CARDIOVASCULAR SYSTEM

Endogenous hydrogen peroxide in paraventricular nucleusmediating cardiac sympathetic afferent reflex and regulatingsympathetic activity

Yang Yu & Ming-Kui Zhong & Jing Li & Xiu-Lan Sun &

Gui-Qing Xie & Wei Wang & Guo-Qing Zhu

Received: 29 January 2007 /Revised: 1 March 2007 /Accepted: 13 March 2007 /Published online: 27 March 2007# Springer-Verlag 2007

Abstract We previously reported that reactive oxygenspecies (ROS) in paraventricular nucleus (PVN) mediatedcardiac sympathetic afferent reflex (CSAR). The presentstudy investigated the role of endogenous hydrogenperoxide (H2O2), a ROS, in the PVN in mediating theCSAR and regulating sympathetic activity. The CSAR wasevaluated by the response of renal sympathetic nerveactivity (RSNA) to epicardial application of bradykinin(BK) in rats. Bilateral microinjection of polyethyleneglycol-catalase (PEG-CAT, an analogue of endogenouscatalase) or polyethylene glycol-superoxide dismutase(PEG-SOD, an analogue of endogenous superoxide dis-mutase) into the PVN abolished the CSAR, decreasedbaseline RSNA and mean arterial pressure (MAP). More-over, pretreatment with PEG-CAT or PEG-SOD blocked theenhanced CSAR and RSNA responses induced by exoge-nous angiotensin II (Ang II) in the PVN. Aminotriazole(ATZ, a catalase inhibitor) alone potentiated the CSAR,increased RSNA and MAP, but failed to augment the AngII-induced CSAR enhancement responses. Pretreated withPEG-SOD, ATZ still increased baseline RSNA and MAPbut inhibited the CSAR and Ang II-induced CSAR andRSNA enhancement responses. These results suggested thatendogenous H2O2 in the PVN mediated both the CSAR andAng II-induced CSAR enhancement responses. H2O2 in the

PVN were involved in regulating sympathetic activity andarterial pressure.

Keywords Cardiac sympathetic afferent reflex .

Reactive oxygen species . Angiotensin II .

Paraventricular nucleus . Sympathetic activity

Introduction

Cardiac sympathetic afferent reflex (CSAR) is enhanced inchronic heart failure (CHF) state, which at least partiallycontributes to the over-excitation of sympathetic nervoussystem [10, 13, 21, 23, 27, 31]. The degree of sympatheticexcitation is prognostic for survival in this disease [7, 11, 12].Paraventricular nucleus (PVN) is an important integrativecenter in the control of cardiovascular activity and sympa-thetic outflow. It was found that microinjection of a GABAA

receptor agonist muscimol into the PVN attenuated theCSAR induced by epicardial application of bradykinin (BK)[29]. Epicardial application of BK increased the c-Fosimmunoreactive cell in the PVN in cats [15]. It was reportedthat AT1 receptors are densely distributed in the PVN [3, 28].Our previous studies indicated that angiotensin II (Ang II)and AT1 receptors in the PVN played important roles in thecentral modulation of CSAR and contributed to the patho-genesis of enhanced CSAR in CHF [16, 27, 30–33].

We recently reported that reduced-form nicotinamideadenine dinucleotide phosphate (NAD(P)H) oxidase-derivedreactive oxygen species (ROS) especially superoxide anionsin the PVN mediated the CSAR and contributed to the effectof Ang II in the PVN on the CSAR in rats [16, 30]. It isknown that superoxide anions are quickly converted tohydrogen peroxide (H2O2) by superoxide dismutase (SOD).H2O2 is a cell-permeant and relative stable ROS and has

Pflugers Arch - Eur J Physiol (2007) 454:551–557DOI 10.1007/s00424-007-0256-9

Y. Yu :M.-K. Zhong : J. Li :X.-L. Sun :G.-Q. Xie :G.-Q. Zhu (*)Department of Physiology,Nanjing Medical University,Nanjing 210029, Chinae-mail: gqzhucn@njmu.edu.cn

W. WangDepartment of Cellular and Integrative Physiology,University of Nebraska College of Medicine,Omaha, NE 68198-5850, USA

been found as an important cellular signaling agent in thebrain and peripheral tissues [1, 20, 22, 26]. However, little isknown about the role of H2O2 in the PVN in the control ofCSAR and sympathetic activity. In the present study, weinvestigated whether endogenous H2O2 in the PVN mediatedthe CSAR and the enhanced CSAR response caused byexogenous Ang II in the PVN. Furthermore, the roles of theendogenous H2O2 in regulating sympathetic activity andarterial pressure were determined.

Materials and methods

Experiments were carried out in 90 male Sprague–Dawleyrats weighing between 300 and 400 g. The procedures wereapproved by the Experimental Animal Care and UseCommittee of Nanjing Medical University and compliedwith the Guide for the Care and Use of Laboratory Animals(NIH publication no. 85-23, revised 1996).

Animal preparation

Each rat was anesthetized with urethane (800 mg/kg, i.p.)and α-chloralose (40 mg/kg, i.p.). Supplemental doses ofanesthesia were administered at 1/10 of the initial dose perhour. The trachea and right carotid artery were cannulatedfor mechanical ventilation and measurement of arterialpressure, respectively. Each vagus was identified in theneck, tied, and sectioned. Baroreceptor denervation wascarried out and identified as previously reported [16, 33].The rat was placed in a stereotaxic instrument (Stoelting,Chicago). The coordinates for PVN were determinedaccording to the Paxinos and Watson rat atlas [25], whichis 1.8 mm caudal from bregma, 0.4 mm lateral to themidline, and 7.9 mm ventral to the dorsal surface. Themicroinjection volume in each side of the PVN was 50 nl.The administration was completed within 1 min.

Renal sympathetic nerve activity (RSNA) was recordedas previously described [16, 32]. The signal was amplifiedwith an AC/DC differential amplifier (Model 3000, A-MSystem) with a low-frequency cutoff at 60 Hz and a high-frequency cutoff at 3 kHz. The amplified and filteredsignals were integrated at time constant of 10 ms. Thebackground noise was determined after sectioning of thecentral end of the renal nerve at the end of the experimentand subtracted from all the integrated values of the RSNA.The raw RSNA, integrated RSNA, arterial pressure, andheart rate were recorded on a PowerLab data acquisitionsystem (8SP, ADInstruments) and stored on hard disk. TheRSNA was expressed as the percent change from control.

At the end of the experiment, 50 nl of Evans blue (2%)was injected into the microinjection site. The microinjection

Fig. 1 Effects of different doses of PEG-CAT (0, 0.002, 0.02, and0.2 units) on the CSAR (a), RSNA (b), and MAP (c). PEG-CATinhibited the CSAR and decreased the RSNA and MAP in a dose-dependent manner. Pretreatment with PEG-CAT inhibited the effectsof microinjection of Ang II into the PVN on the CSAR, RSNA, andMAP in a dose-dependent manner. Values were mean±SE. Asteriskp<0.05 compared with saline, dagger p<0.05 compared with PEG-CAT+Ang II, n=6 for each group

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site was verified histologically. Only the data of rats whosemicroinjection sites were within the boundaries of the PVNwere used for analysis.

Determination of CSAR

The CSAR was elicited by application of a piece of filterpaper (3×3 mm) containing BK (0.3 nmol in 2.0 μl) to theepicardial surface of the anterior wall of the left ventricle.Each piece of paper was applied for 1 min and then removed.The epicardium was rinsed three times with 10 ml of warmnormal saline (38°C). The CSAR was evaluated by theresponse of the RSNA to epicardial application of BK.

Drugs

BK, Ang II, polyethylene glycol-catalase (PEG-CAT, ananalogue of endogenous catalase), polyethylene glycol-superoxide dismutase (PEG-SOD, an analogue of endoge-

nous superoxide dismutase) and aminotriazole (ATZ, acatalase inhibitor) were obtained from Sigma Chemical. Allthe drugs were dissolved in normal saline.

Protocols

In seven groups of rats (n=6 for each), effects of PVNmicroinjection of saline, three doses of PEG-CAT (0.2,0.02, 0.002 units), PEG-SOD (2 units), ATZ (10 nmol), andPEG-SOD (2 units)+ATZ (10 nmol) on the CSAR, RSNA,and mean arterial pressure (MAP) were investigated. In thePEG-SOD+ATZ group, rats were pretreated with PEG-SOD 10 min before the administration of ATZ. The CSARwas, respectively, determined before and 20 min after thePVN microinjection. To exclude the possibility that theeffects of PEG-CAT on the CSAR were caused by diffusionto other brain area or peripheral blood flow, the effects ofeither intravenous injection of PEG-CAT (0.2 units) ormicroinjection of same dose of PEG-CAT into the anterior

Fig. 2 Tracing showing the effects of microinjection of PEG-CAT(0.2 units) into the PVN on the CSAR and the Ang II-induced CSARenhancement in rats. The CSAR was evaluated by the response ofRSNA to epicardial application of BK. PEG-CAT abolished the CSAR

(upper panel). Microinjection of Ang II into the PVN enhanced theCSAR (left panel). Pretreatment with PEG-CAT abolished the effect ofAng II on the CSAR (lower panel)

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hypothalamic area, which is the adjacent area of the PVNon the CSAR, were determined (n=3 for each).

In the other seven groups of rats (n=6 for each), the effectsof pretreatment with PVN microinjection of saline, threedoses of PEG-CAT (0.2, 0.02, 0.002 units), PEG-SOD (2units), ATZ (10 nmol), and PEG-SOD (2 units)+ATZ(10 nmol) on the enhanced CSAR and RSNA responses aswell as pressor response induced by Ang II were investigated.The pretreatment were 20 min earlier than microinjection ofAng II (0.3 nmol) into the PVN. The CSAR was,respectively, determined 2 min after administration of Ang II.

Statistics

Comparisons between two observations in the same animalwere assessed by Student’s paired t test. One-way analysisof variance, followed by the Student’s t test, was used whenmultiple comparisons were made. All statistical analysiswas done using computer software (SigmaStat, SPSS 10.0).All data were expressed as mean±SE. p<0.05 wasconsidered statistically significant.

Results

Effects of different doses of PEG-CAT

Microinjection of PEG-CAT into the PVN inhibited both theCSAR and the enhanced CSAR response induced by Ang IIdose dependently (Fig. 1a). However, intravenous injection ormicroinjection of high dose of PEG-CAT (0.2 units) into theanterior hypothalamic area failed to cause any significanteffects on the CSAR. On the other hand, PEG-CAT decreasedthe RSNA and MAP and inhibited the effect of Ang II on theRSNA and MAP dose dependently (Fig. 1b and c).

Effects of PEG-CAT, PEG-SOD, and ATZ on the CSAR

The representative recordings in the upper panel of Fig. 2showed that microinjection of PEG-CAT into the PVNabolished the CSAR evoked by epicardial application ofBK. Compared with saline, PEG-CAT or PEG-SOD almostcompletely abolished the CSAR. ATZ alone enhanced theCSAR, but PEG-SOD reversed the effect of ATZ on theCSAR (Fig. 3a).

Effects of PEG-CAT, PEG-SOD, and ATZ on the baselineRSNA and MAP

Compared with saline, either PEG-CATor PEG-SOD inducedslow and long-lasting decreases in RSNA and MAP within5 min, peaking at approximately 15 min. ATZ caused rapidand long-lasting increases in RSNA and MAP within 1 min,

peaking at approximately 2 min. Although PEG-SOD aloneinhibited RSNA and decreased MAP, PEG-SOD failed toabolish the effects of ATZ on RSNA and MAP (Fig. 4a).

Effects of PEG-CAT, PEG-SOD, and ATZ on the enhancedCSAR induced by Ang II

Microinjection of Ang II (0.3 nmol) into the PVNsignificantly enhanced the CSAR evoked by epicardialapplication of BK (left panel of Fig. 2), which was similarto our previous reports [32, 33]. The representative record-ings in the lower panel of Fig. 2 showed that pretreatmentwith microinjection of PEG-CAT into the PVN abolishedthe effect of Ang II on the CSAR. Compared with saline,PEG-CAT, PEG-SOD, or PEG-SOD+ATZ almost com-pletely abolished effect of Ang II on the CSAR. However, a

Fig. 3 a Effects of microinjection of saline, PEG-CAT, PEG-SOD, ATZ,and PEG-SOD+ATZ into the PVN on the CSAR. The CSAR wasevaluated by the RSNA response to epicardial application of BK. PEG-CAT or PEG-SOD abolished the CSAR. ATZ enhanced the CSAR, whichwas reversed by pretreatment with PEG-SOD. b Effects of pretreatmentwith saline, PEG-CAT, PEG-SOD, ATZ, or PEG-SOD+ATZ on theenhanced CSAR caused Ang II. Microinjection of Ang II into the PVNenhanced the CSAR, which was abolished by PEG-CAT, PEG-SOD, orPEG-SOD+ATZ. ATZ did not significantly augment the Ang II-inducedCSAR enhancement. Values were mean±SE. Asterisk p<0.05 comparedwith saline, dagger p<0.05 compared with ATZ, n=6 for each group

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catalase inhibitor, ATZ alone failed to augment theenhanced CSAR further (Fig. 3b).

Effects of PEG-CAT, PEG-SOD, and ATZ on the AngII-induced RSNA and MAP change

Microinjection of the Ang II into the PVN significantlyincreased the RSNA and MAP, which was similar to ourprevious reports [32, 33]. PEG-CAT, PEG-SOD, or PEG-SOD+ATZ abolished the effects of Ang II on the RSNAand MAP compared with saline. However, ATZ alone didnot further enhance the effects of Ang II on the RSNA andMAP (Fig. 4b).

Discussion

The primary findings in the present study were thatendogenous H2O2 in the PVN mediated the CSAR and

acted as an important mediator of the Ang II in the PVN inmodulating CSAR. The endogenous H2O2 in the PVN wasinvolved in tonic control of sympathetic activity and arterialpressure.

It is well known that superoxide anions are efficientlyconverted to the tissue-permeant H2O2 by abundant andubiquitous intracellular and extracellular SODs, and CATexclusively catalyzes the conversion of H2O2 to water. SODdecreases the superoxide anions level but increases H2O2

level, whereas CAT decreases the H2O2 level [1, 20, 22,26]. Compared with CAT and SOD, PEG-CAT and PEG-SOD prolong the circulatory half-life of the native enzymesand enhance their intracellular access [19]. ATZ is anirreversible specific catalase inhibitor, and it is found toincrease intracellular levels of H2O2 [5, 9]. In the presentstudy, bilateral microinjection of H2O2 scavenger PEG-CAT into the PVN abolished the CSAR. Conversely,catalase inhibitor ATZ enhanced the CSAR. The PEG-SOD abolished the CSAR, which was consistent with our

Fig. 4 a Effects of microinjection of saline, PEG-CAT, PEG-SOD, ATZ,and PEG-SOD+ATZ into the PVN on the baseline RSNA and MAP.PEG-CAT and PEG-SOD decreased, but ATZ increased baseline RSNAand MAP. PEG-SOD failed to abolish the effects of ATZ on RSNA andMAP (n=6 for each group). b Effects of pretreatment with saline, PEG-CAT, PEG-SOD, ATZ, and PEG-SOD+ATZ into the PVN on the Ang II-

induced RSNA and MAP change. Microinjection of Ang II into the PVNincreased RSNA and MAP, which was completely abolished bypretreatment with PEG-CAT, PEG-SOD, or PEG-SOD+ATZ. ATZ alonedid not further enhance the effects of Ang II significantly. Values weremean±SE. Asterisk p<0.05 compared with saline, dagger p<0.05compared with ATZ, n=6 for each group

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previous findings that either NAD(P)H oxidase inhibitor orsuperoxide anion scavenger inhibited the CSAR in rats [16,30]. These results indicated that both superoxide anion andH2O2 in the PVN were necessary in mediating the CSARand might be pivotal intracellular signal molecules andform a cascade pathway in mediating the CSAR. It wasfound that PEG-SOD reversed the effect of ATZ on theCSAR. We speculated that a very low superoxide anionlevel in the PVN caused by PEG-SOD hardly mediated theCSAR, although the H2O2 level was increased by ATZ.

It was reported that central ROS were involved in thecentral effects of Ang II on cardiovascular and sympatheticnervous activity [4, 17, 34]. Our previous studies showedthat AT1 receptors in the PVN modulated the CSAR innormal rats and contributed to the enhanced CSAR in CHFrats [27, 31, 32]. Microinjection of Ang II (0.3 nmol) intothe PVN increased the CSAR, RSNA, and MAP. Theeffects of Ang II were inhibited by PVN microinjection ofsuperoxide anion scavengers (tempol or tiron) or NAD(P)Hoxidase inhibitors, (apocynin or phenylarsine oxide) butaugmented by a SOD inhibitor (diethyldithiocarbamate)[16, 30]. Either epicardial application of BK or PVNmicroinjection of Ang II increased NAD(P)H oxidaseactivity in the PVN in rats [30]. These findings suggestedthat NAD(P)H oxidase-derived ROS especially superoxideanions in the PVN contributed to the effect of Ang II in thePVN on the CSAR in rats. It was reported that H2O2

mediated AT1 receptor-dependent Ang II signaling invascular cells [14, 24]. The present study found that eitherPEG-CAT or PEG-SOD abolished Ang II-induced effectson the CSAR, RSNA, and MAP. It is more interesting thatPEG-SOD plus ATZ also abolished the effects of Ang II.Theses results indicated that both superoxide anions andH2O2 were necessary in mediating the effects of Ang II inthe PVN on the CSAR, RSNA, and MAP.

It is known that PVN is an important integrative centerin the control of cardiovascular activity and sympatheticoutflow [2, 8]. The present study found that microinjectionof PEG-CAT or PEG-SOD decreased, but ATZ increasedthe baseline RSNA and MAP, which indicated that bothH2O2 and superoxide anion in the PVN were involved intonic control of sympathetic activity and arterial pressure. Areduction in either superoxide anion or H2O2 level in thePVN may result in decreases in RSNA and MAP. However,the increased RSNA and MAP caused by ATZ was notabolished by pretreatment with PEG-SOD, although theenhanced CSAR caused by ATZ was reversed by PEG-SOD. A probable explanation was that the increasedsympathetic activity and arterial pressure were caused byincreased H2O2 level, but little superoxide anion couldmediate the CSAR evoked by epicardial application of BK.Curiously, PEG-SOD decreased sympathetic activity andarterial pressure, although it converted superoxide anions to

H2O2, which was consistent with our previous findings thattempol (which also converts superoxide anions to H2O2)into the PVN decreased sympathetic activity and arterialpressure. These results showed that both superoxide anionsand H2O2 in the PVN were important in regulatingsympathetic activity and arterial pressure. Therefore, therelationship of superoxide anions and H2O2 in the PVN isvery complex and cannot be simply explained by cascadepathway in the tonic control of sympathetic activity andarterial pressure.

A lot of neurotransmitters and neuromodulators in thePVN are involved in the regulation of sympathetic activity.It was found that administration of Ang II into the PVNinduced an increase in NO release, and NO, in turn,inhibited the Ang II-mediated increase in sympathetic nerveactivity [18]. More recently, it was found that Ang IIattenuates GABAergic input to PVN presympathetic neu-rons through ROS, especially superoxide anions [6]. Zahnerand Pan [29] found that microinjection of a GABAA

receptor agonist muscimol into the PVN attenuated theCSAR induced by epicardial application of BK. However,no direct evidence showed the relationship between GABA(or NO) and H2O2 in the control of the CSAR as yet. Thepossible interaction of GABA with H2O2 in the control ofCSAR and sympathetic activity is worthy of furtherinvestigation.

In summary, H2O2 in the PVN played an important rolein mediating the CSAR evoked by epicardial application ofBK in rats. Furthermore, H2O2 in the PVN was involved intonic control of baseline sympathetic activity and arterialpressure. On the other hand, the enhanced CSAR andRSNA responses as well as pressor response caused bymicroinjection of Ang II into the PVN were mediated byH2O2 in the PVN. The superoxide anions in the PVNshowed similar effects to H2O2 in the PVN. The interactionof superoxide anions and H2O2 in the PVN in control of theCSAR, sympathetic activity, and arterial pressure requiresfurther elucidation.

Acknowledgments This study was supported by Chinese NationalNatural Science Fund (30470632 and 30670768).

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