Original Article

Salusin-b in rostral ventrolateral medulla increases sympathetic outflow and blood pressure via superoxide anions in hypertensive rats Ling-Li Zhang a, Lei Ding a, Feng Zhang a, Run Gao a, Qi Chen b, Yue-Hua Li b, Yu-Ming Kang c, and Guo-Qing Zhu a See editorial comment on page 981

Objective: Rostral ventrolateral medulla (RVLM) plays crucial roles in central cardiovascular regulation. Salusin-b is a multifunctional bioactive peptide and is expressed in the brain and various peripheral tissues. The present study was designed to identify the expression of salusin-b in the RVLM, and to determine its roles and mechanisms in regulating sympathetic outflow and blood pressure in hypertension.

glycol superoxide dismutase; PVN, paraventricular nucleus; ROS, reactive oxygen species; RSNA, renal sympathetic nerve activity; RVLM, rostral ventrolateral medulla; Sham, sham-operated; SIgG, antisalusin-b IgG

INTRODUCTION

S

Methods and results: Renovascular hypertension was induced with two-kidney, one-clip (2K1C) method in rats. Acute experiments were carried out 4 weeks after the 2K1C or sham operation under urethane and alpha-chloralose anesthesia. Salusin-b level and the number of salusin-b-like immunopositive neurons in the RVLM were greatly increased in 2K1C rats. Microinjection of salusin-b into the RVLM increased renal sympathetic nerve activity, mean arterial pressure and heart rate, whereas antisalusin-b IgG (SIgG) reduced renal sympathetic nerve activity and mean arterial pressure in 2K1C rats, but not in sham-operated rats. The effects of salusin-b were abolished by the RVLM pretreatment with superoxide anion scavenger tempol or polyethylene glycol superoxide dismutase, or NAD(P)H oxidase inhibitor apocynin, but not attenuated by the RVLM microinjection or intravenous injection of dTyr(CH2)5(Me)AVP, an antagonist of V1 receptors of arginine vasopressin. Salusin-b increased NAD(P)H oxidase activity and superoxide anion level in the RVLM, which were prevented by the pretreatment with SIgG. Microinjection of salusin-b or SIgG into the RVLM had no significant effect on the arginine vasopressin level in the RVLM and plasma.

alusins are translated from an alternatively spliced mRNA of torsin family 2 member A (TOR2A), a gene encoding a protein of the torsion dystonia family and composed of two related bioactive peptides of 28 and 20 amino acids designated salusin-a and salusinb [1]. Salusin-b is shown to have negative inotropic effect on ventricular muscle [2]. The initial 18 amino acids of human salusin-b have high homology with the estimated N-terminal sequence of rat salusin-b [3]. Salusin-b stimulates arginine vasopressin (AVP) release from perfused rat pituitary [1]. Salusin-like immunopositive cells have been found in suprachiasmatic, supraoptic and paraventricular nucleus (PVN), and salusin-positive nerve fibers and their terminals in the posterior pituitary [4]. A recent study in our lab has shown that salusin-b in the PVN increases renal sympathetic nerve activity (RSNA), mean arterial pressure (MAP) and heart rate (HR) in two-kidney, one-clip (2K1C)induced hypertensive rats via both circulating AVP and AVP in rostral ventrolateral medulla (RVLM) in hypertensive rats [5]. It is well known that RVLM plays an important role in maintaining baseline sympathetic vasomotor tone and arterial blood pressure (ABP) [6,7]. RVLM contributes to the elevated blood pressure in a number of hypertension

Conclusion: Salusin-b in the RVLM increases sympathetic outflow, blood pressure and heart rate via NAD(P)H oxidase-derived superoxide anions in renovascular hypertensive rats.

Journal of Hypertension 2014, 32:1059–1067

Keywords: hypertension, rostral ventrolateral medulla, salusin, superoxide anions, sympathetic activity Abbreviations: 2K1C, two-kidney, one-clip; AAVP, dTyr(CH2)5(Me)AVP; ABP, arterial blood pressure; AVP, arginine vasopressin; CIgG, control IgG; DHE, dihydroethidium; DMSO, dimethyl sulfoxide; HR, heart rate; MAP, mean arterial pressure; PEG-SOD, polyethylene

a Key Laboratory of Cardiovascular Disease and Molecular Intervention, Department of Physiology, bDepartment of Pathophysiology, Nanjing Medical University, Nanjing, Jiangsu and cDepartment of Physiology and Pathophysiology, Cardiovascular Research Center, Xi’an Jiaotong University School of Medicine, Xi’an, China

Correspondence to Guo-Qing Zhu, MD, PhD, Professor, Chair, Key Laboratory of Cardiovascular Disease and Molecular Intervention, Department of Physiology, Nanjing Medical University, Nanjing 210029, China. Tel: +86 25 86862885; fax: +86 25 86862885; e-mail: [email protected] Received 21 October 2013 Revised 17 January 2014 Accepted 17 January 2014 J Hypertens 32:1059–1067 ß 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins. DOI:10.1097/HJH.0000000000000143

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models [8]. AVP-immunoreactive fibers were found in the RVLM [9]. Microinjection of AVP V1 receptor antagonist dTyr(CH2)5(Me)AVP (AAVP) into the RVLM abolished the effects of salusin-b in the PVN on the RSNA and HR, but only attenuated the effects of salusin-b on the MAP [5]. However, it is unknown whether salusin-b is expressed in the RVLM, and whether salusin-b in the RVLM is involved in cardiovascular regulation. The present study was designed to determine the roles and the downstream molecular mechanism of salusin-b in the RVLM in the sympathetic activation and hypertension in renovascular hypertensive rats.

METHODS Animals Experiments were carried out in male Sprague–Dawley rats. The procedures were approved by the Experimental Animal Care and Use Committee of Nanjing Medical University and complied with the Guide for the Care and Use of Laboratory Animals (NIH publication, 8th edition, 2011). The rats were caged in an environment under controlled temperature and humidity. Standard laboratory chow and drinking water were made available ad libitum. At the end of experiment, the rat was euthanized with an overdose of pentobarbital sodium [200 mg/kg, intravenous (i.v.)].

Animal model of renovascular hypertension Renovascular hypertension was induced by 2K1C method [5,10]. Briefly, the rat weighing 160–180 g was anesthetized by pentobarbital sodium (60 mg/kg) intraperitoneally. Right renal artery was exposed via a right retroperitoneal flank incision with sterile techniques, and partly occluded with a U-shaped silver clip with an internal diameter of 0.20 mm on the vessel to induce renovascular hypertension. Similar surgeries were made in sham-operated rats (Sham) except the clips were not used. SBP was measured at weekly intervals in a conscious state with a noninvasive computerized tail-cuff system (NIBP, ADInstruments, Sydney, New South Wales, Australia). A SBP of 160 mmHg or greater was accepted as hypertension.

Renal sympathetic nerve activity, mean arterial pressure and heart rate recordings Acute experiments were carried out 4 weeks after surgery under anesthesia with intraperitoneal injection of urethane (800 mg/kg) and a-chloralose (40 mg/kg). The adequacy of anesthesia was determined by the loss of toe pinch reflex [14]. The trachea was cannulated for mechanical ventilation. ABP was monitored with a pressure transducer via a catheter placed into the right carotid artery. HR was counted by triggering from the pulsatile blood pressure. Left renal sympathetic nerve was isolated through a retroperitoneal incision. The renal nerve was cut distally to ensure that afferent activity was not recorded. The nerve was immersed in warm mineral oil and placed on a pair of silver electrodes. The signals were amplified with a four channel AC/DC differential amplifier (DP-304; Warner Instruments, Hamden, Connecticut, USA). The RSNA was filtered with a bandwidth of 100–300 Hz, and integrated at a time constant of 100 ms. At the end of each experiment, the background noise was determined after section of the central end of the nerve and was subtracted from the integrated values of the RSNA [15]. The MAP, HR and RSNA were simultaneously recorded with a PowerLab data acquisition system (8/35, ADInstruments, Sydney, New South Wales, Australia).

Rostral ventrolateral medulla microinjection The coordinates for the RVLM are 4.5–5.0 mm posterior to lambda, 1.8–2.1 mm lateral to midline, and 8.1–8.4 mm below the dorsal surface of cerebellum [16]. The bilateral RVLM microinjections were carried out with two glass micropipettes (50 mm in tip diameter) and completed within 1 min. The microinjection volume was 50 nl for each microinjection site. Functional location of RVLM neurons was carried out at the beginning of each experiment by eliciting a transient increase in systolic arterial pressure (20–30 mmHg) by microinjection of glutamate (2 nmol) [16]. At the end of experiments, 50 nl of 2% Evans blue was injected for marking the injection sites. Then the brains were removed from the skulls, placed in 10% formalin, and sectioned to verify the microinjection sites. The rats with microinjection sites outside the RVLM were excluded from data analysis.

Immunohistochemistry Salusin-b and antisalusin-b antibody Human salusin-b [1,11,12] and antisalusin-b (human) antibody [3,13] were widely used to investigate the function of salusin-b in rats due to the high homology of human salusin-b with rat salusin-b [3]. Rabbit antisalusin-b (human) IgG and rabbit antisalusin-b (human) serum were obtained from Bachem (Bubendorf, Switzerland). The specificity of the purified polyclonal antibody has been identified with radioimmunoassay, and the antisalusin-b IgG (SIgG) had no cross-reaction with salusin-a. Rabbit antisalusin-b (human) IgG is supplied as a lyophilized powder, and is reconstituted by adding 0.01 mol/l PBS (pH 7.4) to get the solution (100 ng in 50 nl), which was used for RVLM microinjection. Rabbit antisalusin-b (human) serum was diluted with 0.01 mol/l PBS and the titer was 1 : 500 for immunohistochemistry. 1060

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Immunohistochemistry for detecting salusin-b-positive neurons in the RVLM was carried out as we previously reported [5]. The number of neurons with salusin-b-like immunoreactivity in the RVLM was determined by averaging the numbers of four sections for each animal.

Immunofluorescent double-labeled staining The brain sections were simultaneously incubated with the rabbit antihuman salusin-b antibody (1 : 500), which was revealed using CY3-labeled antirat antibody (1 : 200; Chemicon International, Temecula, California, USA) and mouse anti-neuronal nuclei (NeuN) antibody (1 : 500; Millipore, Billerica, Massachusetts, USA), which was revealed using fluorescein isothiocyanate (FITC)labeled antimouse antibody (1 : 200; Chemicon International). Immunofluorescence staining was imaged using Volume 32  Number 5  May 2014

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Central salusin-b and hypertension

a fluorescent microscope (BX51; Olympus, Tokyo, Japan). Specific lasers and channels for the emission of fluorescence were used according to each fluorescent compound [17].

Measurement of arginine vasopressin and salusin-b Commercial ELISA kits were used for the measurement of AVP (Enzo Life Sciences, Ann Arbor, Michigan, USA) and salusin-b (Uscn Life Science, Inc., Houston, Texas, USA). According to the manufacturer’s descriptions, the standards or sample diluents were added and incubated in the appropriate well of specific antibody precoated microtiter plate. Conjugate was added and incubated for 1 h at 378C and then washed. The reactions were stopped with stop solution and read at 405 nm for AVP measurement and at 450 nm for salusin-b measurement using a microtiter plate reader (ELX800; BioTek, Winooski, Vermont, USA).

Measurement of NAD(P)H oxidase activity and superoxide anion level Brain was removed and flash-frozen with liquid nitrogen and stored at  708C until being sectioned. Coronal sections of the brain were made with a cryostat microtome (CM1900; Leica, Nussloch, Germany). RVLM areas were punched out with a 15-gauge needle (inner diameter 1.5 mm), homogenized and centrifuged in lysis buffer. Total protein concentration in the homogenate was measured with the Bradford assay (BCA; Pierce, Santa Cruz, California, USA). NAD(P)H oxidase activity and superoxide anion level in the RVLM were measured with lucigenin-derived chemiluminescence method [18–20]. Briefly, the photon emission was started by adding dark-adapted lucigenin for determining the superoxide anion level, by adding both NAD(P)H and dark-adapted lucigenin for determining the NAD(P)H oxidase activity. Light emission was measured for 10 times in 10 min with a luminometer (20/20n; Turner, BioSystems, Sunnyvale, California, USA). The values were averaged, and expressed as mean light unit (MLU) per minute per milligram of protein.

In-situ detection of superoxide anions Dihydroethidium (DHE) fluorescence method was widely used to examine superoxide anion production [18,21]. In brief, the DHE fluorescence in the coronal sections was visualized under a fluorescence microscope (BX51; Olympus, Tokyo, Japan) coupled with an Olympus DP70 digital camera. Detector and laser settings were kept constant among all samples within individual experiment. The control and experimental samples were always processed in parallel. Fluorescence intensity was analyzed and quantified with Image-Pro Plus 6.0 by using the same parameters.

Chemicals Tempol, polyethylene glycol superoxide dismutase (PEGSOD), apocynin, NAD(P)H, dimethyl sulfoxide (DMSO), lucigenin and AAVP were obtained from Sigma Chemical Co (St. Louis, Missouri, USA). Human salusin-b was obtained from Phoenix Pharmaceuticals (California, USA). Apocynin Journal of Hypertension

was dissolved in normal saline containing 1% DMSO. All other chemicals were dissolved in normal saline.

Statistics Comparisons between two groups were made by Student’s t test. Analysis of variance (ANOVA) followed by post-hoc Bonferroni test was used when multiple comparisons were made. All data were expressed as mean  standard error (SE). A value of P less than 0.05 was considered statistically significant.

RESULTS General date The SBP measured in conscious state was higher in 2K1C rats than that in Sham rats (191  2 vs.129  3 mmHg; P < 0.001). Similarly, the MAP measured under anesthesia was higher in 2K1C rats (141  2 vs. 95  3 mmHg; P < 0.001). No significant difference in body weight (356  10 vs. 350  4 g) or HR (360  8 vs. 378  5 bpm) was found between Sham and 2K1C rats.

Salusin-b-like immunoreactivity and salusin-b level in rostral ventrolateral medulla Salusin-b-positive neurons in the RVLM were examined with immunohistochemistry in Sham and 2K1C rats (four sections for each rat, and four rats for each group). The number of salusin-b-like immunopositive neurons in the RVLM areas was significantly increased in 2K1C rats compared with Sham rats (Fig. 1a–c). Salusin-b level in the RVLM of 2K1C rats was three to four times higher than that of Sham rats (Fig. 1d). Double immunofluorescence was performed in the RVLM of tissues from 2K1C rats with cell-specific antibodies. Salusin-b staining overlapped with a neuronal marker NeuN (Fig. 1e–g).

Effects of different doses of salusin-b Representative recordings showed that salusin-b caused immediate increases in the RSNA, MAP and HR, lasting at least 30 min in 2K1C rats (Fig. 2). Microinjections of saline, different doses of salusin-b (1, 10 or 100 pmol) into the RVLM were randomly carried out in Sham and 2K1C rats (n ¼ 6 for each group). The intervals between injections were at least 60 min for a complete recovery. Salusin-b increased RSNA, MAP and HR in a dose-related manner in 2K1C rats, but failed to induce any significant effect in Sham rats (Fig. 3).

Effects of antisalusin-b IgG Antisalusin-b IgG was used to immunoneutralize salusin-b to determine the effects of endogenous salusin-b in the RVLM. Either Sham or 2K1C rats were randomly divided into two groups (n ¼ 6 for each group), which were subjected to the RVLM microinjection of salusin-b (100 pmol) pretreated with control IgG (CIgG; 100 ng) or SIgG (100 ng). Salusin-b was administered 10 min after the pretreatment. Microinjection of SIgG into the RVLM decreased the baseline RSNA and MAP in 2K1C rats, but not in Sham rats. Pretreatment with RVLM microinjection of SIgG almost abolished the effects of salusin-b in the RVLM on the RSNA, www.jhypertension.com

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2K1C

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200 µm FIGURE 1 Salusin-b-positive neurons and salusin-b level in the RVLM in Sham and 2K1C rats. (a, b) Immunohistochemistry for detecting salusin-b-positive neurons in the RVLM. (c) Bar graph showing the average number of neurons with salusin-b-like immunoreactivity per section in the RVLM (four sections for each rat and four rats for each group). (d) Salusin-b level in the RVLM (n ¼ 6 for each group). Values are mean  SE (P < 0.05 vs. Sham). (e–g) Immunofluorescence of salusin-b (e, green), NeuN cells (f, red) and merged image (g) in the RVLM of a 2K1C rat. 2K1C, two-kidney, one-clip; RVLM, rostral ventrolateral medulla; SE, standard error.

MAP and HR in 2K1C rats. Microinjection of CIgG into the RVLM had no significant effect (Fig. 4). Since no significant effect of salusin-b or SIgG was observed in Sham rats, following experiments were only carried out in 2K1C rats.

Effects of superoxide anion scavengers and NAD(P)H oxidase inhibitor The effects of superoxide anion scavengers, PEG-SOD (5 units) and tempol (20 nmol), and NAD(P)H oxidase

inhibitor apocynin (3 nmol) were determined in six groups of 2K1C rats (n ¼ 6 for each group). The rats were subjected to the RVLM microinjection of saline þ saline, saline þ salusin-b, PEG-SOD þ salusin-b, tempol þ salusin-b, DMSO þ salusin-b or apocynin þ salusin-b. The interval between the two microinjections was 10 min. The RVLM microinjection of superoxide anion scavengers PEG-SOD or tempol, or NAD(P)H oxidase inhibitor apocynin reduced the baseline RSNA, MAP and HR in 2K1C rats. The RVLM

Integrated Raw RSNA RSNA mV

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200 150 100 50 200 150 100 50 100 50 0 –50 –100 0.4 0.3 0.2 0.1 0.0

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FIGURE 2 Representative traces showing the effects of RVLM microinjection of salusin-b (100 pmol) on MAP and RSNA in a Sham rat and a 2K1C rat. 2K1C, two-kidney, one-clip; MAP, mean arterial pressure; RSNA, renal sympathetic nerve activity; RVLM, rostral ventrolateral medulla.

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Central salusin-b and hypertension

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FIGURE 3 Effects of RVLM microinjection of saline and different doses of salusin-b (1, 10, 100 pmol) on the RSNA, MAP and HR in Sham and 2K1C rats.  P < 0.05 vs. Sham; yP < 0.05 vs. saline. Values are mean  SE. n ¼ 6 for each group. 2K1C, two-kidney, one-clip; HR, heart rate; MAP, mean arterial pressure; RSNA, renal sympathetic nerve activity; RVLM, rostral ventrolateral medulla; SE, standard error.

pretreatment with PEG-SOD, tempol or apocynin abolished the RSNA, MAP and HR responses to salusin-b in the RVLM in 2K1C rats (Fig. 5). 24

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The rats were sacrificed for the detection of superoxide anions 10 min after the RVLM microinjection of saline or salusin-b (100 pmol) in 2K1C rats (n ¼ 4 for each group). Compared with the RVLM microinjection of saline, salusinb increased the superoxide anion level in the RVLM detected with fluorogenic probe DHE in 2K1C rats (Fig. 6b).



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Microinjection of salusin-b into the RVLM increased the superoxide anion level and NAD(P)H oxidase activity in the RVLM in 2K1C rats, which were prevented by the pretreatment with SIgG (n ¼ 6 for each group). Microinjection of SIgG into the RVLM had a tendency in reducing the superoxide anion level and NAD(P)H oxidase activity in the RVLM, but the effects did not reach a significant level (Fig. 6a).

In-situ detection of superoxide anions † ‡ † † ‡ ‡ ‡

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Rats subjected to two-kidney, one-clip method were randomly divided into four groups (n ¼ 6 for each group), which were subjected to the RVLM microinjection of saline or V1 receptor antagonist AAVP (40 pmol), or salusin-b (100 pmol) pretreated with RVLM microinjection of saline or AAVP. Salusin-b was administered 10 min after the pretreatment. RVLM microinjection of AAVP had no significant effects on the baseline RSNA, MAP and HR, as well as the RSNA, MAP and HR responses to salusin-b in the RVLM in 2K1C rats (Fig. 7a).

Effects of intravenous infusion of dTyr(CH2)5(Me)AVP

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(min) FIGURE 4 Effects of RVLM microinjection of control IgG (CIgG) and antisalusin-b IgG (SIgG) on the RSNA, MAP and HR, as well as the RSNA, MAP and HR responses to salusin-b in the RVLM in Sham and 2K1C rats. Salusin-b was administered 10 min after the pretreatment. Arrows show the time of the PVN microinjections. P < 0.05 vs. values before salusin-b; yP < 0.05 vs. Sham with same treatment; zP < 0.05 vs. 2K1C/CIgG þ salusin-b. Values are mean  SE. n ¼ 6 for each group. 2K1C, two-kidney, one-clip; HR, heart rate; MAP, mean arterial pressure; PVN, paraventricular nucleus; RSNA, renal sympathetic nerve activity; RVLM, rostral ventrolateral medulla; SE, standard error.

Journal of Hypertension

Rats subjected to two-kidney, one-clip method were randomly divided into four groups (n ¼ 6 for each group), which were subjected to the intravenous infusion of saline (20 ml/min), AAVP (4.0 nmol/min), or intravenous infusion of saline or AAVP and the RVLM microinjection of salusin-b (100 pmol). Salusin-b was administered 10 min after the intravenous infusion. Intravenous infusion of AAVP decreased the baseline RSNA, MAP and HR in 2K1C rats, but had no significant effects on the RSNA, MAP and HR responses to salusin-b in the RVLM (Fig. 7b).

Arginine vasopressin levels Rats subjected to two-kidney, one-clip method were randomly divided into four groups (n ¼ 6 for each group), which www.jhypertension.com

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FIGURE 5 Effects of RVLM microinjection of PEG-SOD or tempol (a) or apocynin (b) on the RSNA, MAP and HR responses to salusin-b in the RVLM in 2K1C rats. The pretreatment was carried out 10 min before salusin-b administration. Arrows show the time of the PVN microinjections. P < 0.05 vs. values before salusin-b; yP < 0.05 vs. saline þ saline. zP < 0.05 vs. saline þ salusin-b; #P < 0.05 vs. DMSO þ salusin-b. Values are mean  SE. n ¼ 6 for each group. 2K1C, two-kidney, one-clip; DMSO, dimethyl sulfoxide; HR, heart rate; MAP, mean arterial pressure; PVN, paraventricular nucleus; RSNA, renal sympathetic nerve activity; RVLM, rostral ventrolateral medulla; SE, standard error.

were subjected to the RVLM microinjection of saline, salusin-b (100 pmol), CIgG (100 ng) or SIgG (100 ng). Ten minutes later, AVP levels in both RVLM and plasma were determined in 2K1C rats (n ¼ 6 for each group). The RVLM microinjection of salusin-b had no significant effect on plasma AVP level (þ8.2  1.7 vs. þ7.3  0.9 pg/ml) or AVP level in the RVLM (þ3.1  2.2 vs. þ3.0  1.5 pg/mg protein) in 2K1C rats compared with saline. Similarly, SIgG had no significant effect on the plasma AVP level (þ8.0  2.5 vs. þ7.5  1.9 pg/ml) or AVP level in the RVLM (þ3.4  1.1 vs. þ3.6  1.8 pg/mg protein) in 2K1C rats compared with CIgG.

Effects of salusin-b in spontaneously hypertensive rats All experiments mentioned above were carried out in 2K1C-induced hypertensive rats. In this experiment, effects of the RVLM microinjection of saline or salusin-b (100 pmol) on the RSNA and MAP were determined in SHRs (n ¼ 3 for each group). Compared with saline, microinjection of salusin-b into the RVLM of SHRs significantly increased 1064

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RSNA (þ18.4  3.8 vs. 0.2  3.4%; P < 0.05) and MAP (þ8.3  2.0 vs. þ0.7  1.9 mm Hg; P < 0.05).

DISCUSSION Excessive sympathetic activity plays a pathogenic role in triggering and sustaining the hypertensive state and contributes to the pathogenesis of hypertension and progression of organ damage [22]. Intervention of the enhanced sympathetic activity has been considered as an antihypertensive strategy [23–25]. RVLM is critical for regulating sympathetic outflow and blood pressure [6]. Increased activity of neurons in the RVLM contributes to experimental and essential hypertension [8]. We found that salusin-b-like immunopositive neurons and salusin-b level in the RVLM were greatly increased in 2K1C rats compared with Sham rats. Exogenous salusin-b in the RVLM increased the RSNA, MAP and HR in a dose-dependent manner in 2K1C rats, but not in Sham rats. Since salusin receptor antagonist has not been found yet, antisalusin antibody was used to investigate the effects of endogenous salusins [13,26,27]. The affinity-purified antibody recognizes rat Volume 32  Number 5  May 2014

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Central salusin-b and hypertension

60 Saline Salusin-β ClgG SlgG

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FIGURE 6 Superoxide anion level and NAD(P)H oxidase activity in the RVLM in 2K1C rats. (a) Effects of RVLM microinjection of saline, salusin-b, control (CIgG) or antisalusin-b IgG (SIgG) on superoxide anion level and NAD(P)H oxidase activity in the RVLM in 2K1C rats. (b) Effects of microinjection of saline or salusin-b into the RVLM of the 2K1C rats on the superoxide anion levels in the RVLM, indicated by DHE fluorescence intensity. P < 0.05 vs. saline. yP < 0.05 vs. salusin-b or CigG þ salusin-b. Values are mean  SE. n ¼ 6 for each group. 2K1C, two-kidney, one-clip; DHE, dihydroethidium; RVLM, rostral ventrolateral medulla; SE, standard error.

salusin in the hypothalamo-pituitary system, and the specificity of the salusin-b staining is assessed by preabsorption of the antibody with the full-length human salusin-b completely abolished salusin-b staining [4]. In the present study, SIgG that neutralizes endogenous salusin-b almost abolished the effects of salusin-b in the RVLM, suggesting that the dose of SIgG was high enough to block the effects of endogenous salusin-b. Importantly, SIgG reduced the RSNA, MAP and HR in 2K1C rats, but not in Sham rats. These results indicate that salusin-b in the RVLM contributes to hypertension and sympathetic activation in 2K1C rats. The possibility that the effects of salusin-b was caused by diffusion into peripheral circulation can be excluded

because intravenous salusin-b administration to intact anesthetized rats caused hypotension and bradycardia [1], which were abolished by pretreatment with atropine, a muscarinic receptor antagonist [12]. We recently found that salusin-b in the PVN increased RSNA, MAP and HR in 2K1C rats [5]. Accumulated evidences indicate that the projection from PVN to RVLM is involved in regulating sympathetic outflow and blood pressure [28,29]. Although AVP V1 receptor antagonist AAVP in the RVLM abolished the RSNA and HR responses to salusin-b in the PVN, and intravenous infusion of AAVP abolished the MAP response to salusin-b in the PVN [5], the same dose of AAVP in the RVLM or intravenous infusion of same dose of

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FIGURE 7 Effects of RVLM microinjection or intravenous injection of V1 receptor antagonist AAVP on the RSNA, MAP and HR, as well as the RSNA, MAP and HR responses to salusin-b in the RVLM in 2K1C rats. (a) Saline or AAVP was administered into the RVLM 10 min before the RVLM microinjection of salusin-b. (b) Intravenous infusion of saline or AAVP started 10 min before the RVLM microinjection of salusin-b. P < 0.05 vs. saline. Values are mean  SE. n ¼ 6 for each group. 2K1C, two-kidney, one-clip; AAVP, dTyr(CH2)5(Me)AVP; AVP, arginine vasopressin; HR, heart rate; MAP, mean arterial pressure; RSNA, renal sympathetic nerve activity; RVLM, rostral ventrolateral medulla; SE, standard error.

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Zhang et al.

AAVP failed to attenuate the effects of salusin-b in the RVLM. Microinjection of salusin-b or SIgG into the RVLM had no significant effect on AVP levels in the RVLM and plasma. These results indicate that circulating AVP or AVP in the RVLM is not involved in the sympatho-excitatory and pressor effects of salusin-b in the RVLM. Although the sympathoexcitatory and pressor effects of salusin-b in the RVLM are similar to the effects of salusin-b in the PVN, the mechanism is not the same. Salusin-b in the RVLM does not stimulate the AVP release from both RVLM and neurohypophysis, but activates the presympathetic neurons in the RVLM, which in turn increases RSNA, MAP and HR. However, salusin-b in the PVN not only increases the plasma AVP level which partially contributes to the pressor response, but also increases the AVP release from the RVLM via the projection from PVN to RVLM, which contributes to the sympathetic activation and its related pressor effect and increased HR. It has been found that elevated superoxide anion level in the RVLM in SHRs is associated with hypertension [30]. Enhanced oxidative stress within the RVLM systemically plays a major role in maintaining high ABP and sympathetic drive in hypertensive rats [31–33]. Reactive oxygen species (ROS) in the RVLM increased the sympatho-excitatory input to the RVLM from the PVN in SHRs [34]. PEG-SOD is widely used to scavenge the intracellular superoxide anions which shows a longer circulatory half-life than uncoupled SOD [35,36]. PEG-SOD can penetrate into cells rapidly, whereas SOD has limited cellular penetration [37]. Tempol is another kind of membrane-permeable superoxide anion scavenger [38]. Blood pressure reduction induced by chronic tempol treatment was related to attenuated sympathetic vasoconstriction [39]. We found that RVLM microinjection of PEG-SOD or tempol abolished the effects of salusin-b on the RSNA, MAP and HR in 2K1C rats, and salusin-b increased the oxide anion level in the RVLM in 2K1C rats. These results indicate that superoxide anions in the RVLM mediate the effects of salusin-b in the RVLM on the RSNA, MAP and HR in 2K1C rats, which was supported by the findings that the ROS production was increased in the RVLM in hypertensive rats, which enhanced sympathetic outflow, and thereby led to hypertension [40]. NAD(P)H oxidase is a multicomponent enzyme complex, and one of the major origins of the superoxide anions [41]. NAD(P)H oxidasederived superoxide was involved in the pressor effect of apelin-13 in the RVLM [42]. In the present study, RVLM microinjection of NAD(P)H oxidase inhibitor apocynin abolished the effects of salusin-b on the RSNA, MAP and HR, and salusin-b in the RVLM increased the NAD(P)H oxidase activity in 2K1C rats. These results suggest that NAD(P)H oxidase is a major source of salusin-b-induced superoxide anion production in the RVLM, which is supported by the finding that the mRNA expression of a NADPH oxidase family member Nox2 is increased by treatment with salusin-b in cultured human umbilical vein endothelial cells [13]. It is noted that angiotensin II potentiates the activity of RVLM neurons, whereas angiotensin II receptor blocker reduces RVLM activities [43]. A more recent study shows that AT1 receptor antagonist losartan reduces oxidative stress within the RVLM of 2K1C rats [21]. The possible interaction of salusin-b and renin–angiotensin system in the RVLM needs further investigation. 1066

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In summary, our study provides new information that salusin-b in the RVLM increases sympathetic outflow, blood pressure and HR, and endogenous salusin-b in the RVLM contributes to sympathetic activation and hypertension in renovascular hypertensive rats, but not in Sham rats. The numbers of salusin-b-like immunopositive neurons and salusin-b level in the RVLM areas were greatly increased in 2K1C rats compared with Sham rats. Furthermore, the effects of salusin-b in the RVLM are mediated by the NAD(P)H oxidase-derived superoxide anions in the RVLM, rather than by AVP and its V1 receptors in the RVLM and peripheral tissues. These findings provide new insights into the signaling mechanisms underlying increased sympathetic activity and blood pressure in hypertension.

ACKNOWLEDGEMENTS The work was supported by National Natural Science Foundation of China (81300194, 31171095 and 31271213) and National Basic Research Program of China (973 Program, No. 2012CB517805). The authors gratefully acknowledge the generous support of the Collaborative Innovation Center for Cardiovascular Disease Translational Medicine. G.-Q.Z. is currently receiving grants (31171095 and 31271213) from National Natural Science Foundation of China. F.Z. is currently receiving a grant (81300194) from National Natural Science Foundation of China. Y.-M.K. is currently receiving a grant (2012CB517805) from National Basic Research Program of China

Conflicts of interest There are no conflicts of interest.

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Reviewers’ Summary Evaluations Reviewer 1 The strengths of the study are the novelty, the appropriate methodological approach, the elegance of the protocols that are clear, and the conclusion of the study that is well supported by the data. The limitation is the lack of immunohistological identification of the rostral ventrolateral medulla neurons using a specific marker.

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Reviewer 2 The present study demonstrated that salusin-beta acts at the rostral ventrolateral medulla to activate sympathetic nervous system and to increase blood pressure in renovascular hypertensive rats. NAD(P)H oxidase-derived superoxide anions were considered to be involved in this activation of sympathetic nervous system by salusinbeta. Unfortunately, the detailed mechanisms involved in NAD(P)H oxidase-derived superoxide anions and salusinbeta, and interactions between brain renin–angiotensin system and salusin-beta have not been determined. www.jhypertension.com

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Salusin-β in rostral ventrolateral medulla increases sympathetic outflow and blood pressure via superoxide anions in hypertensive rats.

Rostral ventrolateral medulla (RVLM) plays crucial roles in central cardiovascular regulation. Salusin-β is a multifunctional bioactive peptide and is...
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