| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Department of Anatomy and Physiology, Kansas State University, Manhattan, Kansas 66506
Submitted 13 January 2004 ; accepted in final form 16 July 2004
 |
ABSTRACT |
|---|
 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
superoxide dismutase; angiotensin II; 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl; intracerebroventricular
Although these data support a role for ANG II in central SND regulation, brain signaling mechanisms that mediate SND responses to central ANG II administration are not well established. A recent study by Zimmerman et al. (54) demonstrated that overexpression of superoxide dismutase (SOD) in brain tissue of adult mice eliminates the arterial blood pressure, heart rate (HR), and dipsogenic responses to icv administration of ANG II, which thereby established a role for superoxide in mediating the central effects of ANG II. This finding is consistent with the results of previous studies, which demonstrated that the signaling mechanism used by ANG II in peripheral tissues includes superoxide and other reactive oxygen species (10, 24, 31, 37, 38). Zimmerman et al. (54) hypothesized that central neural reactive oxygen species play an important role in cardiovascular function and that altered regulation of central redox mechanisms may be involved in heart failure and hypertension, disease states that are characterized by activation of the sympathetic nervous system (5, 7, 8, 25). However, the role of superoxide in mediating the effects of brain ANG II on efferent SND remains poorly defined.
The membrane-permeable 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (Tempol) is a SOD mimetic; its peripheral administration affects cardiovascular and sympathetic nerve regulation (41, 45-47). However, little is known concerning the effect of central Tempol administration on SND regulation (41, 47). Therefore, the first goal of the present study was to establish dose-response relationships between icv Tempol administration and the level of renal sympathetic nerve activity in anesthetized, baroreceptor-denervated, normotensive rats. The second goal was to test the hypotheses that icv Tempol pretreatment would attenuate centrally mediated changes in SND produced by icv ANG II administration and that brain superoxide levels would be higher in rats that received icv ANG II administration alone compared with rats pretreated with icv Tempol before central ANG II administration.
|
METHODS |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
The Institutional Animal Care and Use Committee approved the experimental procedures and protocols used in the present study, and all procedures were performed in accordance with the American Physiological Society's "Guiding Principles for Research Involving Animals" (1). Experiments were performed on Sprague-Dawley rats (348 ± 5 g body wt). Anesthesia was initiated with the administration of 
-chloralose (80 mg/kg ip) and urethane (800 mg/kg ip). Rats received isoflurane (3% induction with subsequent 1.5–2.5%) during surgical procedures. After the surgical procedures were complete, maintenance doses of -chloralose were infused (femoral vein, 35–45 mg·kg–1 · h–1 iv). The trachea was cannulated with a polyethylene-240 catheter. Femoral arterial pressure was monitored using a pressure transducer connected to a blood pressure analyzer. HR was derived from the pulsatile arterial pressure output of the blood pressure analyzer. Colonic temperature (Tc) was measured with a thermistor probe inserted 
5 cm into the colon. Tc was maintained between 37.8 and 38.0°C by a homeothermic blanket during surgical and experimental procedures (except during the heating protocol).
Sinoaortic Denervation
Bilateral denervation of the aortic arch was completed in anesthetized rats by cutting the superior laryngeal nerve near its junction with the vagus nerve and removing the superior cervical ganglion (23). Bilateral carotid sinus denervation was completed by removing the adventitia from the carotid sinus bifurcation (23). Sinoaortic denervation was considered complete by the loss of coherence between the arterial pulse and SND (14, 21). Experiments were completed in sinoaortic-denervated rats to eliminate the influence of baroreceptor afferent feedback mechanisms that may influence SND responses of central origin.
Lateral Ventricular Cannulation
A lateral ventricular cannula was surgically implanted after the general surgical procedures were completed. Anesthetized rats were placed in a stereotaxic frame with the head level between lambda and bregma, and a small hole was made in the skull (1.2–1.4 mm lateral to midline and 0.8–1.0 mm posterior to bregma). A 10-mm stainless steel guide cannula (22 gauge) was lowered 4 mm below the surface of the skull and fixed in place using cranioplastic cement. A stainless steel injector was introduced through the guide cannula to protrude 0.5 mm beyond the tip of the guide cannula.
Sympathetic Nerve Recordings
After the sinoaortic denervation and lateral ventricular cannulation procedures were complete, the anesthetized rats were prepared for renal SND recordings. Renal sympathetic nerve activity was recorded biphasically with a platinum bipolar electrode after preamplification of the signal (band pass, 30–3,000 Hz). Renal nerves were isolated using a lateral approach, and nerve-electrode preparations were covered with silicone gel. Filtered neurograms were routed to an oscilloscope and a nerve traffic analyzer to allow monitoring during the experiment and for subsequent data analysis. Sympathetic nerve potentials were full-wave rectified, integrated (time constant, 10 ms), and quantified as volts x seconds (17–21). The level of renal sympathetic nerve activity was corrected for background noise after ganglionic blockade with 10–15 mg/kg iv trimethaphan camsylate (17–21).
Experimental Protocols
Nine experimental groups [icv artificial cerebrospinal fluid (aCSF) + icv ANG II; icv losartan + icv ANG II; icv Tempol + icv ANG II; icv aCSF alone; icv Tempol alone; icv aCSF + heating; icv Tempol + heating; iv ANG II alone; and iv Tempol + icv ANG II] were used to complete four protocols. The icv infusions were completed using an injector that was connected via polyethylene tubing to a 100-µl microsyringe driven by a micropump (1 µl/min for 10 min). MAP, SND, and HR values were recorded continuously during a 60-min stabilization period before initiation of each experimental protocol.
Protocol I. After control (basal) data were collected, rats were treated with 10-min icv Tempol infusions (5, 50, 75, or 100 µmol/kg; 10 µl). SND, MAP, and HR values were recorded during and 10 min after Tempol infusions.
Protocol II. After control (basal) data were collected, rats were pretreated with 10-min icv infusions of aCSF (10 µl), losartan (15 µg/kg; 10 µl), or Tempol (5, 50, or 75 µmol/kg; 10 µl). Ten minutes after cessation of pretreatment infusions, icv infusions of ANG II (150 ng/kg; 10 µl) or aCSF (10 µl) were initiated and maintained for 10 min. MAP, SND, and HR values were recorded for 25 min after icv infusions of ANG II and aCSF. In a separate set of experiments, SND, MAP, and HR values were recorded before, during, and 45 min after the icv administration of Tempol only (5, 50, or 75 µmol/kg; 10 µl infused over a 10-min period). Control experiments for central ANG II administration were completed by recording SND and MAP values before and 25 min after iv (femoral vein) ANG II administration (150 ng/kg; 10 µl infused over a 10-min period). Control experiments for central Tempol administration were completed by recording SND, MAP, and HR values before and 10 min after rats were pretreated with Tempol (75 µmol/kg iv infused over 10 min), which was followed 10 min later by ANG II infusion (150 ng/kg icv infused over 10 min).
Protocol III. Brain superoxide anion levels were estimated in three separate groups of rats (n = 3 rats/group: icv aCSF alone; icv aCSF + icv ANG II; 100 µmol/kg icv Tempol + icv ANG II) using dihydroethidium (DHE) staining in accordance with previously established protocols (11, 46, 52). Brains were removed and flash frozen in liquid nitrogen-cooled 2-methylbutane. Unfixed frozen regions of the PVN and RVLM were cut into 30-µm sections using a cryostat and transferred to glass slides. Brain slices were topically treated in one of three ways: 0.3 mol/l Tempol + 2 µmol/l DHE; 2 µmol/l DHE only; or no treatment (background control). Each treatment was completed twice per brain region for a total of six slides per region. After treatment, slides were covered, sealed, and incubated at 37°C for 30 min in a light-protected, humidified chamber. The presence of superoxide anions was observed using a Leica DMR microscope (Heerbrugg) and a Leica N2.1 filter set (excitation wavelengths, 515–560 nm; emission long pass 590 nm). A series of exposure times (1–25 s) were tested to determine the working range before saturation of the fluorescent signal occurred. Images of DHE fluorescence were captured after 15 s of exposure using QCapture software 2.60 (Quantitative Imaging). Gain and offset values were identical for all images and arbitrary units of fluorescence intensity were assigned using Adobe Photoshop software after correction for background fluorescence. Fluorescence intensity of topical DHE only and topical Tempol + DHE treatments were pooled after statistical analysis revealed no fluorescence intensity differences between groups.
Protocol IV. After control data were collected, rats were pretreated with 10-min icv infusions of aCSF (10 µl) or Tempol (75 µmol/kg; 10 µl). Ten minutes after cessation of pretreatment infusions, Tc was increased at a rate of 0.1°C/min from 38 to 40.5°C (25-min heating period) using a heat lamp (17–20). Previous studies have established that heating provides a potent stimulus to the sympathetic nervous system (17–20).
Brain Histology
Fluorescent latex microspheres (50 nm in diameter) were injected into the lateral ventricle. Rats received an overdose of methohexital sodium (150 mg/kg iv) and were transcardially perfused first with 0.15 M NaCl (that contained 3 IU/ml heparin) and then by a fixative solution that consisted of 10% buffered neutral formalin (pH 7.4). Brains were removed, blocked, postfixed in buffered neutral formalin for at least 2 h, and placed in 20% sucrose for cryoprotection. Brains were frozen sectioned at 30 µm in the coronal plane, collected into phosphate-buffered saline, and mounted on slides in serial sequence. The sections were rinsed in distilled water and air dried. Lateral ventricular injection sites were confirmed by observing fluorescent microspheres in the ventricular system using bright-field or epifluorescence microscopy.
Data and Statistical Analysis
Values are means ± SE. Control values of SND were considered as 0%. Statistical analysis of SND, MAP, and HR data was completed using repeated-measures ANOVA techniques with Bonferroni post hoc tests. Population means of fluorescence intensity between groups were compared using Student's t-test. The overall level of statistical significance was P < 0.05.
|
RESULTS |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
Figure 1 summarizes the renal SND, MAP, and HR responses during (0–10 min) and 10 min after icv administration of four doses of Tempol (5, 50, 75, and 100 µmol/kg). There were dose-dependent decreases in renal SND, MAP, and HR values in response to icv administration of Tempol.
|
Figure 2 summarizes the renal SND, MAP, and HR values before (control, –20 min), during (–20 to –10 min), and after (–10 to 0 min) icv administration of Tempol (3 doses) or aCSF and during (0–10 min) and after (10–35 min) icv administration of ANG II. Renal SND values were significantly reduced from control values during icv pretreatment with Tempol (5, 50, and 75 µmol/kg) and were unchanged from control values during icv pretreatment with aCSF. Renal SND values were significantly increased (compared with time 0) after icv ANG II infusion in rats pretreated with aCSF or Tempol at doses of 5 and 50 µmol/kg but not in rats pretreated with Tempol at a dose of 75 µmol/kg. Renal SND values were significantly higher after ANG II infusion in rats pretreated with aCSF or Tempol at 5 and 50 µmol/kg compared with rats pretreated with Tempol at 75 µmol/kg. MAP and HR values were reduced during pretreatment with 50 and 75 µmol/kg Tempol doses and remained unchanged during pretreatment with aCSF or 5 µmol/kg Tempol. During and after ANG II infusion, MAP values remained unchanged (compared with time 0) in rats pretreated with 50 and 75 µmol/kg Tempol doses but were increased in rats pretreated with aCSF or 5 µmol/kg Tempol. HR values remained unchanged (compared with time 0) during and after ANG II infusion in rats pretreated with aCSF or 50 and 75 µmol/kg Tempol and were increased after ANG II infusion in rats pretreated with 5 µmol/kg Tempol.
|
|
Rats were treated with iv ANG II (150 ng/kg infused over 10 min) in three experiments. MAP (preinfusion, 105 ± 3 mmHg; immediately after ANG II infusion, 102 ± 4 mmHg; 25 min after cessation of ANG II infusion, 101 ± 6 mmHg) and SND (immediately after ANG II infusion, –3 ± 4%; 25 min after cessation of ANG II infusion, 7 ± 5%) remained unchanged from preinfusion levels for 25 min after iv ANG II administration.
Protocol III: Effects of Tempol on Brain Superoxide Anion Levels
DHE staining of brain slices from the RVLM and PVN of the hypothalamus was completed to determine the effects of Tempol on brain superoxide anion levels. Figure 4A shows representative images of DHE-treated brain slices from the RVLM. Increased fluorescence levels, which represent higher superoxide anion levels, were present in the brain slices from rats treated with aCSF + ANG II and Tempol + ANG II compared with rats treated with aCSF only. Figure 4B summarizes fluorescence imaging data from the RVLM. The level of DHE fluorescence in the RVLM was significantly higher in aCSF + ANG II-treated and Tempol + ANG II-treated rats compared with aCSF-treated rats (P < 0.05). Fluorescence in the RVLM did not differ in aCSF + ANG II-treated compared with Tempol + ANG II-treated rats. Fluorescence in the PVN did not differ between aCSF + ANG II-treated (85 ± 17 units), Tempol + ANG II-treated (80 ± 11 units), and aCSF-treated (61 ± 9 units) rats.
|
Figure 5 summarizes renal SND, MAP, and HR values at control, during 10-min infusion, and for 10 min after icv administration of Tempol (75 µmol/kg) or aCSF and during heating (Tc increased from 38 to 40.5°C in 25 min). Tc was maintained at 38°C during the control, infusion, and postinfusion periods. Renal SND was significantly reduced from control values during icv infusion of Tempol but remained unchanged during aCSF infusion. Renal SND was progressively and significantly increased (compared with the postinfusion period) during acute heating in rats pretreated with aCSF or Tempol and did not differ between groups. MAP and HR values were significantly reduced after treatment with Tempol but not aCSF. MAP measurements were significantly increased during heating (compared with the postinfusion period) in rats treated with Tempol but not aCSF, whereas HR values were significantly increased during heating in rats treated with Tempol or aCSF.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
It is well established that ANG II plays a role in numerous physiological responses including arterial blood pressure regulation, fluid balance and thirst regulation, and vasopressin release (16, 32, 36). The cellular effects of ANG II are initiated after interaction of the peptide with specific membrane receptors including AT1 and AT2 receptors (30, 44). Recent studies have established that signal transduction steps that mediate cellular responses to binding of ANG II to AT1 receptors in peripheral vascular tissue involve superoxide and other reactive oxygen species (24, 37). Zimmerman et al. (54) reported that overexpression of central nervous system SOD in mice virtually eliminated the arterial blood pressure and HR responses to icv ANG II, which implicated superoxide in the signaling pathway mediating cardiovascular responses to central ANG II administration. The present study was designed to extend this finding by testing the hypothesis that central administration of a SOD mimetic (Tempol) would attenuate sympathetic nerve responses to central ANG II administration and thereby implicate superoxide in the signaling pathway mediating sympathetic nerve responses to central ANG II administration.
The present results demonstrate that in a dose-dependent manner, icv Tempol administration reduced central sympathetic nerve outflow and attenuated sympathoexcitatory responses to central ANG II administration. Moreover, icv ANG II increased brain superoxide levels, and this effect was not significantly reduced by icv pretreatment with Tempol. Taken together, the results of the present study demonstrate that icv Tempol administration exerts prominent sympathetic and cardiovascular effects; however, a role for the antioxidant properties of this drug in mediating these changes remains uncertain. Previous studies aimed at determining the effects of peripheral Tempol administration on arterial blood pressure provide additional support for this conclusion. Xu et al. (45–47) reported that iv administration of Tempol reduced MAP values in normotensive rats and in sham and deoxycorticosterone acetate (DOCA)-salt-treated rats, although the effect of Tempol in reducing vascular DHE-induced fluorescence measurements in DOCA-salt-treated rats was inconsistent (46, 47). Tempol administration (via drinking water) prevented ANG II-induced hypertension and prevented or attenuated increases in renal and systemic isoprostanes (33), whereas it reversed ACTH-induced hypertension but had no effect on F2-isoprostane concentrations (51).
The results of recent studies suggest that ANG II, nitric oxide (NO), reactive oxygen species, and IL-1
interact to influence central sympathetic neural networks (4, 26). The icv infusion of ANG II increases arterial blood pressure and renal SND and reduces the abundance of IL-1 and NO synthase mRNA in the posterior hypothalamus, PVN, and the locus coeruleus (4). Because NO can tonically inhibit SND (12, 40), and because the central administration of IL-1 antibody increases renal and splenic SND (28), it is reasonable to speculate that sympathoexcitatory responses to icv ANG II administration may be mediated by decreased central expression of IL-1 and NO. In addition, it is known that ANG II is capable of generating oxidative stress (35) and that superoxide can interact rapidly with NO to reduce its bioavailability (9). Consistent with these findings, administration of SOD into the RVLM has been shown to decrease renal SND and arterial blood pressure (49). Taken together, these findings suggest complex interactions between various neurotransmitters and/or neuromodulators in mediating brain ANG II-sympathetic neural interactions.
Why study brain ANG II sympathetic neural interactions? Heart failure and hypertension, two prominent cardiovascular pathologies, are each characterized by significant alterations in the renin-angiotensin and sympathetic nervous systems (13, 27, 34, 43). Regarding heart failure, muscle SND is increased in human patients (8, 25), renal SND has been reported to be higher in rats with heart failure compared with sham controls (7), acute lateral ventricle injection of losartan decreases levels of resting renal SND in rats with chronic heart failure (6), and chronic central AT1 receptor blockade normalizes the enhanced sympathoexcitation, reduced sympathoinhibition, and desensitized baroreflex responses observed in congestive heart failure in rats after myocardial infarction (50). Regarding hypertension, Huang and Leenen (15) reported that brain ANG II contributes to the renal sympathoexcitation and hypertension observed in Dahl salt-sensitive rats fed a high-salt diet, whereas Ye et al. (48) reported that central administration of losartan reduced the excitatory effects of intrarenal phenol injection (a model of neurogenic hypertension) on renal SND and norepinephrine release from the posterior hypothalamus. These findings support a role for central ANG II sympathetic neural interactions in the pathophysiology of heart failure and hypertension and thereby provide rationale for studying signaling mechanisms that mediate ANG II effects on central sympathetic neural circuits.
In contrast with the effects of Tempol administration on ANG II-induced central sympathoexcitatory responses, SND was increased in response to acute heating in rats pretreated with icv Tempol, which demonstrates specificity in the effect of Tempol to attenuate sympathoexcitation. This is consistent with the findings of Zimmerman et al. (54), who reported that cardiovascular responses to central carbachol administration were not altered in mice that overexpressed SOD.
There are at least three limitations to the present study. First, anesthesia may influence SND responses to central administration of ANG II. Although this cannot be entirely discounted, in the present study, rats were anesthetized with a combination of -chloralose and urethane, which is an anesthetic regimen used widely in experimental protocols concerned with studying autonomic and cardiovascular regulation. Second, icv infusions provide limited information concerning specific central sites that mediate SND responses to ANG II. Because CSF provides a diffusion medium whereby substances can act as volume-transmission signals to the brain (22), we chose to use icv infusions as an initial experimental strategy to determine interactions between ANG II and superoxide in SND regulation. Based on the present findings, additional studies can be completed to determine specific central sites that mediate the observed responses. Finally, the purpose of the present study was to determine the effects of icv Tempol administration on central sympathoexcitatory actions of ANG II; these effects were determined using a relatively short experimental protocol. Consequently, the present findings provide no information concerning the duration of ANG II-induced sympathoexcitatory responses.
|
GRANTS |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
|
ACKNOWLEDGMENTS |
|---|
Present address of N. Lu: Department of Physiology, Fudan University, Shanghai Medical College, Shanghai, China.
|
FOOTNOTES |
|---|
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 |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
mediates angiotensin II-dependent stimulation of sympathetic nerve activity. Hypertension 39: 519–524, 2002. antibody increases renal and splenic sympathetic nerve discharge. Am J Physiol Heart Circ Physiol 284: H1536–H1541, 2003.
B are critically involved in reactive oxygen species-mediated induction of IL-6 by angiotensin II in cardiac fibroblasts. Circ Res 89: 661–669, 2001.This article has been cited by other articles:
|
|
 |
|
  B. Xue, Y. Zhao, A. K. Johnson, and M. Hay Central estrogen inhibition of angiotensin II-induced hypertension in male mice and the role of reactive oxygen species Am J Physiol Heart Circ Physiol, September 1, 2008; 295(3): H1025 - H1032. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Biaggioni Should We Target the Sympathetic Nervous System in the Treatment of Obesity-Associated Hypertension? Hypertension, February 1, 2008; 51(2): 168 - 171. [Full Text] [PDF]
|
|
|
|
|
|
M. Fujita, K. Ando, A. Nagae, and T. Fujita Sympathoexcitation by Oxidative Stress in the Brain Mediates Arterial Pressure Elevation in Salt-Sensitive Hypertension Hypertension, August 1, 2007; 50(2): 360 - 367. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. N. Mayorov Brain superoxide as a key regulator of the cardiovascular response to emotional stress in rabbits Exp Physiol, May 1, 2007; 92(3): 471 - 479. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Xu, X. Bian, S. W. Watts, and A. Hlavacova Activation of Vascular BK Channel by Tempol in DOCA-Salt Hypertensive Rats Hypertension, November 1, 2005; 46(5): 1154 - 1162. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Sun, K. W. Sellers, C. Sumners, and M. K. Raizada NAD(P)H Oxidase Inhibition Attenuates Neuronal Chronotropic Actions of Angiotensin II Circ. Res., April 1, 2005; 96(6): 659 - 666. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
P < 0.05 for renal SND and HR, significantly different from 5 µmol/kg Tempol; for MAP, data points at 5, 10, 15, and 20 min for Tempol at 50, 75, and 100 µmol/kg significantly different from 5 µmol/kg; 
P < 0.05, 100 µmol/kg Tempol significantly different from 50 and 75 µmol/kg. Tempol at 5 and 50 µmol/kg: n = 11 for renal SND, MAP, and HR. Tempol at 75 µmol/kg: n = 14 for renal SND and n = 22 for MAP and HR. Tempol at 100 µmol/kg: n = 6 for renal SND and n = 11 for MAP and HR.
