Oxidative Stress Mediates Angiotensin II–Dependent Stimulation of Sympathetic Nerve Activity
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-1β (IL-1β) 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-1β and nNOS mRNA in the PH Ang II raised BP, NE secretion from the PH, and RSNA and reduced the abundance of IL-1β 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.
Substantial 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-1β (IL-1β) 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-1β 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 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-2,2,6,6-tetramethyl 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-1β and nNOS in brain nuclei involved in the noradrenergic control of BP, including the PH, PVN, and LC.
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 PEG-SOD, 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
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 μL 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 μL 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 high-impedance 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 μg IV) and during sinoaortic baroreceptor loading with the injection of NE (5 μg 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 μL/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/μL) 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 μg/μL 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 μg/μL 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-1β 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 μL 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.
We used a highly sensitive microradioenzymatic assay.16 A total of 10 μL of dialysate was added to 5 μL of reaction mixture containing 1 μL of 3.7 mol/L Tris base (with 0.37 mol/L EGTA and 1.8 mol/L MgCl2, pH 8.2), 0.06 μL of 36 mmol/L benzoxylamine, 1.5 μL of S-[methyl-3H]adenosyl-l-methionine, and 2.4 μL 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-1β 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-μm 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-1β 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-1β 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 β-actin (GenBank accession No. Joo691) and primers for either nNOS ( GenBank accession No. X59949) or IL-1β (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 β-actin.
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-μm 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.
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.
Effects of Ang II on BP, NE Secretion From the PH, and RSNA
Ang II infused intracerebroventricularly at a rate of 1.67 ng/μL 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 intracerebro ventricular 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
Tempol given in increasing doses of 10, 30, 50, and 100 μg/mL per μL/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 μg/mL per μL/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 μg/mL per μL/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 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.
Effects of Tempol, PEG-SOD, and Ang II on nNOS and IL-1β Abundance in the PH, PVN, and LC
Ang II significantly (P<0.01) reduced the abundance of IL-1β 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-1β and nNOS mRNA. PEG-SOD (320 U/kg) increased the abundance of nNOS and IL-1β and abolished the effects of Ang II on nNOS and IL-1β in the PH, PVN, and LC (Figure 3C and 3D).
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.
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 μmol/kg IV) lowered mean BP and renal SNS activity in urethane-anesthetized deoxycorticosterone acetate (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 μg/μL 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 μg/1 μL 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-l-arginine 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 al27 observed that tempol (300 μmol/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-1β and nNOS in the PH, PVN, and LC in rats.36 These data support the hypothesis that oxygen radicals may modulate central SNS activation 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, PEG-SOD 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-1β may play a role in the physiological role of the SNS in the regulation of BP. Administration of IL-1β in the lateral ventricle of 5/6 nephrectomized and control rats, respectively, caused a dose-dependent 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-1β 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-1β and nNOS mRNA in the PH, PVN, and LC. Tempol and PEG-SOD raised the abundance of IL-1β 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-1β. This suggests that ROS may inhibit IL-1β 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.
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.
Support for these studies was provided by National Institutes of Health grant R01 HL070027, and the National Kidney Foundation of Southern California.
- Received December 14, 2004.
- Revision received January 6, 2005.
- Accepted July 13, 2005.
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