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Superoxide anions involved in sympathoexcitation and pressor effects of salusin-b in paraventricular nucleus in hypertensive rats H.-J. Sun,1 L.-L. Zhang,1 Z.-D. Fan,2 D. Chen,1 L. Zhang,1 X.-Y. Gao,1 Y.-M. Kang3 and G.-Q. Zhu1 1 Key Laboratory of Cardiovascular Disease and Molecular Intervention, Department of Physiology, Nanjing Medical University, Nanjing, China 2 Department of Rheumatology and Immunology, Nanjing Children’s Hospital Affiliated to Nanjing Medical University, Nanjing, China 3 Department of Physiology and Pathophysiology, Cardiovascular Research Center, Xi’an Jiaotong University School of Medicine, Xi’an, China
Received 24 June 2013, revision requested 18 July 2013, revision received 5 October 2013, accepted 23 October 2013 Correspondence: G.-Q. Zhu, MD, PhD, Key Laboratory of Cardiovascular Disease and Molecular Intervention, Department of Physiology, Nanjing Medical University, 140 Hanzhong Road, Nanjing 210029, China. E-mail: [email protected]
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Abstract Aims: Salusin-b in paraventricular nucleus (PVN) increases renal sympathetic nerve activity (RSNA), mean arterial pressure (MAP), heart rate (HR) and arginine vasopressin (AVP) release in hypertensive rats but not in normal rats. The present study was designed to investigate the downstream molecular mechanism of salusin-b in the PVN in hypertension. Method: Renovascular hypertension was induced by two-kidney, one-clip (2K1C) in male SD rats. Acute experiments were carried out 4 weeks after 2K1C or sham operation under anaesthesia. Results: MrgA1 mRNA expression and salusin-b level in the PVN as well as plasma salusin-b level were increased in 2K1C rats. Bilateral PVN microinjection of salusin-b increased the RSNA, MAP and HR in 2K1C rats, which were abolished by the pre-treatment with polyethylene glycol– superoxide dismutase (PEG-SOD), the superoxide anion scavenger tempol, the NAD(P)H oxidase inhibitor apocynin or the protein kinase C (PKC) inhibitor chelerythrine chloride (CLC), but not affected by the AT1 receptor antagonist losartan, the Mas receptor antagonist A-779, the NOS inhibitor L-NAME or the GABAA and GABAB receptor antagonists gabazine+CGP-35348. Salusin-b-induced increases in superoxide anion level and NAD(P)H oxidase activity in the PVN were abolished by the PVN pre-treatment with CLC. Salusin-b increased AVP levels in rostral ventrolateral medulla and plasma, which were prevented by the pre-treatment with PEG-SOD, apocynin or CLC in 2K1C rats. Salusin-b augmented the enhanced activity of PKC in the PVN in 2K1C rats. Conclusion: Protein kinase C-NAD(P)H oxidase-superoxide anions pathway in the PVN is involved in salusin-b-induced sympathetic activation, pressor response and AVP release in renovascular hypertension. Keywords hypertension, salusin, superoxide anions, sympathetic nerve activity, vasopressin.
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Salusins were originally identified from full-length human cDNAs by bioinformatics analyses, composing of two related bioactive peptides of 28 and 20 amino acids designated salusin-a and salusin-b (Shichiri et al. 2003). Salusins are translated from an alternatively spliced mRNA of TOR2A, a gene encoding a protein of the torsion dystonia family. The initial 18 amino acids of human salusin-b have high homology with the estimated N-terminal sequence of rat salusin (Suzuki et al. 2007). In the rat brain, the salusin-like immunopositive cells were distributed in suprachiasmatic, supraoptic and paraventricular nucleus (PVN) including both parvocellular and magnocellular neurones, and many salusin-positive nerve fibres and their terminals were identified in the internal layer of the median eminence and posterior pituitary. Most of the salusin-like immunoreactivity was detected in vasopressin, but not in oxytocin-containing neurones in these nuclei (Takenoya et al. 2005). Furthermore, salusin-b stimulated the arginine vasopressin (AVP) release from perfused rat pituitary (Shichiri et al. 2003). Paraventricular nucleus is an important integrative site in control of cardiovascular activity and sympathetic outflow (Pyner 2009). Sympathetic outflow was attenuated by the PVN lesion and augmented by the PVN activation in spontaneously hypertensive rats (SHR) (Takeda et al. 1991). AT1 receptors of angiotensin (Ang) II and Mas receptors of Ang-(1-7) in the PVN contributed to the sympathetic activation in renovascular hypertension (Chen et al. 2011, Sun et al. 2012). Artificial microRNA interference targeting AT1a receptors in the PVN attenuated hypertension in SHR (Fan et al. 2012). Superoxide anions in the PVN mediated the effects of Ang II in the PVN in renovascular hypertension (Han et al. 2011a). Overexpression of Cu/ZnSOD in the PVN attenuated hypertension and normalized the sympathetic activity in SHR. These results suggest that superoxide anions in the PVN are involved in sympathetic activation and hypertension. We recently found that microinjection of salusin-b into the PVN increased renal sympathetic nerve activity (RSNA), mean arterial pressure (MAP) and heart rate (HR), while anti-salusin-b IgG decreased RSNA and MAP in two-kidney, one-clip (2K1C)-induced hypertensive rats, but not in control rats. Intravenous injection of the AVP V1 receptor antagonist dTyr (CH2)5(Me)AVP (AAVP) decreased baseline RSNA and MAP and abolished the effects of salusin-b in 2K1C rats. Microinjection of AAVP into the rostral ventrolateral medulla (RVLM) abolished the effects of salusin-b in the PVN on RSNA and HR. Microinjection of salusin-b into the PVN increased plasma AVP level in 2K1C rats, but not in control rats. These results indicate that salusin-b in the PVN plays an important role in sympathetic activation, AVP release
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and hypertension in 2K1C rats (Chen et al. 2013). However, the downstream molecular mechanism of salusin-b is unknown. The present study was designed to determine whether superoxide anions are involved in the effects of salusin-b in the PVN on RSNA, MAP and AVP release in renovascular hypertensive rats. Furthermore, the mechanism of salusin-b-induced increase in superoxide anions is investigated.
Materials and methods The experiments were carried out in male rats. The study is conformed with Good Publishing Practice in Physiology (Persson & Henriksson 2011). The rats were housed in a temperature-controlled and humidity-controlled room with a 12-h/12-h light–dark cycle with standard chow and tap water ad libitum.
Hypertension animal models Renovascular hypertension was induced in Sprague– Dawley rat weighing 160–180 g with two-kidney oneclip (2K1C) method as we previously reported (Han et al. 2011b). Briefly, the rat was anaesthetized by pentobarbital sodium (60 mg kg 1) intraperitoneally. The adequacy of anaesthesia was evaluated by the loss of pedal withdrawal reflex. A right retroperitoneal flank incision was performed with sterile techniques. Right renal artery was exposed, and partly occluded with a U-shaped silver clip with an internal diameter of 0.20 mm. Sham-operated Sprague–Dawley rats (Sham) received similar surgical process except that the clip was not used. Thirteen-week-old male SHR and normotensive Wistar rats were purchased from Vital River Laboratory Animal Technology (Beijing, China). Systolic arterial pressure (SBP) of tail artery was measured weekly in a conscious state with a noninvasive computerized tail-cuff system (NIBP, ADInstruments, Sydney, Australia) as we previously reported (Xiong et al. 2012). The criterion of hypertension was set at systolic arterial pressure (SBP) ≥ 160 mmHg in a conscious state.
General procedures of acute experiments Acute experiments were carried out at the end of the 4th week after surgery. Rats were anesthetized with urethane (800 mg kg 1) and a-chloralose (40 mg kg 1) intraperitoneally. Supplemental doses of anaesthesia were used during the experiment to maintain an appropriate level of anaesthesia, which was assessed by the absence of corneal reflexes and paw withdrawal response to a noxious pinch. A midline incision in the neck was made to expose the trachea and carotid artery. The trachea was intubated for mechanical
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ventilation. RSNA, MAP and HR were simultaneously recorded with a PowerLab data acquisition system (8/35, ADInstruments, Castle Hill, Australia).
PVN microinjection The PVN stereotaxic coordinates were 1.8 mm caudal from bregma, 0.4 mm lateral to the midline and 7.9 mm ventral to the dorsal surface (Shi et al. 2011). The bilateral PVN microinjections were carried out with two glass micropipettes (about 50 lm tip diameter) and completed within 1 min. Microinjection volume was 50 nL for each microinjection site. At the end of the experiment, same volume of Evans Blue (2%) was injected into the microinjection site for histological identification. Rats with microinjection sites outside the PVN or at the margin of the PVN were excluded from data analysis (Fig. 1).
RSNA recording Left renal sympathetic nerve was isolated through a retroperitoneal incision and was cut distally to eliminate its afferent activity. The nerve was immersed in warm mineral oil and placed on a pair of silver electrodes. The RSNA was amplified with a four channel AC/DC differential amplifier (DP-304, Warner Instruments, Hamden, CT, USA) with a high pass filter at 100 Hz and a low pass filter at 3000 Hz. The RSNA was integrated at a time constant of 100 ms. At the end of each experiment, background noise was determined after section of the central end of the nerve and was subtracted from the integrated values of the RSNA (Cui et al. 2013).
In situ detection of superoxide anions Specific fluorogenic probe dihydroethidium (DHE) was used to detect in situ superoxide anions in the PVN as we previously reported (Shi et al. 2009). Simply, the DHE fluorescence in the coronal sections was visualized under a fluorescence microscope (BX51, Olympus, Tokyo, Japan). Images were collected by using an Olympus BX51 microscope coupled with an
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Olympus DP70 digital camera at 9100. 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 from 2K1C or sham rats was analysed and quantified with IMAGE-PRO PLUS 6.0 by using the same parameters.
Measurement of superoxide anions, NAD(P)H oxidase activity and PKC activity Brain was removed and flash-frozen with liquid nitrogen and stored at 70 °C until being sectioned. Coronal sections of the brain were made with a cryostat microtome (CM1900, Leica, Nussloch, Germany). A 450-lm-thick coronal section was cut through the hypothalamus and incorporated the PVN area, and then, the PVN 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, CA, USA). Superoxide anion level and NAD(P)H oxidase activity in the PVN were measured with lucigenin-derived chemiluminescence method as we previously reported (Shi et al. 2009, Han et al. 2011a, Rosc-Schluter et al. 2012). 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, USA). The values were averaged and expressed as mean light unit (MLU) per minute per milligram of protein. Protein kinase C activity in the supernatants was measured with a solid-phase enzyme-linked immunoabsorbent assay kit (Enzo Life Sciences, Ann Arbor, Michigan, USA). A specific synthetic peptide was used as a substrate of PKC, and a polyclonal antibody that recognizes the phosphorylated form of the substrate was used in a solution phase. The substrate pre-coated on the wells of PKC substrate microtiter plate was phosphorylated by PKC, followed by adding ATP into
Figure 1 Schematic representations of serial sections from the rostral ( 1.44 mm) to the caudal ( 2.16 mm) extent of the region of the PVN. Dots or open circles represent the sites of termination of the microinjections. Dots are considered to be within the PVN. Open circles are considered to be outside of the PVN or at the margin of the PVN, which were excluded for data analysis. 3V, third ventricle.
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the wells to initiate the kinase reaction and incubating for 90 min at 30 °C, the reactions were terminated by emptying the contents of each well and the phosphospecific substrate antibody bounded specifically to the phosphorylated peptide substrate was added into wells and subsequently bound by a peroxidase conjugated secondary antibody. The colour development was initiated with tetramethylbenzidine substrate (TMB) in proportion to PKC phosphotransferase activity and stopped with acid stop solution. The intensity of the colour was measured in a microplate reader (ELX800, BioTek, Vermont, USA) at 450 nm (Triantafyllou et al. 2011, Chang et al. 2012, Ji et al. 2012). Measurement of AVP and salusin-b. Commercial ELISA kits were used for the measurement of AVP (Enzo Life Sciences, Ann Arbor, MI, USA) or salusin-b (Uscn Life Science, Houston, TX, USA). According to the manufacturer’s descriptions, the standards or samples diluent were added and incubated in the appropriate well of specific antibody pre-coated microtiter plate. Conjugate was added and incubated for 1 h at 37 °C and then washed. The reactions were stopped with stop solution and read at 405 nm for the AVP measurements or 450 nm for the salusin-b measurements using a microtiter plate reader (ELX800, BioTek, Vermont, USA) (Vega et al. 2010). Measurement of MrgA1 mRNA with Real-time PCR. The mRNA expression of Mas-like G proteincoupled receptors (Mas-related gene A1, MrgA1) in the PVN was measured using a fluorescence quantitative PCR system (Roche, Basel, Sweden). Simply, total RNA was extracted by TRIzol (Ambion, Austen, Texas, USA) and evaluated with SYBR Green I fluorescence. The rat forward primer and reverse primer of MrgA1 are 5′-AAGAGGAATGGGGGAAAGCA-3′ and 5′-AAGGCGTTCCTGTGCAAACA-3′, respectively. The rat forward primer and reverse primer of b-actin are 5′-ATGTGGATCAGCAAGCAGGA-3′ and 5′-AAGGGTGTAAAACGCAGCTCA-3′, respectively. After being denatured at 94 °C for 5 min, the solution underwent PCR for MrgA1 and b-actin at 94 °C for 20 s, 62 °C for 30 s and 72 °C for 45 s for 45 cycles. All processes were performed in triplicate, and the average cycle thresholds (Ct) were used to determine fold-change. The relative quantification of gene expression was reported as a relative quantity to the control value.
Experimental design We recently found that microinjection of salusin-b into the PVN increased RSNA, MAP and HR in a dose-related manner in 2K1C rats, but did not cause
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any significant changes in Sham rats (Chen et al. 2013). According to the findings, a dose of salusin-b (100 pmol) was used in the present study. Most experiments were carried out only in 2K1C and/or Sham rats except that the Experiment 8 was carried out in normotensive Wistar rats and SHR.
Chemicals Tempol, NAD(P)H, polyethylene glycol-superoxide dismutase (PEG-SOD), apocynin, dimethyl sulfoxide (DMSO), losartan, lucigenin, NG-nitro-L-arginine methyl ester (L-NAME), gabazine and CGP-35348 were obtained from Sigma Chemical (St. Louis, MO, USA). Salusin-b, D-Alanine-Ang-(1-7) (A-779) and chelerythrine chloride (CLC) were obtained from Phoenix Pharmaceuticals (CA, USA), Bachem (Bubendorf, Switzerland) and Tocris Bioscience (Bristol, United Kingdom), respectively. Apocynin and CLC were dissolved in normal saline containing 1% of DMSO. All other chemicals were dissolved in normal saline.
Experimental design Experiment 1, pre-treatment with microinjection of saline or three different doses of PEG-SOD (0.2, 1 and 5 units) into the PVN was randomly carried out in 2K1C rats (n = 6). Then, the RSNA, MAP and HR responses to the microinjection of salusin-b into the PVN (100 pmol) were determined 8 min after the pretreatment. The intervals between injections were at least 60 min for a complete recovery. Experiment 2, pre-treatment with microinjection of saline, the superoxide anion scavenger tempol (20 nmol), the AT1 receptor antagonist losartan (50 nmol), the Mas receptor antagonist A-779 (3 nmol), the non-specific NOS inhibitor L-NAME (200 nmol) or the GABAA receptor antagonist gabazine (0.1 nmol) plus GABAB receptor antagonist CGP35348 (10 nmol) into the PVN was carried out in 2K1C rats. Then, the RSNA, MAP and HR responses to the microinjection of salusin-b (100 pmol) into the PVN were determined 8 min after the pre-treatment (n = 6 for each group). Experiment 3, pre-treatment with the microinjection of DMSO (1%), the NAD(P)H oxidase inhibitor apocynin (1 nmol) or the protein kinase C (PKC) inhibitor CLC (5 nmol) into the PVN was carried out in 2K1C rats. Then, the RSNA, MAP and HR responses to the microinjection of salusin-b (100 pmol) into the PVN were determined 8 min after the pre-treatment (n = 6 for each group). Experiment 4, microinjection of saline or salusin-b (100 pmol) into the PVN was carried out in Sham or 2K1C rats for in situ detection of superoxide anions
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with specific fluorogenic probe DHE (n = 4 for each group). The rats were killed for the detection of superoxide anions in the PVN 10 min after the microinjection. Experiment 5, effects of microinjection of saline or salusin-b (100 pmol) into the PVN on the superoxide anion level, NAD(P)H oxidase activity and PKC activity in the PVN were determined in Sham or 2K1C rats (n = 6 for each group). The rats were killed for the measurements 10 min after the microinjection. Experiment 6, microinjection of saline or salusin-b (100 pmol) into the PVN was carried out 8 min after the pre-treatment with saline, PEG-SOD (5 units), DMSO, apocynin (1 nmol) or CLC (5 nmol) in 2K1C rats (n = 6 for each group). The rats were killed for the measurement of superoxide anion level and NAD (P)H oxidase activity in the PVN, AVP levels in the RVLM and plasma 10 min after microinjection of salusin-b. Experiment 7, MrgA1 mRNA expression and salusin-b level in the PVN as well as plasma salusin-b level were determined in Sham rats and 2K1C rats (n = 6 for each group). Experiment 8, the effects of PVN microinjection of saline or salusin-b (100 pmol) on the RSNA, MAP and HR were determined in Wistar rats and SHR (n = 6 for each group).
Statistical analysis Comparisons between two groups were made by Student’s t test. ANOVA followed by post hoc Bonferroni test was used when multiple comparisons were made. All data were expressed as mean SE. A value of P < 0.05 was considered statistically significant.
Table 1 Body weight, SBP, baseline MAP and baseline HR at the end of the fourth week Variables
Sham
n Body weight, g SBP, mm Hg Baseline MAP, mm Hg Baseline HR, bmp
26 346 126 92 348
2K1C
5 2 3 9
149 339 191 136 360
2 2* 7* 4
Systolic blood pressure (SBP) of tail artery was measured in conscious state by use of a non-invasive computerized tailcuff system. MAP and HR were measured under anaesthesia with a pressure transducer through a catheter placed in right carotid artery. Values are mean SE. *P < 0.05 compared with the Sham.
Results General data There was no significant difference in body weight or HR between Sham and 2K1C rats at the end of the fourth week. However, both SBP and MAP in 2K1C rats were significantly higher than those in Sham rats (Table 1).
Effects of different doses of PEG-SOD Microinjection of the superoxide anion scavenger PEG-SOD into the PVN decreased the RSNA and MAP, while salusin-b increased RSNA, MAP and HR in 2K1C rats (Table 2). Pre-treatment with PEG-SOD attenuated the effects of salusin-b on the RSNA, MAP and HR in 2K1C rats, and high dose of PEG-SOD almost abolished the effects of salusin-b (Fig. 2).
Table 2 Changes of baseline RSNA, MAP and HR Groups
RSNA (%)
Saline PEG-SOD, 0.2 units PEG-SOD, 1 units PEG-SOD, 5 units Tempol, 20 nmol Losartan, 50 nmol A-779, 3 nmol L-NAME, 200 nmol Gabazine, 0.1 nmol + CGP-35348, 10 nmol 1% DMSO Apocynin, 1 nmol CLC, 5 nmol
0.3 2.6 7.6 9.0 8.3 10.7 13.7 15.7 19.2 0.5 8.1 5.6
1.9 2.2 2.7* 2.3* 2.6* 2.2* 2.3* 3.1* 4.8* 2.3 3.0* 2.0*
MAP (mm Hg) 0.2 2.2 6.0 6.2 6.5 7.8 7.0 5.6 15.2 0.5 6.2 7.7
1.5 0.8 1.4* 1.1* 1.7* 1.6* 1.5* 1.2* 3.1* 1.5 2.3* 2.2*
HR (bpm) 0.8 1.7 3.3 3.5 4.0 1.8 1.4 0.2 0.8 0.7 0.3 0.2
1.4 1.1 1.4 1.6 1.1 1.9 2.4 2.1 1.4 1.8 1.8 2.4
Values are mean SE. *P < 0.05 vs. Saline or DMSO.
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(a)
(b)
Figure 2 Effects of PVN pre-treatment with saline, three doses of PEG-SOD, superoxide anion scavenger tempol, AT1 receptor antagonist losartan, Mas receptor antagonist A-779, NOS inhibitor L-NAME or GABAA receptor antagonist gabazine plus GABAB receptor antagonist CGP-35348 on the RSNA, MAP and HR responses to the microinjection of salusin-b into the PVN in 2K1C rats. Representative tracings in the lower panel showing that pre-treatment with microinjection of PEG-SOD (5 units) into the PVN abolished the RSNA, MAP and HR responses to salusin-b. Values are mean SE. *P < 0.05 vs. Saline+Saline. †P < 0.05 vs. Saline + Salusin-b. n = 6 for each group.
Effects of tempol, losartan, A-779, L-NAME and gabazine plus CGP-35348. Microinjection of the superoxide anion scavenger tempol, the AT1 receptor antagonist losartan or the Mas receptor antagonist A-779 into the PVN decreased RSNA and MAP, while the non-specific NOS inhibitor L-NAME or the GABAA receptor antagonist gabazine plus the GABAB receptor antagonist CGP-35348 decreased RSNA and MAP in 2K1C rats (Table 2). Pre-treatment with tempol almost abolished the effects of salusin-b in 2K1C rats, but pre-treatment with losartan, A-779, L-NAME or gabazine plus CGP-35348 had no
significant effect on the RSNA, MAP and HR responses to salusin-b in 2K1C rats (Fig. 2).
Effects of apocynin and CLC Microinjection of the NAD(P)H oxidase inhibitor apocynin or the PKC inhibitor CLC into the PVN decreased baseline RSNA and MAP in 2K1C rats (Table 2). Pre-treatment with apocynin or CLC almost abolished the effects of salusin-b on the RSNA, MAP and HR in 2K1C rats (Fig. 3).
Figure 3 Effects of PVN pre-treatment with the NAD(P)H oxidase inhibitor apocynin, or the PKC inhibitor chelerythrine chloride (CLC) on the RSNA, MAP and HR responses to microinjection of salusin-b into the PVN in 2K1C rats. Values are mean SE. *P < 0.05 vs. DMSO+Saline. †P < 0.05 vs. DMSO+Salusin-b. n = 6 for each group. © 2013 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.12188
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(a)
(b)
(c)
Figure 4 Superoxide anion level and NAD(P)H oxidase activity in the PVN in Sham and 2K1C rats. (a) representative photos showing the effects of microinjection of saline or salusin-b into the PVN on the superoxide anion levels in the PVN, indicated by DHE fluorescence intensity at the levels of 1.44, 1.80 and 2.16 mm caudal from bregma.3V, third ventricle; (b) effects of microinjection of saline or salusin-b into the PVN on the DHE fluorescence intensity in the PVN; (c) effects of microinjection of saline or salusin-b on superoxide anion level and NAD(P)H oxidase activity in the PVN measured with lucigenin-derived chemiluminescence method. Values are mean SE. *P < 0.05 vs. Saline. †P < 0.05 vs. Sham. n = 6 for each group.
Superoxide anions and NAD(P)H oxidase activity Superoxide anions detected with fluorogenic probe DHE were increased in the PVN in 2K1C rats, and PVN microinjection of salusin-b further increased the superoxide anions in the PVN in 2K1C rats, but not in Sham rats. The most obvious change was observed at the level of 1.80 mm caudal from bregma (Fig. 4a, b). Both superoxide anion level and NAD(P)H oxidase activity in the PVN measured with a chemiluminescence method were higher in 2K1C rats than those in (a)
Sham rats. Microinjection of salusin-b into the PVN increased the superoxide anion level and NAD(P)H oxidase activity in 2K1C rats but not in Sham rats (Fig. 4c).
PKC activity Protein kinase C activity in the PVN was enhanced in 2K1C rats, and microinjection of salusin-b into the PVN further enhanced the PKC activity in 2K1C rats, but not in Sham rats (Fig. 5a).
(b)
Figure 5 PKC activity in the PVN and effects of PKC inhibitor CLC. (a) effects of PVN microinjection of saline or salusin-b on PKC activity in the PVN in Sham and 2K1C rats. *P < 0.05 vs. Saline; †P < 0.05 vs. Sham. (b) effects of PVN pre-treatment with DMSO or CLC on the superoxide anion level and NAD(P)H oxidase activity responses to the PVN microinjection of salusin-b in the PVN in 2K1C rats. Values are mean SE. *P < 0.05 vs. Saline; †P < 0.05 vs. Sham. ‡P < 0.05 vs. DMSO+Salusin-b. n = 6 for each group.
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Discussion
Figure 6 Effects of pre-treatment with microinjection of saline, PEG-SOD, DMSO, apocynin or CLC on the AVP response to the microinjection of salusin-b into the PVN in 2K1C rats. Values are mean SE. *P < 0.05 vs. Saline+ Saline; †P < 0.05 vs. Saline+Salusin-b; ‡P < 0.05 vs. DMSO+Salusin-b. n = 6 for each group.
Effects of CLC on the responses of superoxide anions and NAD(P)H oxidase activity to salusin-b After microinjection of salusin-b into the PVN, the superoxide anion level and NAD(P)H oxidase activity in the PVN were lower in the rats pre-treated with PKC inhibitor than those rats pre-treated with 1% of DMSO (Fig. 5b).
RVLM and Plasma AVP levels Microinjection of salusin-b into the PVN increased both RVLM and plasma AVP levels in 2K1C rats, which were prevented by PEG-SOD, apocynin or CLC (Fig. 6).
Salusin-b levels and MrgA1 mRNA expression Salusin-b levels in both plasma and PVN, as well as the MrgA1 mRNA expression in the PVN were increased in 2K1C rats (Fig. 7).
Effects of Salusin-b in SHR Microinjection of salusin-b into the PVN increased RSNA, MAP and HR in SHR, but not in normotensive Wistar rats (Fig. 8).
We recently found that exogenous salusin-b in the PVN increased RSNA, MAP, HR via both circulating AVP and AVP in the RVLM in 2K1C rats, but not in Sham rats (Chen et al. 2013). Similarly, microinjection of salusin-b into the PVN increased RSNA, MAP and HR in SHR, but not in normotensive Wistar rats. The primary new findings in the present study were that the PKC-NAD(P)H oxidase-superoxide anions pathway in the PVN was involved in the effects of salusin-b on RSNA, MAP, HR and AVP release in 2K1C-induced renovascular hypertensive rats. PEG-SOD shows a longer circulatory half-life than native SOD and is widely used to scavenge the intracellular superoxide anions. It binds to cell membranes and rapidly penetrates into cells, whereas SOD has limited cellular penetration (Beckman et al. 1988). Tempol is another kind of membrane-permeable superoxide anion scavenger (Yamada et al. 2003). Blood pressure reduction induced by chronic tempol treatment was related to attenuated sympathetic vasoconstriction (Vaneckova et al. 2013). In the present study, PEG-SOD or tempol almost abolished the effects of salusin-b. Superoxide anion level was increased in 2K1C rats, which was further increased by microinjection of salusin-b into the PVN. These results indicate that superoxide anions in the PVN are required for the effects of salusin-b on the RSNA, MAP and HR in 2K1C rats. It is known that Ang II is involved in hypertension (Grisk 2011). NAD(P)H oxidase is a multi-component enzyme complex and one of major origins of the superoxide anions (Cai et al. 2003). NAD(P)H oxidase-derived superoxide anions in the PVN mediated the angiotensin II-induced sympathetic activation and pressor effects in 2K1C rats (Han et al. 2011a). Oxidant signalling in the PVN contributed to the pathogenesis of renovascular hypertension (Burmeister et al. 2011). In the present study, both NAD(P)H oxidase activity and superoxide anion level in the PVN were higher in 2K1C rats than those in Sham rats, and salusin-b further increased the NAD(P)H oxidase activity and
Figure 7 Salusin-b levels in plasma and PVN, and MrgA1 mRNA expression in the PVN in Sham rats and 2K1C rats. Values are mean SE. *P < 0.05 vs. Sham. n = 6 for each group. © 2013 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.12188
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Figure 8 Effects of microinjection of saline or salusin-b into the PVN on the RSNA, MAP and HR in Wistar rats and SHR. Values are mean SE. *P < 0.05 vs. Saline; †P < 0.05 vs. Wistar. n = 6 for each group.
superoxide anion level in 2K1C rats rather than in Sham rats. The PVN microinjection of NAD(P)H oxidase inhibitor apocynin almost abolished the effects of salusin-b on the RSNA, MAP and HR in 2K1C rats. These results suggest that NAD(P)H oxidase is a major source of salusin-b-induced superoxide anion production in the PVN, which is supported by the finding that the mRNA expression of NADPH oxidase family member Nox2 was increased by treatment with salusin-b in cultured human umbilical vein endothelial cells (HUVECs) (Koya et al. 2012). It has been found that PKC is involved in the salusin-b-induced improvement in calcium uptake and protein synthesis in neonatal rat cardiomyocytes (Yu et al. 2004). We found that PKC activity was higher in 2K1C rats than that in Sham rats, and salusin-b further increased PKC activity in 2K1C rats but not in Sham rats. Microinjection of the PKC inhibitor CLC into the PVN not only almost abolished the effects of salusin-b on the RSNA, MAP and HR, but also prevented salusin-b-induced increases in NAD(P)H oxidase activity and superoxide anion level in 2K1C rats. These results suggest that the NAD(P)H oxidasederived superoxide anions are dependent upon the activation of PKC. It was found that salusin-b increased intracellular Ca2+ in the rat vascular smooth muscle cells and fibroblasts in a concentration-dependent manner, which were attenuated by pre-treatment with calcium channel antagonist nicardipine and were nearly abolished by calcium chelator EGTA (Shichiri et al. 2003). Salusin-b increased intracellular Ca2+ in freshly isolated single nerve terminals from the neurohypophysis devoid of pars intermedia, which were blocked by high-voltage-activated Ca2+ channel blockers (Saito et al. 2008). Ca2+ is one of critical factors for the activation of PKC (Tanaka & Saito 1992). PKC is involved in the intracellular regulation of redox balance and oxidative stress levels (De et al. 2013). These results suggest that increased intracellular Ca2+ may be a possible mechanism of salusin-binduced PKC activation. On the other hand, the present study showed that Ang II, Ang-(1-7), NO and GABA were not involved in the effects of salusin-b. 542
Arginine vasopressin is primarily synthesized in PVN and supraoptic nuclei and secreted from neurohypophysis to the systemic circulation, which causes powerful constriction in a variety of vascular regions via AVP V1 receptors (Noguera et al. 1997). Salusin-b, not salusin-a, stimulated the AVP release from peri-fused rat pituitary (Shichiri et al. 2003). Salusin-b-like immunopositive cells were found in the parvocellular and magnocellular part of the PVN, especially in those AVP-containing neurones (Takenoya et al. 2005). The neurones in the magnocellular part of PVN project to the neurohypophysis and contribute to the production of posterior pituitary hormones including AVP; the neurones in the parvocellular part of PVN project to RVLM and spinal cord and contribute to the sympathetic activation (Pyner 2009). AVP-immunoreactive fibres were found in the RVLM (Gomez et al. 1993). After microinjection of retrograde tracer cholera toxin b subunit (CT-b) into the RVLM, 14.6% of CT-b-labelled PVN neurones were double-labelled with AVP, and most of labelled neurones were in the regions of the PVN that are involved in autonomic control (Kc et al. 2010). Microinjection of AVP into the RVLM increased blood pressure (Gomez et al. 1993). Blockade of V1a receptors of AVP in the RVLM attenuated the PVN-evoked increases in RVLM unit discharge, and AVP acted like glutamate as an excitatory neurotransmitter on RVLM neurones (Yang et al. 2001, Yang & Coote 2003). Chronic intermittent hypoxia (CIH) up-regulated AVPV1a receptor signalling in the RVLM, thus increasing blood pressure (Kc et al. 2010). These results indicate that AVP neurones in the PVN project to the RVLM, which are involved in regulating blood pressure. Microinjection of salusin-b into the PVN increased the plasma AVP level in 2K1C rats, but not in Sham rats. Intravenous injection of AVP V1 receptor antagonist decreased RSNA and MAP and abolished the effects of salusin-b in 2K1C rats. Microinjection of AAVP into RVLM, but not into the PVN, abolished the effects of salusin-b on RSNA and HR. These results suggest that the effects of salusin-b in the PVN on RSNA, MAP and HR are mediated by both circulating AVP and AVP in the RVLM in 2K1C rats
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(Chen et al. 2013). In the present study, microinjection of salusin-b into the PVN increased plasma AVP level in 2K1C rats, which was consist with our previous findings (Chen et al. 2013). Pre-treatment with PEG-SOD, apocynin or CLC prevented the salusin-b-induced increases in both RVLM and plasma AVP levels in 2K1C rats. These results suggest that PKC, NAD(P)H oxidase-originated superoxide anions in the PVN are necessary for the AVP release from neurohypophysis, which contribute to the increased plasma AVP level and blood pressure, and from RVLM which may contribute to the sympathetic activation in 2K1C rats. The latter is supported by the finding that AVP may act as excitatory neurotransmitters at synapses of paraventricular neurones on the RVLM vasomotor neurones (Yang et al. 2001). It is very interesting that microinjection of salusin-b into the PVN increased sympathetic activity, MAP, HR and AVP release, and neutralization endogenous salusin-b with anti-salusin-b IgG caused opposite effects in 2K1C rats, but not in Sham rats (Chen et al. 2013). Similarly, salusin-b in the PVN elicited sympathoexcitation and pressor effects in SHR, but not in normotensive Wistar rats. These results suggest that salusin-b in the PVN may not be involved in the tonic control of sympathetic outflow, blood pressure and AVP release in physiological state, but contributes to the pathogenesis of hypertension. There is a possibility that salusin-b may contribute to the maintenance of blood pressure via sympathetic activation and AVP release in haemorrhage or other hypotensive states. We are interested to test the hypothesis in the rat model of hypotension in the near future study. MrgA1 have been shown to bind a wide variety of ligands from peptides to amino acid derivatives. Human salusin-b can activate the mouse orphan G protein-coupled receptor, mMrgA1, and may be a surrogate ligand of the MrgA1 (Wang et al. 2006). We found that salusin-b level and MrgA1 mRNA expression in the PVN were greatly increased in 2K1C rats compared with Sham rats, which may be responsible for the involvement of salusin-b in the PVN in the pathogeneses of hypertension in 2K1C rats. It is noted that intravenous administration of salusin-b caused a temporary rapid decrease in aortic blood flow concomitantly with hypotension and bradycardia without affecting systemic vascular resistance by facilitating vagal outflows to the heart and its negative inotropic and chronotropic actions on the heart (Izumiyama et al. 2005). The greatly increased plasma salusin-b level in 2K1C rats may be a compensatory mechanism for attenuating hypertension. A limitation in the present study was that the experiments were performed in acute experiments with anaesthesia. The results may not reflect the actual situation in conscious rats.
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In conclusion, salusin-b in the PVN activates PKC, which in turn activates NAD(P)H oxidase in 2K1C rats. The enhanced NAD(P)H oxidase activity contributes to the increase in superoxide anions in the PVN. The higher superoxide anion level in the PVN increases the AVP levels in the plasma and the RVLM, which are responsible for sympathetic activation and hypertension.
Conflict of interest The authors declared no conflict of interest. This work was supported by National Natural Science Foundation of China (31171095 & 31271213), 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.
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medulla of the hypertensive rat. Auton Neurosci 104, 83– 87. Yang, Z., Bertram, D. & Coote, J.H. 2001. The role of glutamate and vasopressin in the excitation of RVL neurones by paraventricular neurones. Brain Res 908, 99– 103. Yu, F., Zhao, J., Yang, J., Gen, B., Wang, S., Feng, X., Tang, C. & Chang, L. 2004. Salusins promote cardiomyocyte growth but does not affect cardiac function in rats. Regul Pept 122, 191–197.
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Superoxide anions involved in sympathoexcitation and pressor effects of salusin-b in paraventricular nucleus in hypertensive rats H.-J. Sun,1 L.-L. Zhang,1 Z.-D. Fan,2 D. Chen,1 L. Zhang,1 X.-Y. Gao,1 Y.-M. Kang3 and G.-Q. Zhu1 1 Key Laboratory of Cardiovascular Disease and Molecular Intervention, Department of Physiology, Nanjing Medical University, Nanjing, China 2 Department of Rheumatology and Immunology, Nanjing Children’s Hospital Affiliated to Nanjing Medical University, Nanjing, China 3 Department of Physiology and Pathophysiology, Cardiovascular Research Center, Xi’an Jiaotong University School of Medicine, Xi’an, China
Received 24 June 2013, revision requested 18 July 2013, revision received 5 October 2013, accepted 23 October 2013 Correspondence: G.-Q. Zhu, MD, PhD, Key Laboratory of Cardiovascular Disease and Molecular Intervention, Department of Physiology, Nanjing Medical University, 140 Hanzhong Road, Nanjing 210029, China. E-mail: [email protected]
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Abstract Aims: Salusin-b in paraventricular nucleus (PVN) increases renal sympathetic nerve activity (RSNA), mean arterial pressure (MAP), heart rate (HR) and arginine vasopressin (AVP) release in hypertensive rats but not in normal rats. The present study was designed to investigate the downstream molecular mechanism of salusin-b in the PVN in hypertension. Method: Renovascular hypertension was induced by two-kidney, one-clip (2K1C) in male SD rats. Acute experiments were carried out 4 weeks after 2K1C or sham operation under anaesthesia. Results: MrgA1 mRNA expression and salusin-b level in the PVN as well as plasma salusin-b level were increased in 2K1C rats. Bilateral PVN microinjection of salusin-b increased the RSNA, MAP and HR in 2K1C rats, which were abolished by the pre-treatment with polyethylene glycol– superoxide dismutase (PEG-SOD), the superoxide anion scavenger tempol, the NAD(P)H oxidase inhibitor apocynin or the protein kinase C (PKC) inhibitor chelerythrine chloride (CLC), but not affected by the AT1 receptor antagonist losartan, the Mas receptor antagonist A-779, the NOS inhibitor L-NAME or the GABAA and GABAB receptor antagonists gabazine+CGP-35348. Salusin-b-induced increases in superoxide anion level and NAD(P)H oxidase activity in the PVN were abolished by the PVN pre-treatment with CLC. Salusin-b increased AVP levels in rostral ventrolateral medulla and plasma, which were prevented by the pre-treatment with PEG-SOD, apocynin or CLC in 2K1C rats. Salusin-b augmented the enhanced activity of PKC in the PVN in 2K1C rats. Conclusion: Protein kinase C-NAD(P)H oxidase-superoxide anions pathway in the PVN is involved in salusin-b-induced sympathetic activation, pressor response and AVP release in renovascular hypertension. Keywords hypertension, salusin, superoxide anions, sympathetic nerve activity, vasopressin.
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Salusins were originally identified from full-length human cDNAs by bioinformatics analyses, composing of two related bioactive peptides of 28 and 20 amino acids designated salusin-a and salusin-b (Shichiri et al. 2003). Salusins are translated from an alternatively spliced mRNA of TOR2A, a gene encoding a protein of the torsion dystonia family. The initial 18 amino acids of human salusin-b have high homology with the estimated N-terminal sequence of rat salusin (Suzuki et al. 2007). In the rat brain, the salusin-like immunopositive cells were distributed in suprachiasmatic, supraoptic and paraventricular nucleus (PVN) including both parvocellular and magnocellular neurones, and many salusin-positive nerve fibres and their terminals were identified in the internal layer of the median eminence and posterior pituitary. Most of the salusin-like immunoreactivity was detected in vasopressin, but not in oxytocin-containing neurones in these nuclei (Takenoya et al. 2005). Furthermore, salusin-b stimulated the arginine vasopressin (AVP) release from perfused rat pituitary (Shichiri et al. 2003). Paraventricular nucleus is an important integrative site in control of cardiovascular activity and sympathetic outflow (Pyner 2009). Sympathetic outflow was attenuated by the PVN lesion and augmented by the PVN activation in spontaneously hypertensive rats (SHR) (Takeda et al. 1991). AT1 receptors of angiotensin (Ang) II and Mas receptors of Ang-(1-7) in the PVN contributed to the sympathetic activation in renovascular hypertension (Chen et al. 2011, Sun et al. 2012). Artificial microRNA interference targeting AT1a receptors in the PVN attenuated hypertension in SHR (Fan et al. 2012). Superoxide anions in the PVN mediated the effects of Ang II in the PVN in renovascular hypertension (Han et al. 2011a). Overexpression of Cu/ZnSOD in the PVN attenuated hypertension and normalized the sympathetic activity in SHR. These results suggest that superoxide anions in the PVN are involved in sympathetic activation and hypertension. We recently found that microinjection of salusin-b into the PVN increased renal sympathetic nerve activity (RSNA), mean arterial pressure (MAP) and heart rate (HR), while anti-salusin-b IgG decreased RSNA and MAP in two-kidney, one-clip (2K1C)-induced hypertensive rats, but not in control rats. Intravenous injection of the AVP V1 receptor antagonist dTyr (CH2)5(Me)AVP (AAVP) decreased baseline RSNA and MAP and abolished the effects of salusin-b in 2K1C rats. Microinjection of AAVP into the rostral ventrolateral medulla (RVLM) abolished the effects of salusin-b in the PVN on RSNA and HR. Microinjection of salusin-b into the PVN increased plasma AVP level in 2K1C rats, but not in control rats. These results indicate that salusin-b in the PVN plays an important role in sympathetic activation, AVP release
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and hypertension in 2K1C rats (Chen et al. 2013). However, the downstream molecular mechanism of salusin-b is unknown. The present study was designed to determine whether superoxide anions are involved in the effects of salusin-b in the PVN on RSNA, MAP and AVP release in renovascular hypertensive rats. Furthermore, the mechanism of salusin-b-induced increase in superoxide anions is investigated.
Materials and methods The experiments were carried out in male rats. The study is conformed with Good Publishing Practice in Physiology (Persson & Henriksson 2011). The rats were housed in a temperature-controlled and humidity-controlled room with a 12-h/12-h light–dark cycle with standard chow and tap water ad libitum.
Hypertension animal models Renovascular hypertension was induced in Sprague– Dawley rat weighing 160–180 g with two-kidney oneclip (2K1C) method as we previously reported (Han et al. 2011b). Briefly, the rat was anaesthetized by pentobarbital sodium (60 mg kg 1) intraperitoneally. The adequacy of anaesthesia was evaluated by the loss of pedal withdrawal reflex. A right retroperitoneal flank incision was performed with sterile techniques. Right renal artery was exposed, and partly occluded with a U-shaped silver clip with an internal diameter of 0.20 mm. Sham-operated Sprague–Dawley rats (Sham) received similar surgical process except that the clip was not used. Thirteen-week-old male SHR and normotensive Wistar rats were purchased from Vital River Laboratory Animal Technology (Beijing, China). Systolic arterial pressure (SBP) of tail artery was measured weekly in a conscious state with a noninvasive computerized tail-cuff system (NIBP, ADInstruments, Sydney, Australia) as we previously reported (Xiong et al. 2012). The criterion of hypertension was set at systolic arterial pressure (SBP) ≥ 160 mmHg in a conscious state.
General procedures of acute experiments Acute experiments were carried out at the end of the 4th week after surgery. Rats were anesthetized with urethane (800 mg kg 1) and a-chloralose (40 mg kg 1) intraperitoneally. Supplemental doses of anaesthesia were used during the experiment to maintain an appropriate level of anaesthesia, which was assessed by the absence of corneal reflexes and paw withdrawal response to a noxious pinch. A midline incision in the neck was made to expose the trachea and carotid artery. The trachea was intubated for mechanical
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ventilation. RSNA, MAP and HR were simultaneously recorded with a PowerLab data acquisition system (8/35, ADInstruments, Castle Hill, Australia).
PVN microinjection The PVN stereotaxic coordinates were 1.8 mm caudal from bregma, 0.4 mm lateral to the midline and 7.9 mm ventral to the dorsal surface (Shi et al. 2011). The bilateral PVN microinjections were carried out with two glass micropipettes (about 50 lm tip diameter) and completed within 1 min. Microinjection volume was 50 nL for each microinjection site. At the end of the experiment, same volume of Evans Blue (2%) was injected into the microinjection site for histological identification. Rats with microinjection sites outside the PVN or at the margin of the PVN were excluded from data analysis (Fig. 1).
RSNA recording Left renal sympathetic nerve was isolated through a retroperitoneal incision and was cut distally to eliminate its afferent activity. The nerve was immersed in warm mineral oil and placed on a pair of silver electrodes. The RSNA was amplified with a four channel AC/DC differential amplifier (DP-304, Warner Instruments, Hamden, CT, USA) with a high pass filter at 100 Hz and a low pass filter at 3000 Hz. The RSNA was integrated at a time constant of 100 ms. At the end of each experiment, background noise was determined after section of the central end of the nerve and was subtracted from the integrated values of the RSNA (Cui et al. 2013).
In situ detection of superoxide anions Specific fluorogenic probe dihydroethidium (DHE) was used to detect in situ superoxide anions in the PVN as we previously reported (Shi et al. 2009). Simply, the DHE fluorescence in the coronal sections was visualized under a fluorescence microscope (BX51, Olympus, Tokyo, Japan). Images were collected by using an Olympus BX51 microscope coupled with an
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Olympus DP70 digital camera at 9100. 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 from 2K1C or sham rats was analysed and quantified with IMAGE-PRO PLUS 6.0 by using the same parameters.
Measurement of superoxide anions, NAD(P)H oxidase activity and PKC activity Brain was removed and flash-frozen with liquid nitrogen and stored at 70 °C until being sectioned. Coronal sections of the brain were made with a cryostat microtome (CM1900, Leica, Nussloch, Germany). A 450-lm-thick coronal section was cut through the hypothalamus and incorporated the PVN area, and then, the PVN 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, CA, USA). Superoxide anion level and NAD(P)H oxidase activity in the PVN were measured with lucigenin-derived chemiluminescence method as we previously reported (Shi et al. 2009, Han et al. 2011a, Rosc-Schluter et al. 2012). 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, USA). The values were averaged and expressed as mean light unit (MLU) per minute per milligram of protein. Protein kinase C activity in the supernatants was measured with a solid-phase enzyme-linked immunoabsorbent assay kit (Enzo Life Sciences, Ann Arbor, Michigan, USA). A specific synthetic peptide was used as a substrate of PKC, and a polyclonal antibody that recognizes the phosphorylated form of the substrate was used in a solution phase. The substrate pre-coated on the wells of PKC substrate microtiter plate was phosphorylated by PKC, followed by adding ATP into
Figure 1 Schematic representations of serial sections from the rostral ( 1.44 mm) to the caudal ( 2.16 mm) extent of the region of the PVN. Dots or open circles represent the sites of termination of the microinjections. Dots are considered to be within the PVN. Open circles are considered to be outside of the PVN or at the margin of the PVN, which were excluded for data analysis. 3V, third ventricle.
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the wells to initiate the kinase reaction and incubating for 90 min at 30 °C, the reactions were terminated by emptying the contents of each well and the phosphospecific substrate antibody bounded specifically to the phosphorylated peptide substrate was added into wells and subsequently bound by a peroxidase conjugated secondary antibody. The colour development was initiated with tetramethylbenzidine substrate (TMB) in proportion to PKC phosphotransferase activity and stopped with acid stop solution. The intensity of the colour was measured in a microplate reader (ELX800, BioTek, Vermont, USA) at 450 nm (Triantafyllou et al. 2011, Chang et al. 2012, Ji et al. 2012). Measurement of AVP and salusin-b. Commercial ELISA kits were used for the measurement of AVP (Enzo Life Sciences, Ann Arbor, MI, USA) or salusin-b (Uscn Life Science, Houston, TX, USA). According to the manufacturer’s descriptions, the standards or samples diluent were added and incubated in the appropriate well of specific antibody pre-coated microtiter plate. Conjugate was added and incubated for 1 h at 37 °C and then washed. The reactions were stopped with stop solution and read at 405 nm for the AVP measurements or 450 nm for the salusin-b measurements using a microtiter plate reader (ELX800, BioTek, Vermont, USA) (Vega et al. 2010). Measurement of MrgA1 mRNA with Real-time PCR. The mRNA expression of Mas-like G proteincoupled receptors (Mas-related gene A1, MrgA1) in the PVN was measured using a fluorescence quantitative PCR system (Roche, Basel, Sweden). Simply, total RNA was extracted by TRIzol (Ambion, Austen, Texas, USA) and evaluated with SYBR Green I fluorescence. The rat forward primer and reverse primer of MrgA1 are 5′-AAGAGGAATGGGGGAAAGCA-3′ and 5′-AAGGCGTTCCTGTGCAAACA-3′, respectively. The rat forward primer and reverse primer of b-actin are 5′-ATGTGGATCAGCAAGCAGGA-3′ and 5′-AAGGGTGTAAAACGCAGCTCA-3′, respectively. After being denatured at 94 °C for 5 min, the solution underwent PCR for MrgA1 and b-actin at 94 °C for 20 s, 62 °C for 30 s and 72 °C for 45 s for 45 cycles. All processes were performed in triplicate, and the average cycle thresholds (Ct) were used to determine fold-change. The relative quantification of gene expression was reported as a relative quantity to the control value.
Experimental design We recently found that microinjection of salusin-b into the PVN increased RSNA, MAP and HR in a dose-related manner in 2K1C rats, but did not cause
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any significant changes in Sham rats (Chen et al. 2013). According to the findings, a dose of salusin-b (100 pmol) was used in the present study. Most experiments were carried out only in 2K1C and/or Sham rats except that the Experiment 8 was carried out in normotensive Wistar rats and SHR.
Chemicals Tempol, NAD(P)H, polyethylene glycol-superoxide dismutase (PEG-SOD), apocynin, dimethyl sulfoxide (DMSO), losartan, lucigenin, NG-nitro-L-arginine methyl ester (L-NAME), gabazine and CGP-35348 were obtained from Sigma Chemical (St. Louis, MO, USA). Salusin-b, D-Alanine-Ang-(1-7) (A-779) and chelerythrine chloride (CLC) were obtained from Phoenix Pharmaceuticals (CA, USA), Bachem (Bubendorf, Switzerland) and Tocris Bioscience (Bristol, United Kingdom), respectively. Apocynin and CLC were dissolved in normal saline containing 1% of DMSO. All other chemicals were dissolved in normal saline.
Experimental design Experiment 1, pre-treatment with microinjection of saline or three different doses of PEG-SOD (0.2, 1 and 5 units) into the PVN was randomly carried out in 2K1C rats (n = 6). Then, the RSNA, MAP and HR responses to the microinjection of salusin-b into the PVN (100 pmol) were determined 8 min after the pretreatment. The intervals between injections were at least 60 min for a complete recovery. Experiment 2, pre-treatment with microinjection of saline, the superoxide anion scavenger tempol (20 nmol), the AT1 receptor antagonist losartan (50 nmol), the Mas receptor antagonist A-779 (3 nmol), the non-specific NOS inhibitor L-NAME (200 nmol) or the GABAA receptor antagonist gabazine (0.1 nmol) plus GABAB receptor antagonist CGP35348 (10 nmol) into the PVN was carried out in 2K1C rats. Then, the RSNA, MAP and HR responses to the microinjection of salusin-b (100 pmol) into the PVN were determined 8 min after the pre-treatment (n = 6 for each group). Experiment 3, pre-treatment with the microinjection of DMSO (1%), the NAD(P)H oxidase inhibitor apocynin (1 nmol) or the protein kinase C (PKC) inhibitor CLC (5 nmol) into the PVN was carried out in 2K1C rats. Then, the RSNA, MAP and HR responses to the microinjection of salusin-b (100 pmol) into the PVN were determined 8 min after the pre-treatment (n = 6 for each group). Experiment 4, microinjection of saline or salusin-b (100 pmol) into the PVN was carried out in Sham or 2K1C rats for in situ detection of superoxide anions
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with specific fluorogenic probe DHE (n = 4 for each group). The rats were killed for the detection of superoxide anions in the PVN 10 min after the microinjection. Experiment 5, effects of microinjection of saline or salusin-b (100 pmol) into the PVN on the superoxide anion level, NAD(P)H oxidase activity and PKC activity in the PVN were determined in Sham or 2K1C rats (n = 6 for each group). The rats were killed for the measurements 10 min after the microinjection. Experiment 6, microinjection of saline or salusin-b (100 pmol) into the PVN was carried out 8 min after the pre-treatment with saline, PEG-SOD (5 units), DMSO, apocynin (1 nmol) or CLC (5 nmol) in 2K1C rats (n = 6 for each group). The rats were killed for the measurement of superoxide anion level and NAD (P)H oxidase activity in the PVN, AVP levels in the RVLM and plasma 10 min after microinjection of salusin-b. Experiment 7, MrgA1 mRNA expression and salusin-b level in the PVN as well as plasma salusin-b level were determined in Sham rats and 2K1C rats (n = 6 for each group). Experiment 8, the effects of PVN microinjection of saline or salusin-b (100 pmol) on the RSNA, MAP and HR were determined in Wistar rats and SHR (n = 6 for each group).
Statistical analysis Comparisons between two groups were made by Student’s t test. ANOVA followed by post hoc Bonferroni test was used when multiple comparisons were made. All data were expressed as mean SE. A value of P < 0.05 was considered statistically significant.
Table 1 Body weight, SBP, baseline MAP and baseline HR at the end of the fourth week Variables
Sham
n Body weight, g SBP, mm Hg Baseline MAP, mm Hg Baseline HR, bmp
26 346 126 92 348
2K1C
5 2 3 9
149 339 191 136 360
2 2* 7* 4
Systolic blood pressure (SBP) of tail artery was measured in conscious state by use of a non-invasive computerized tailcuff system. MAP and HR were measured under anaesthesia with a pressure transducer through a catheter placed in right carotid artery. Values are mean SE. *P < 0.05 compared with the Sham.
Results General data There was no significant difference in body weight or HR between Sham and 2K1C rats at the end of the fourth week. However, both SBP and MAP in 2K1C rats were significantly higher than those in Sham rats (Table 1).
Effects of different doses of PEG-SOD Microinjection of the superoxide anion scavenger PEG-SOD into the PVN decreased the RSNA and MAP, while salusin-b increased RSNA, MAP and HR in 2K1C rats (Table 2). Pre-treatment with PEG-SOD attenuated the effects of salusin-b on the RSNA, MAP and HR in 2K1C rats, and high dose of PEG-SOD almost abolished the effects of salusin-b (Fig. 2).
Table 2 Changes of baseline RSNA, MAP and HR Groups
RSNA (%)
Saline PEG-SOD, 0.2 units PEG-SOD, 1 units PEG-SOD, 5 units Tempol, 20 nmol Losartan, 50 nmol A-779, 3 nmol L-NAME, 200 nmol Gabazine, 0.1 nmol + CGP-35348, 10 nmol 1% DMSO Apocynin, 1 nmol CLC, 5 nmol
0.3 2.6 7.6 9.0 8.3 10.7 13.7 15.7 19.2 0.5 8.1 5.6
1.9 2.2 2.7* 2.3* 2.6* 2.2* 2.3* 3.1* 4.8* 2.3 3.0* 2.0*
MAP (mm Hg) 0.2 2.2 6.0 6.2 6.5 7.8 7.0 5.6 15.2 0.5 6.2 7.7
1.5 0.8 1.4* 1.1* 1.7* 1.6* 1.5* 1.2* 3.1* 1.5 2.3* 2.2*
HR (bpm) 0.8 1.7 3.3 3.5 4.0 1.8 1.4 0.2 0.8 0.7 0.3 0.2
1.4 1.1 1.4 1.6 1.1 1.9 2.4 2.1 1.4 1.8 1.8 2.4
Values are mean SE. *P < 0.05 vs. Saline or DMSO.
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(a)
(b)
Figure 2 Effects of PVN pre-treatment with saline, three doses of PEG-SOD, superoxide anion scavenger tempol, AT1 receptor antagonist losartan, Mas receptor antagonist A-779, NOS inhibitor L-NAME or GABAA receptor antagonist gabazine plus GABAB receptor antagonist CGP-35348 on the RSNA, MAP and HR responses to the microinjection of salusin-b into the PVN in 2K1C rats. Representative tracings in the lower panel showing that pre-treatment with microinjection of PEG-SOD (5 units) into the PVN abolished the RSNA, MAP and HR responses to salusin-b. Values are mean SE. *P < 0.05 vs. Saline+Saline. †P < 0.05 vs. Saline + Salusin-b. n = 6 for each group.
Effects of tempol, losartan, A-779, L-NAME and gabazine plus CGP-35348. Microinjection of the superoxide anion scavenger tempol, the AT1 receptor antagonist losartan or the Mas receptor antagonist A-779 into the PVN decreased RSNA and MAP, while the non-specific NOS inhibitor L-NAME or the GABAA receptor antagonist gabazine plus the GABAB receptor antagonist CGP-35348 decreased RSNA and MAP in 2K1C rats (Table 2). Pre-treatment with tempol almost abolished the effects of salusin-b in 2K1C rats, but pre-treatment with losartan, A-779, L-NAME or gabazine plus CGP-35348 had no
significant effect on the RSNA, MAP and HR responses to salusin-b in 2K1C rats (Fig. 2).
Effects of apocynin and CLC Microinjection of the NAD(P)H oxidase inhibitor apocynin or the PKC inhibitor CLC into the PVN decreased baseline RSNA and MAP in 2K1C rats (Table 2). Pre-treatment with apocynin or CLC almost abolished the effects of salusin-b on the RSNA, MAP and HR in 2K1C rats (Fig. 3).
Figure 3 Effects of PVN pre-treatment with the NAD(P)H oxidase inhibitor apocynin, or the PKC inhibitor chelerythrine chloride (CLC) on the RSNA, MAP and HR responses to microinjection of salusin-b into the PVN in 2K1C rats. Values are mean SE. *P < 0.05 vs. DMSO+Saline. †P < 0.05 vs. DMSO+Salusin-b. n = 6 for each group. © 2013 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.12188
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(a)
(b)
(c)
Figure 4 Superoxide anion level and NAD(P)H oxidase activity in the PVN in Sham and 2K1C rats. (a) representative photos showing the effects of microinjection of saline or salusin-b into the PVN on the superoxide anion levels in the PVN, indicated by DHE fluorescence intensity at the levels of 1.44, 1.80 and 2.16 mm caudal from bregma.3V, third ventricle; (b) effects of microinjection of saline or salusin-b into the PVN on the DHE fluorescence intensity in the PVN; (c) effects of microinjection of saline or salusin-b on superoxide anion level and NAD(P)H oxidase activity in the PVN measured with lucigenin-derived chemiluminescence method. Values are mean SE. *P < 0.05 vs. Saline. †P < 0.05 vs. Sham. n = 6 for each group.
Superoxide anions and NAD(P)H oxidase activity Superoxide anions detected with fluorogenic probe DHE were increased in the PVN in 2K1C rats, and PVN microinjection of salusin-b further increased the superoxide anions in the PVN in 2K1C rats, but not in Sham rats. The most obvious change was observed at the level of 1.80 mm caudal from bregma (Fig. 4a, b). Both superoxide anion level and NAD(P)H oxidase activity in the PVN measured with a chemiluminescence method were higher in 2K1C rats than those in (a)
Sham rats. Microinjection of salusin-b into the PVN increased the superoxide anion level and NAD(P)H oxidase activity in 2K1C rats but not in Sham rats (Fig. 4c).
PKC activity Protein kinase C activity in the PVN was enhanced in 2K1C rats, and microinjection of salusin-b into the PVN further enhanced the PKC activity in 2K1C rats, but not in Sham rats (Fig. 5a).
(b)
Figure 5 PKC activity in the PVN and effects of PKC inhibitor CLC. (a) effects of PVN microinjection of saline or salusin-b on PKC activity in the PVN in Sham and 2K1C rats. *P < 0.05 vs. Saline; †P < 0.05 vs. Sham. (b) effects of PVN pre-treatment with DMSO or CLC on the superoxide anion level and NAD(P)H oxidase activity responses to the PVN microinjection of salusin-b in the PVN in 2K1C rats. Values are mean SE. *P < 0.05 vs. Saline; †P < 0.05 vs. Sham. ‡P < 0.05 vs. DMSO+Salusin-b. n = 6 for each group.
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Discussion
Figure 6 Effects of pre-treatment with microinjection of saline, PEG-SOD, DMSO, apocynin or CLC on the AVP response to the microinjection of salusin-b into the PVN in 2K1C rats. Values are mean SE. *P < 0.05 vs. Saline+ Saline; †P < 0.05 vs. Saline+Salusin-b; ‡P < 0.05 vs. DMSO+Salusin-b. n = 6 for each group.
Effects of CLC on the responses of superoxide anions and NAD(P)H oxidase activity to salusin-b After microinjection of salusin-b into the PVN, the superoxide anion level and NAD(P)H oxidase activity in the PVN were lower in the rats pre-treated with PKC inhibitor than those rats pre-treated with 1% of DMSO (Fig. 5b).
RVLM and Plasma AVP levels Microinjection of salusin-b into the PVN increased both RVLM and plasma AVP levels in 2K1C rats, which were prevented by PEG-SOD, apocynin or CLC (Fig. 6).
Salusin-b levels and MrgA1 mRNA expression Salusin-b levels in both plasma and PVN, as well as the MrgA1 mRNA expression in the PVN were increased in 2K1C rats (Fig. 7).
Effects of Salusin-b in SHR Microinjection of salusin-b into the PVN increased RSNA, MAP and HR in SHR, but not in normotensive Wistar rats (Fig. 8).
We recently found that exogenous salusin-b in the PVN increased RSNA, MAP, HR via both circulating AVP and AVP in the RVLM in 2K1C rats, but not in Sham rats (Chen et al. 2013). Similarly, microinjection of salusin-b into the PVN increased RSNA, MAP and HR in SHR, but not in normotensive Wistar rats. The primary new findings in the present study were that the PKC-NAD(P)H oxidase-superoxide anions pathway in the PVN was involved in the effects of salusin-b on RSNA, MAP, HR and AVP release in 2K1C-induced renovascular hypertensive rats. PEG-SOD shows a longer circulatory half-life than native SOD and is widely used to scavenge the intracellular superoxide anions. It binds to cell membranes and rapidly penetrates into cells, whereas SOD has limited cellular penetration (Beckman et al. 1988). Tempol is another kind of membrane-permeable superoxide anion scavenger (Yamada et al. 2003). Blood pressure reduction induced by chronic tempol treatment was related to attenuated sympathetic vasoconstriction (Vaneckova et al. 2013). In the present study, PEG-SOD or tempol almost abolished the effects of salusin-b. Superoxide anion level was increased in 2K1C rats, which was further increased by microinjection of salusin-b into the PVN. These results indicate that superoxide anions in the PVN are required for the effects of salusin-b on the RSNA, MAP and HR in 2K1C rats. It is known that Ang II is involved in hypertension (Grisk 2011). NAD(P)H oxidase is a multi-component enzyme complex and one of major origins of the superoxide anions (Cai et al. 2003). NAD(P)H oxidase-derived superoxide anions in the PVN mediated the angiotensin II-induced sympathetic activation and pressor effects in 2K1C rats (Han et al. 2011a). Oxidant signalling in the PVN contributed to the pathogenesis of renovascular hypertension (Burmeister et al. 2011). In the present study, both NAD(P)H oxidase activity and superoxide anion level in the PVN were higher in 2K1C rats than those in Sham rats, and salusin-b further increased the NAD(P)H oxidase activity and
Figure 7 Salusin-b levels in plasma and PVN, and MrgA1 mRNA expression in the PVN in Sham rats and 2K1C rats. Values are mean SE. *P < 0.05 vs. Sham. n = 6 for each group. © 2013 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.12188
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Figure 8 Effects of microinjection of saline or salusin-b into the PVN on the RSNA, MAP and HR in Wistar rats and SHR. Values are mean SE. *P < 0.05 vs. Saline; †P < 0.05 vs. Wistar. n = 6 for each group.
superoxide anion level in 2K1C rats rather than in Sham rats. The PVN microinjection of NAD(P)H oxidase inhibitor apocynin almost abolished the effects of salusin-b on the RSNA, MAP and HR in 2K1C rats. These results suggest that NAD(P)H oxidase is a major source of salusin-b-induced superoxide anion production in the PVN, which is supported by the finding that the mRNA expression of NADPH oxidase family member Nox2 was increased by treatment with salusin-b in cultured human umbilical vein endothelial cells (HUVECs) (Koya et al. 2012). It has been found that PKC is involved in the salusin-b-induced improvement in calcium uptake and protein synthesis in neonatal rat cardiomyocytes (Yu et al. 2004). We found that PKC activity was higher in 2K1C rats than that in Sham rats, and salusin-b further increased PKC activity in 2K1C rats but not in Sham rats. Microinjection of the PKC inhibitor CLC into the PVN not only almost abolished the effects of salusin-b on the RSNA, MAP and HR, but also prevented salusin-b-induced increases in NAD(P)H oxidase activity and superoxide anion level in 2K1C rats. These results suggest that the NAD(P)H oxidasederived superoxide anions are dependent upon the activation of PKC. It was found that salusin-b increased intracellular Ca2+ in the rat vascular smooth muscle cells and fibroblasts in a concentration-dependent manner, which were attenuated by pre-treatment with calcium channel antagonist nicardipine and were nearly abolished by calcium chelator EGTA (Shichiri et al. 2003). Salusin-b increased intracellular Ca2+ in freshly isolated single nerve terminals from the neurohypophysis devoid of pars intermedia, which were blocked by high-voltage-activated Ca2+ channel blockers (Saito et al. 2008). Ca2+ is one of critical factors for the activation of PKC (Tanaka & Saito 1992). PKC is involved in the intracellular regulation of redox balance and oxidative stress levels (De et al. 2013). These results suggest that increased intracellular Ca2+ may be a possible mechanism of salusin-binduced PKC activation. On the other hand, the present study showed that Ang II, Ang-(1-7), NO and GABA were not involved in the effects of salusin-b. 542
Arginine vasopressin is primarily synthesized in PVN and supraoptic nuclei and secreted from neurohypophysis to the systemic circulation, which causes powerful constriction in a variety of vascular regions via AVP V1 receptors (Noguera et al. 1997). Salusin-b, not salusin-a, stimulated the AVP release from peri-fused rat pituitary (Shichiri et al. 2003). Salusin-b-like immunopositive cells were found in the parvocellular and magnocellular part of the PVN, especially in those AVP-containing neurones (Takenoya et al. 2005). The neurones in the magnocellular part of PVN project to the neurohypophysis and contribute to the production of posterior pituitary hormones including AVP; the neurones in the parvocellular part of PVN project to RVLM and spinal cord and contribute to the sympathetic activation (Pyner 2009). AVP-immunoreactive fibres were found in the RVLM (Gomez et al. 1993). After microinjection of retrograde tracer cholera toxin b subunit (CT-b) into the RVLM, 14.6% of CT-b-labelled PVN neurones were double-labelled with AVP, and most of labelled neurones were in the regions of the PVN that are involved in autonomic control (Kc et al. 2010). Microinjection of AVP into the RVLM increased blood pressure (Gomez et al. 1993). Blockade of V1a receptors of AVP in the RVLM attenuated the PVN-evoked increases in RVLM unit discharge, and AVP acted like glutamate as an excitatory neurotransmitter on RVLM neurones (Yang et al. 2001, Yang & Coote 2003). Chronic intermittent hypoxia (CIH) up-regulated AVPV1a receptor signalling in the RVLM, thus increasing blood pressure (Kc et al. 2010). These results indicate that AVP neurones in the PVN project to the RVLM, which are involved in regulating blood pressure. Microinjection of salusin-b into the PVN increased the plasma AVP level in 2K1C rats, but not in Sham rats. Intravenous injection of AVP V1 receptor antagonist decreased RSNA and MAP and abolished the effects of salusin-b in 2K1C rats. Microinjection of AAVP into RVLM, but not into the PVN, abolished the effects of salusin-b on RSNA and HR. These results suggest that the effects of salusin-b in the PVN on RSNA, MAP and HR are mediated by both circulating AVP and AVP in the RVLM in 2K1C rats
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(Chen et al. 2013). In the present study, microinjection of salusin-b into the PVN increased plasma AVP level in 2K1C rats, which was consist with our previous findings (Chen et al. 2013). Pre-treatment with PEG-SOD, apocynin or CLC prevented the salusin-b-induced increases in both RVLM and plasma AVP levels in 2K1C rats. These results suggest that PKC, NAD(P)H oxidase-originated superoxide anions in the PVN are necessary for the AVP release from neurohypophysis, which contribute to the increased plasma AVP level and blood pressure, and from RVLM which may contribute to the sympathetic activation in 2K1C rats. The latter is supported by the finding that AVP may act as excitatory neurotransmitters at synapses of paraventricular neurones on the RVLM vasomotor neurones (Yang et al. 2001). It is very interesting that microinjection of salusin-b into the PVN increased sympathetic activity, MAP, HR and AVP release, and neutralization endogenous salusin-b with anti-salusin-b IgG caused opposite effects in 2K1C rats, but not in Sham rats (Chen et al. 2013). Similarly, salusin-b in the PVN elicited sympathoexcitation and pressor effects in SHR, but not in normotensive Wistar rats. These results suggest that salusin-b in the PVN may not be involved in the tonic control of sympathetic outflow, blood pressure and AVP release in physiological state, but contributes to the pathogenesis of hypertension. There is a possibility that salusin-b may contribute to the maintenance of blood pressure via sympathetic activation and AVP release in haemorrhage or other hypotensive states. We are interested to test the hypothesis in the rat model of hypotension in the near future study. MrgA1 have been shown to bind a wide variety of ligands from peptides to amino acid derivatives. Human salusin-b can activate the mouse orphan G protein-coupled receptor, mMrgA1, and may be a surrogate ligand of the MrgA1 (Wang et al. 2006). We found that salusin-b level and MrgA1 mRNA expression in the PVN were greatly increased in 2K1C rats compared with Sham rats, which may be responsible for the involvement of salusin-b in the PVN in the pathogeneses of hypertension in 2K1C rats. It is noted that intravenous administration of salusin-b caused a temporary rapid decrease in aortic blood flow concomitantly with hypotension and bradycardia without affecting systemic vascular resistance by facilitating vagal outflows to the heart and its negative inotropic and chronotropic actions on the heart (Izumiyama et al. 2005). The greatly increased plasma salusin-b level in 2K1C rats may be a compensatory mechanism for attenuating hypertension. A limitation in the present study was that the experiments were performed in acute experiments with anaesthesia. The results may not reflect the actual situation in conscious rats.
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In conclusion, salusin-b in the PVN activates PKC, which in turn activates NAD(P)H oxidase in 2K1C rats. The enhanced NAD(P)H oxidase activity contributes to the increase in superoxide anions in the PVN. The higher superoxide anion level in the PVN increases the AVP levels in the plasma and the RVLM, which are responsible for sympathetic activation and hypertension.
Conflict of interest The authors declared no conflict of interest. This work was supported by National Natural Science Foundation of China (31171095 & 31271213), 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.
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