 5 minutes for
the entire duration of the experiments.
Renal SNS Activity
For renal nerve recording, we prepared rats according to the method
of Lundin and Thoren,14 as modified by DiBona et al.15 We exposed
retroperitoneally the left kidney, left renal artery, and abdominal
aorta via flank incision and dissected a renal nerve branch, usually
found in the angle between the aorta and the renal artery, free from
fat and connective tissue for the length of ⬇10 mm. We left the renal
branch intact and placed it on thin (0.005 inch) bipolar platinum
electrodes (Cooner Wire Company) and connected to a highimpedance probe Grass HIP 511 (Grass Instrument Co.). RSNA was
amplified (⫻10 000 to 50 000) and filtered (low 30; high 3000) with
a Grass 511 bandpass amplifier. The amplified and filtered signal
was channeled to a 5113 oscilloscope (Tektronix, Inc.) for visual
evaluation, to an audio amplifier/loud speaker (Grass model Am 8
audio monitor) for auditory evaluation, and to a rectifying voltage
integrator (Grass model 7P 10). The output signal of the Grass 7P 10
was then displayed on a Grass polygraph. The quality of the renal
nerve activity was assessed during operation by examining the
magnitude of changes in recorded RSNA during sinoaortic baroreceptor unloading with injection of acetylcholine (1 ␮g IV) and
during sinoaortic baroreceptor loading with the injection of NE (5 ␮g
IV). When an optimal recording was achieved, the nerve on the
electrode was isolated with silicone rubber (Wacker Sil-Gel 604;
Wacker Inc.). Throughout the experiments, animals were kept warm
under heated lamps and received an intravenous infusion of 30
␮L/min of 5% dextrose in normal saline. Arterial pressure, heart rate,
and RSNA were monitored continuously. A postmortem background
signal was determined and the experimental data corrected for this.
Effect of Intracerebroventricular Ang II on BP,
NE Secretion From the PH, and RSNA
We dissolved Ang II in aCSF (1.67 ng/␮L) and infused it intracerebroventricularly (a dose of 1.67 ng/kg body weight/min per 60
minutes) by an infusion pump (K.D. Scientific) and recorded the
effects on BP, NE secretion from the PH, and RSNA. At the end of
the experiment, rats were decapitated and the brains were immediately frozen and stored at ⫺70°C until assay. A group of rats
received only intracerebroventricular aCSF and served as control.
Effects of Tempol and PEG-SOD on BP, NE
Secretion From the PH, and RSNA
To determine whether tempol by itself lowers BP, we evaluated the
effects of increasing doses of tempol (10, 30, 50, and 100 ␮g/␮L per
kg/min infused ICV for 60 minutes) on BP. To test the hypothesis
that ROS mediate the effects of Ang II on SNS activity, we infused
tempol intracerebroventricularly (50 ␮g/␮L per kg body weight per
minute⫻60 minutes) along with Ang II through a homemade
Y-shaped cannula and measured BP, NE secretion from the PH,
RSNA, and the abundance of nNOS and IL-1␤ mRNA in the PH,
PVN, and LC. Control animals received the same volume of aCSF as
rats that received tempol and Ang II.
To test the specificity of tempol effects, in separate groups of rats,
we evaluated the effects of a different SOD mimetic, PEG-SOD (in
doses of 80 to 160 and 320 U/kg dissolved in 10 ␮L of aCSF, infused
over 10 minutes) on BP, NE secretion from the PH, and RSNA when
given alone or when given in combination with Ang II.
NE Microassay
We used a highly sensitive microradioenzymatic assay.16 A total of
10 ␮L of dialysate was added to 5 ␮L of reaction mixture containing
1 ␮L of 3.7 mol/L Tris base (with 0.37 mol/L EGTA and 1.8 mol/L
MgCl2, pH 8.2), 0.06 ␮L of 36 mmol/L benzoxylamine, 1.5 ␮L of
S-[methyl-3H]adenosyl-L-methionine, and 2.4 ␮L of partially purified catechol-O-methyltransferase and incubated for 60 minutes at
37°C. The sensitivity of this method is 0.5 pg.
Determination of nNOS and IL-1␤ mRNA
Abundance in the Brain
At the end of the experiments, rats were euthanized by decapitation
and brains immediately removed, frozen in dry ice, and stored at
⫺80°C until assay but for no longer than 3 weeks. Brains were cut
into consecutive 200-␮m sections in a cryostat at ⫺20°C and
bilateral micropunches 0.5 mm in diameter from several brain nuclei
obtained as described previously.12
The coordinates for the PH were A-P from ⫺3.5 to ⫺4.1 mm;
lateral ⫾0.4 mm; V⫽8 mm; coordinates for the PVN were A-P from
⫺1.4 to ⫺2.0 mm; lateral ⫾ 0.3 mm; V⫽7.9 mm; and for the LC
were A-P from ⫺9.8 mm to ⫺10.2 mm; lateral ⫾1.4 mm;
V⫽7.2 mm. The nuclei so isolated were used to measure IL-1␤ and
nNOS mRNA gene expression. We selected those 3 nuclei because
they are all involved in the noradrenergic control of BP.
Total RNA extraction and reverse transcription (RT) were performed by methods well established in our laboratory. Polymerase
chain reaction (PCR) was performed on the RT product using
specific oligonucleotide primers for either neural NOS or IL-1␤
derived from cDNAs cloned from rat brain17 or rat liver.18 A master
mix of PCR reagents was made for duplex reactions containing
primers for the “housekeeping” gene ␤-actin (GenBank accession
No. Joo691) and primers for either nNOS (GenBank accession No.
X59949) or IL-1␤ (GenBank accession No. M98820).
The RT-PCR products were quantified by the method of Higuchi
et al.19 Fluorescence was measured in a fluorescence spectrofluorometer (F-2000; Hitachi Ltd.). Excitation was at 280 nm and
emitted light was selected at 590 nm. Results were expressed as a
ratio of the resultant optical densities for the specific gene to ␤-actin.
Campese et al
Ang II, Oxidative Stress, and Sympathetic Activity
535
Random hexamers, dithiothretol (DTT), Super Scrip Super reverse
transcriptase with reaction buffer (5⫻; 20 mmol/L Tris-HCl,
10 mmol/L NaCl, 0.1 mmol/L EDTA, 1 mmol/L DTT, 0.01%
Nonidet P-40, and 50% glycerol), TaqDNA polymerase with reaction buffer (10⫻; 50 mmol/L Tris-HCl, 10 mmol/L NaCl,
0.1 mmol/L EDTA, 5 mmol/L DTT, 50% glycerol, and 1.0% Triton
X-100), deoxynucleotide mixture (2⬘-deoxynucleoside 5⬘triphosphate), and MgCl2 were purchased from GIBCO/BRL.
Location of Probes
At the end of the experiments, while rats were still anesthetized, the
dialysis probes were removed and rats were euthanized by decapitation. Brains were immediately removed, frozen in dry ice, and
stored at ⫺70°C. Later, brains were sliced in 200-␮m sections and
the proper location of the lesion in the PH identified. Only rats with
probes implanted properly in the PH were considered for further
analysis. Approximately 15% to 20% of animals were eliminated
because of improper position of the probes.
Statistical Analyses
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Data were analyzed by 1-way ANOVA and by the Fisher test when
indicated. A 2-way ANOVA was used to examine interactions
between the effects of Ang II, tempol, and PEG-SOD. The computer
program Prism (GraphPad Software) was used for the analyses.
Results are expressed as mean⫾SEM.
Results
Effects of Ang II on BP, NE Secretion From the
PH, and RSNA
Ang II infused intracerebroventricularly at a rate of 1.67
ng/␮L per kg/min⫻60 minutes raised mean BP from
100⫾1.6 to 123⫾1.2 mm Hg (P⬍0.01), NE secretion from
the PH from 158⫾2.9 to 209⫾1.6 pg/mL (P⬍0.01), or by
32% (Figure 1A and 1B), and RSNA by ⬇41% compared
with control rats (Figure 1C). In contrast, the intracerebroventricular administration of aCSF caused no change in BP,
NE secretion from the PH, and RSNA.
Effects of Tempol and Ang II on BP, NE secretion
from the PH, and RSNA
Figure 1. A, The line graphs with closed circles show the levels
of arterial BP in rats after the injection of Ang II (1.6 ng/␮L per
kg body weight/min⫻60 minutes), or vehicle (open circles) in the
lateral ventricle (intracerebroventricular). Closed squares indicate
BP levels in rats pretreated with tempol (50 ␮g/␮L per kg body
weight in the lateral ventricle) 15 minutes before the administration of Ang II. Closed triangles indicate the effects of tempol (50
␮g/␮L per kg body weight in the lateral ventricle) given without
Ang II. The number of rats was 5 in each group. B, The line
graphs show the concentration of NE in the dialysate collected
from the PH of rats after the injection of Ang II or vehicle in the
lateral ventricle. Closed squares indicate NE secretion from the
PH of rats pretreated with tempol 15 minutes before the administration of Ang II. Closed triangles indicate the effects of tempol
given without Ang II. C, The line graphs show the changes in
RSNA of rats after the injection of Ang II or vehicle in the lateral
ventricle. Closed squares indicate RSNA in rats that received
tempol along with Ang II 15 minutes before the administration of
Ang II. Closed triangles indicate the effects of tempol given
without Ang II. The number of rats was 5 in each group. Values
Tempol given in increasing doses of 10, 30, 50, and 100
␮g/mL per ␮L/kg per minute for 60 minutes caused a
dose-related decrease in mean arterial pressure. For the
purpose of these studies, we selected the dose of 50 tempol
␮g/mL per ␮L/kg per minute. This dose of tempol significantly reduced BP, NE secretion from the PH, and RSNA
(Figure 1B). When given in concomitance with Ang II,
tempol completely abolished the effects of Ang II on BP, NE
secretion from the PH, and RSNA (Figure 1A through 1C).
There was a highly statistically significant interaction
(P⬍0.0001⫻2-way ANOVA) between tempol, Ang II and
BP, NE secretion from the PH, and RSNA.
Effects of PEG-SOD and Ang II on BP, NE
Secretion From the PH, and RSNA
Because at the dose of 50 ␮g/mL per ␮L/kg per minute,
tempol reduced BP, NE secretion from the PH, and RSNA,
this might indicate that under the experimental conditions of
the study, ROS are modulating BP. Alternatively, this might
are expressed as mean⫾SEM. *Significant statistical difference
(P⬍0.001 by ANOVA) vs controls; #significant statistical difference (P⬍0.001 by ANOVA) vs tempol⫹Ang II.
536
Hypertension
September 2005
suggest that the ability of tempol to block the effects of Ang
II may not necessarily depend on interference with Ang
II–mediated activation of ROS. Instead, Ang II and tempol
could have independent and opposite effects on BP.
To partially deal with this possibility, we used a different
SOD agonist: PEG-SOD. PEG-SOD given alone without Ang
II in doses of 80 and 160 U/kg body weight had no direct
effects on BP, NE secretion from the PH, and RSNA but
significantly attenuated the effects of Ang II on BP, NE
secretion from the PH, and RSNA. A dose of PEG-SOD of
320 U/kg caused a further decrease in BP, NE secretion from
the PH, and RSNA, but this dose of PEG-SOD by itself
reduced BP (Figure 2A through 2C). There was a highly
statistically significant interaction (P⬍0.0001⫻2-way
ANOVA) between PEG-SOD, Ang II and BP, NE secretion
from the PH, and RSNA.
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Effects of Tempol, PEG-SOD, and Ang II on
nNOS and IL-1␤ Abundance in the PH, PVN,
and LC
Ang II significantly (P⬍0.01) reduced the abundance of
IL-1␤ in the PH (from 68.0⫾1.78 to 33.4⫾1.49), PVN (from
64.4⫾2.68 to 33.1⫾1.27), and LC (from 66.0⫾2.26 to
26.3⫾0.67) compared with control rats (Figure 3A). Ang II
also reduced (P⬍0.01) the abundance of nNOS mRNA in the
PH (from 64.8⫾2.52 to 43.7⫾1.32), PVN (from 64.0⫾2.88
to 42.4⫾1.44), and LC (from 62.8⫾1.48 to 50.4⫾2.28;
Figure 3B). Tempol abolished the effects of Ang II on IL-1␤
and nNOS mRNA. PEG-SOD (320 U/kg) increased the
abundance of nNOS and IL-1␤ and abolished the effects of
Ang II on nNOS and IL-1␤ in the PH, PVN, and LC (Figure
3C and 3D).
Discussion
These studies have shown that tempol and PEG-SOD, 2 SOD
mimetics, abolish the effects of Ang II on central and
peripheral SNS activity. The studies are consistent with ROS
mediating the effects of Ang II on central and peripheral SNS
activity.
It is well established that the effects of Ang II on BP are
mediated in part by ROS. Infusion of Ang II into rats is
associated with increased vascular superoxide production,
and this effect appears not to be BP mediated because doses
of catecholamines that raised BP did not affect ROS.20 Ang II
stimulates oxidative stress through NADH/NADPH-oxidase
activation, and chronic infusion of Ang II raises the concentration of oxidative markers such as prostaglandin.21 Antioxidants, such as tempol and vitamin E, prevent Ang II–induced
hypertension in rats.22 Limited data are available concerning
the effects of Ang II on oxidative stress in the brain and the
role this might play in SNS-mediated regulation of cardiovascular function.
Zimmerman et al12 observed that the effects of intracerebroventricular Ang II on BP and heart rate were abolished by
pretreatment with AsSOD in mice. Zanzinger et al23 showed
that removal of extracellular superoxide or reactive nitrogen
species within the rostral ventrolateral medulla by microinjection of SOD reduced SNS activity.
Figure 2. A, The line graphs with closed circles show the levels of
arterial BP in rats after the injection of Ang II (1.6 ng/␮L per kg body
weight/min⫻60 minutes) in the lateral ventricle (intracerebroventricular)
with or without PEG-SOD (80, 160, and 320 U/kg body weight in the
lateral ventricle). Open squares show the effects of PEG-SOD (320
U/kg body weight in the lateral ventricle) without Ang II, and open circles show the effects of vehicle. B, The line graphs show the concentration of NE in the dialysate collected from the PH of rats after the
injection in the lateral ventricle of Ang II with or without PEG-SOD.
Open squares show the effects of PEG-SOD (320 U/kg body weight
ICV) without Ang II, and open circles show the effects of vehicle. C,
The line graphs show the changes in RSNA of rats after the injection
in the lateral ventricle (intracerebroventricular) of Ang II with or without
PEG-SOD. Open squares show the effects of PEG-SOD (320 U/kg
body weight ICV) without Ang II, and open circles show the effects of
vehicle. The number of rats was 5 for controls and for rats treated
with Ang II alone, and 4 each in groups treated with PEG-SOD. Values are expressed as mean⫾SEM. *Significant statistical difference
(P⬍0.001 by ANOVA) vs controls.
Large doses of tempol given intravenously have been
shown to acutely lower BP in normotensive24 and hypertensive.25,26 Xu et al observed that intravenous administration of
tempol (300 ␮mol/kg IV) lowered mean BP and renal SNS
activity in urethane-anesthetized deoxycorticosterone acetate
Campese et al
Ang II, Oxidative Stress, and Sympathetic Activity
537
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Figure 3. A, The bar graphs show levels of IL-1␤ mRNA measured in the PH, PVN, and LC of rats that received intracerebroventricularly
vehicle alone, Ang II alone (1.6 ng/␮L per kg body weight/min⫻60 minutes), pretreatment with tempol (50 ␮g/kg body weight in the lateral
ventricle) 15 minutes before the administration of Ang II, or tempol without Ang II. B, The bar graphs show levels of nNOS mRNA measured
in the PH, PVN, and LC of rats that received in the lateral ventricle (intracerebroventricular) vehicle alone, Ang II alone (1.6 ng/␮L per kg body
weight/min⫻60 minutes), pretreatment with tempol (50 ␮g/kg body weight in the lateral ventricle) 15 minutes before the administration of Ang
II, or tempol without Ang II. The number of rats was 5 in each group. C, The bar graphs show levels of IL-1␤ measured in the PH, PVN, and
LC of rats that received intracerebroventricularly vehicle alone, Ang II (1.6 ng/␮L per kg body weight/min⫻60 minutes) alone, or in combination with PEG-SOD (80, 160, and 320 U/kg body weight) or PEG-SOD (320 U/kg body weight in the lateral ventricle) without Ang II. D, The
bar graphs show levels of nNOS mRNA measured in the PH, PVN, and LC of rats that received intracerebroventricularly vehicle alone, Ang II
(1.6 ng/␮L per kg body weight/min⫻60 minutes) alone or in combination with PEG-SOD (80, 160, and 320 U/kg body weight), or PEG-SOD
(320 U/kg body weight in the lateral ventricle) without Ang II. The number of rats was 5 in each group. Values are expressed as mean⫾SEM.
*Significant statistical difference (P⬍0.001 by ANOVA) vs controls.
(DOCA)–salt hypertensive rats,27 but it did not reduce O2⫺ in
the aorta and vena cava. Moreover, intravenous administration of PEG-SOD and apocynin, an NAD(P)H oxidase
inhibitor, did not alter BP, suggesting that acute treatment
with antioxidants does not lower BP via a reduction in O2⫺ in
DOCA-salt hypertensive rats.
More controversial are the effects of tempol on BP and
SNS activity when given intracerebroventricularly. We observed a reduction in BP, NE secretion from the PH, and
RSNA when tempol was continuously infused intracerebroventricularly in a large dose (50 ␮g/␮L per kg body weight/
min⫻60 minutes). In contrast, Shokoji et al28 observed no
effects of intracerebroventricular tempol on BP and RSNA
when infused in bolus dose of 300 ␮g/1 ␮L in spontaneously
hypertensive rats (SHR) and Wistar-Kyoto rats. Differences
in dosing, type of administration, and animal model could
explain the difference in results among these studies.
Tempol is a membrane-permeable and metal-independent
SOD mimetic that has been widely used for the removal of
intracellular and extracellular O2⫺29 and has proven antioxidative activity in various tissues.30 In coronary arteries, O2⫺
has been shown to inactivate NO,31 and O2⫺ is important in
the decomposition of NO to peroxynitrite.32 Tempol increases
the half-life of NO and results in vasodilation, hypotension,
and reflex activation of the SNS. Consistent with the NO
hypothesis is the observation that tempol reduces BP and
renal vascular resistance in SHR but not in the Wistar-Kyoto
rats, and this response is blocked by nitro-L-arginine methyl
ester but not by NE.33
Not all evidence supports the notion that tempol causes
vasodilation through NO-mediated mechanisms. NG-nitro-Larginine methyl ester, an inhibitor of NOS and the enzyme
involved in the production of NO from arginine as substrate,34,35 increased BP but failed to prevent the hypotensive
action of tempol. Xu et al 27 observed that tempol
(300 ␮mol/kg IV bolus) decreases BP and RSNA in anesthetized DOCA-salt and sham rats, and these effects were
blocked by ganglionic blockade with hesamethonium but not
by NG-nitro-L-arginine, an NOS inhibitor. The authors suggested that tempol-induced depressor responses might be
largely mediated by NO-independent sympathoinhibition. We
have shown previously that tempol injected intracerebroventricularly reduces NE secretion from the PH and BP and
increases the abundance of IL-1␤ and nNOS in the PH, PVN,
and LC in rats.36 These data support the hypothesis that
oxygen radicals may modulate central SNS activation
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September 2005
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through local NO production. This hypothesis is further
supported by studies showing that Ang II reduced nNOS
abundance in the PH and NOx concentration in the dialysate
collected from the PH of normal rats,37 and tempol abolished
these effects of Ang II.
The fact that tempol reduced BP, NE secretion from the
PH, and RSNA suggests that under the experimental conditions of the study, ROS might be modulating BP. Alternatively, this might suggest that the ability of tempol to block
the effects of Ang II may not necessarily depend on interference
with Ang II–mediated activation of ROS. Instead, Ang II and
tempol could have independent and opposite effects on BP.
To further substantiate a role of O2⫺ in Ang II–mediated
effects on central SNS activity and BP, we evaluated the
effects of a different SOD agonist: PEG-SOD. In doses of 80
and 160 U/kg body weight, PEG-SOD had no effect on BP
but significantly reduced or abolished the effects of Ang II on
BP, NE secretion, and RSNA. In doses of 320 U/kg, PEGSOD reduced BP, but this effect occurred slowly over time,
whereas the effects of Ang II on BP occurred immediately
after initiation of the infusion. This suggests that direct effects
of PEG-SOD on BP cannot fully explain the inhibitory action
on Ang II–induced effects on BP and SNS activity and is
against the hypothesis that ROS modulates BP under baseline
conditions. Moreover, the data suggest that the hypotensive
action of tempol may not necessarily be linked to ROS
inhibition.
NOS is present in specific areas of the brain involved in
modulation of the neurogenic control of BP and the cardiovascular system, and it is an important component of transduction pathways that tonically inhibit SNS activity.38 NO
actively reacts with superoxide (O2⫺) and other ROS to
produce peroxynitrite, a highly cytotoxic reactive nitrogen
species. Increased production of ROS could enhance oxidation/inactivation of NO and result in activation of the SNS.
The current studies support this possibility.
We have shown that IL-1␤ may play a role in the
physiological role of the SNS in the regulation of BP.
Administration of IL-1␤ in the lateral ventricle of 5/6
nephrectomized and control rats, respectively, caused a dosedependent decrease in BP and NE secretion from the PH and
an increase in nNOS mRNA abundance in several brain
nuclei.39 In contrast, intracerebroventricular infusion of a
specific anti-rat IL-1␤ antibody decreased NOS mRNA
expression in the PH, PVN, and LC and raised BP and NE
secretion from the PH in these rats.
In the present studies, Ang II reduced the abundance of
IL-1␤ and nNOS mRNA in the PH, PVN, and LC. Tempol
and PEG-SOD raised the abundance of IL-1␤ and nNOS in
the PH, PVN, and LC, and pretreatment with tempol and
PEG-SOD abolished the effects of Ang II on nNOS and
IL-1␤. This suggests that ROS may inhibit IL-1␤ and nNOS
production at a transcriptional level.
In conclusion, these studies have shown that SOD mimetics administered intracerebroventricularly abrogate the effects of Ang II on BP and SNS activity, supporting the
hypothesis that the effects of Ang II on central SNS activation
are mediated by increased oxidative stress in brain regions
involved in the noradrenergic control of BP. This hypothesis
refers only to the effects of centrally administered Ang II and
cannot be generalized to peripheral effects of Ang II.
Perspectives
It is well recognized that Ang II enhances SNS activity
centrally and peripherally, but the exact mechanisms of this
activation are not well established. These studies have shown
that Ang II may activate central noradrenergic pathways
involved in the control of BP via increased oxidative stress
and downregulation of NO. Future studies are needed to
ascertain whether specific Ang II receptor antagonists or
agents that inhibit oxidative stress block the effects of Ang II
on central noradrenergic activation.
Acknowledgments
Support for these studies was provided by National Institutes of
Health grant R01 HL070027, and the National Kidney Foundation of
Southern California.
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Vito M. Campese, Ye Shaohua and Zhong Huiquin
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Hypertension. 2005;46:533-539; originally published online August 22, 2005;
doi: 10.1161/01.HYP.0000179088.57586.26
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