Cardiovasc Toxicol DOI 10.1007/s12012-015-9330-2
Salusin b Within the Nucleus Tractus Solitarii Suppresses Blood Pressure Via Inhibiting the Activities of Presympathetic Neurons in the Rostral Ventrolateral Medulla in Spontaneously Hypertensive Rats Hong-Bao Li1 • Yan Lu2 • Jin-Jun Liu1 • Yu-Wang Miao1 • Tian-Zhen Zheng3 • Qing Su1 • Jie Qi1 • Hong Tan1 • Zu-Yi Yuan4 • Guo-Qing Zhu5 • Yu-Ming Kang1
Ó Springer Science+Business Media New York 2015
Abstract Salusin b is a newly identified bioactive peptide, which shows peripheral hypotensive, mitogenic and proatherosclerotic effects. The present study was undertaken to investigate the role of salusin b within the nucleus tractus solitarii (NTS) and the underlying mechanism in regulating blood pressure and heart rate (HR) in spontaneously hypertensive rats (SHR). Our results showed that bilateral or unilateral microinjection of salusin b (0.4–40 pmol) into the NTS in SHR decreased mean arterial pressure and HR in a dose-dependent manner. Bilateral microinjection of salusin b (4 pmol) within NTS improved baroreflex sensitivity functions in SHR. Pretreatment with glutamate receptors antagonist kynurenic acid (5 nmol) into the NTS in SHR did not alter the salusin b (4 pmol)
induced hypotension and bradycardia. Likewise, bilateral vagotomy also did not alter the salusin b (4 pmol) induced hypotension and bradycardia. However, pretreatment with GABAA receptors agonist muscimol (100 pmol) within the rostral ventrolateral medulla (RVLM) in SHR almost completely abolished the hypotension and bradycardia evoked by intra-NTS salusin b (4 pmol). Our findings suggested that microinjection of salusin b into the NTS produced hypotension and bradycardia, as well as improved baroreflex sensitivity functions, via inhibiting the activities of presympathetic neurons in the RVLM in SHR.
Keywords Salusin Nucleus tractus solitarii Rostral ventrolateral medulla Baroreflex Presympathetic neuron & Yan Lu [email protected] & Yu-Ming Kang [email protected] 1
Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Xi’an Jiaotong University Health Science Center, Xi’an 710061, China
2
Department of Clinical Laboratory, SanAiTang Hospital, Lanzhou 730030, China
3
Department of Physiology and Psychology, School of Basic Medical Sciences, Lanzhou University, Lanzhou 730000, China
4
Department of Cardiovascular Medicine, First Affiliated Hospital of Medical College of Xi’an Jiaotong University, Xi’an 710061, China
5
Key Laboratory of Cardiovascular Disease and Molecular Intervention, Department of Physiology, Nanjing Medical University, Nanjing 210029, China
Introduction Salusins derive from the prosalusin precursor molecule and regulate hemodynamics, mitogenesis and atherogenesis. Salusins are considered to be the source of two bioactive peptides, salusin a and salusin b, which comprise 28 and 20 amino acid residues, respectively. Salusins induce immediate early response genes and stimulate proliferation of human vascular smooth muscle cells (VSMCs) [1]. They can be concomitantly biosynthesized from their precursor, prosalusin, after alternative splicing and subsequent processing of the human torsion dystonia-related gene (TOR2A) [1]. Preprosalusin and its mRNA are widely expressed in both human tissues and rat tissues, including vasculature, central nervous system (CNS), lung and kidney [1–3]. Salusin b was also abundantly expressed in the CNS in both human and rats [3, 4].
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Previous studies show that salusins exert multiple functions in the cardiovascular, endocrine and immune systems [1, 5, 6]. Bolus intravenous administration of salusin b in rats produces hypotension, bradycardia and cardiac dysfunction, which is mainly mediated by the cholinergic mechanism [7]. In additional, salusins also stimulate the proliferation of VSMCs, cardiomyocytes and fibroblasts, participate in the formation of atherosclerosis, modulate the intracellular signaling pathways and may be a potential survival factor against serum deprivation-induced myocardial cell death in cardiomyocytes [2, 8, 9]. Preprosalusin and mRNA have been reported to exist abundantly in the hypothalamus and pituitary [3, 4]. Salusin b in the paraventricular nucleus (PVN) increases blood pressure (BP) and sympathetic outflow via vasopressin in hypertensive rats [10]. Salusin b causes potent hypotensive/ bradycardic effects [1], stimulates arginine vasopressin secretion from the neurohypophysis [1, 11] and exerts antiapoptotic and growth-promoting effects on cardiovascular cells [8]. Hypertension is characterized by elevated levels of BP and sympathetic tone, which are closely related to a poor prognosis in this disease [12]. It is well known that the nucleus tractus solitarii (NTS), which is situated at the dorsomedial part of medulla oblongata, serves as a putative gateway to various visceral sensory [13, 14] and plays a pivotal role in maintaining arterial baroreflex (ABR) and resting BP in cardiovascular system [14, 15]. Stimulation of baroreceptor afferents is generally thought to activate excitatory amino acid receptors within the NTS. The NTS at first integrates afferent signals and sends an excitatory transmitter, glutamic acid, to the caudal ventrolateral medulla (CVLM) and stimulates a GABAergic inhibitory pathway to the rostral ventrolateral medulla (RVLM) [16– 18]. However, it is unknown whether salusin b in the NTS is involved in regulating BP and baroreflex sensitivity functions. The present study was designed to determine whether salusin b in the NTS attenuates BP and improves baroreflex sensitivity functions, and the mechanisms of exogenous salusin b regulating cardiovascular activity in spontaneously hypertensive rats.
National Institutes of Health Guide for the Care and Use of Laboratory Animals. Salusin b and Anti-salusin b Serum Human salusin b was obtained from Phoenix Pharmaceuticals (CA, USA). Rabbit anti-salusin b (human) serum was obtained from Bachem (Bubendorf, Switzerland). The function of salusin b in rats was investigated by human salusin b and rabbit anti-salusin b (human) serum because human salusin b has been proved to be highly homologous to the rat salusin b [10, 19]. Rabbit anti-salusin b (human) serum was diluted in 0.01 M PBS (1:400) for immunohistochemistry or 5 % goat serum (1:1000) for western blot. Immunofluorescence Studies Immunofluorescence labeling was performed in floating sections as described previously [20–22] to identify salusin b expressions. Briefly, rats (n = 6 per group) were anaesthetized by a ketamine (80 mg/kg) and xylazine (10 mg/ kg) mixture (ip). The transcardiac perfusion was performed with 200 ml of 1 % phosphate buffer saline (PBS), followed by 400 ml of 4 % buffered paraformaldehyde (pH 7.4). Brains were removed and fixed in 4 % buffered paraformaldehyde for 48 h. Free-floating sections (18 lm) were incubated with 0.3 % Triton-X for 20 min and with 5 % goat serum for 30 min. The brain sections were then incubated with salusin b serum diluted in 0.01 M PBS at 4 °C overnight followed by incubation with Alexa 488-labeled anti-rabbit secondary antibody (1:200 green fluorescence) or Alexa 594-labeled anti-rabbit secondary antibody (1:200 red fluorescence) (Invitrogen, CA) for 60 min at 37 °C. The salusin b positive neurons in the RVLM, NTS and PVN were observed using a fluorescence microscope (Nikon, Tokyo, Japan). The number of neurons with salusin b immunoreactivity in the RVLM, NTS and PVN was determined by averaging the numbers of four sections for each animal. Western Blot Analysis
Materials and Methods Animals One hundred and fifty-six ten-week-old male normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) were used in this study. All animal and experimental procedures in this study were approved by the Animal Care and Use Committees of Xi’an Jiaotong University and were conducted in accordance with the US
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Proteins extracted from the PVN, NTS and RVLM were used for the measurement of salusin b expression by western blot. The procedures of western blot were described previously [23, 24]. The protein concentration was measured and loaded onto a SDS-PAGE gel and then transferred to a polyvinylidene fluoride membrane. The membrane was probed with salusin b serum and secondary antibody (1:2000, Santa Cruz Biotechnology). The protein bands were visually detected and analyzed. Protein loading was controlled by probing all blots with b-actin antibody
Cardiovasc Toxicol
(Santa Cruz Biotechnology) and normalizing salusin b protein intensity to that of b-actin. The bands were quantified using NIH Image J software.
solution was injected into the NTS and RVLM to mark the site. Baroreflex Sensitivity (BRSHP)
General Experimental Procedure The methods for animal preparation and microinjection were similar to those described previously [25, 26]. Briefly, rats were anesthetized with a a-chloralose (80 mg/kg) and urethane (500 mg/kg) mixture (ip). Then, the right femoral artery and vein were cannulated with polyethylene catheters for the measurement of arterial BP and drug administration, respectively. The catheters were prior filled with heparinized saline (50 U/ml) or 0.9 % NaCl. BP and (heart rate) HR were measured with an analysis software (RM6240, China) by a computer, and heart period (HP) was monitored by the differential signal of BP pulse. A trachea was cannulated and connected to animal ventilator (WJ-2000, China). a-Chloralose (25 mg/kg) was intravenously supplemented when necessary. Adequacy of anesthesia was assessed by monitoring the stability of BP and HP. The anesthetized rats were fixed in the stereotaxic apparatus (MP-8003, China). The dorsal surface of medulla oblongata was exposed by removing the partial occipital and cerebellum. In this process, an infrared heating lamp was utilized to keep rat body temperature at 37 °C. Microinjection Procedure According to stereotaxic coordinates, the multi-barreled micropipette (tip diameter 20–30 lm) was inserted into the NTS (0.4–0.5 mm rostral to the calamus scriptorius, 0.5–0.6 mm lateral to the midline and 0.4–0.5 mm below the dorsal surface of the medulla) or RVLM (2.0–2.5 mm rostral to the calamus scriptorius, 1.8–2.0 mm lateral to the midline and 2.8–3.2 mm below the dorsal surface of the medulla). The micropipette was filled with L-glutamate, salusin b, glutamate receptors antagonist kynurenic acid (KYN) or GABAA receptors agonist muscimol. KYN was initially dissolved in 10 % sodium hydroxide (NaOH) and finally diluted with artificial cerebrospinal fluid (aCSF, in mM: 133.3 NaCl, 3.4 KCl, 1.3 CaCl2, 1.2 MgCl2, 0.6 NaH2PO4, 32.0 NaHCO3 and 3.4 glucose, pH 7.4). The other drugs were dissolved into aCSF. The dose of salusin b, KYN, and muscimol were based on previous studies [10, 27]. The bilateral or unilateral microinjections were completed within 10–30 s, and the microinjection volume was 100 nl for each microinjection site. Functional location of NTS or RVLM on either side was carried out at the beginning of each experiment by eliciting a transient decrease or increase in mean arterial pressure (MAP) (15–25 mmHg) by microinjection of glutamate (2 nmol), respectively [26]. Finally, 20 nl of 2 % pontamine sky blue
Rats were anesthetized with a-chloralose (80 mg/kg, ip) and urethane (500 mg/kg, ip). The bradycardiac response to phenylephrine was measured by using previously described method [28]. A rapid bolus injection of phenylephrine was used to induce a BP elevation and then baroreceptor activation. The dose of phenylephrine (2–10 lg/kg) was adjusted to elevate systolic blood pressure (SBP) between 20 and 40 mmHg. There exists a delay between the elevation of BP (stimulus) and the prolongation of heart period HP (response) for ABR. In rat, HR is about 7 per second. HP was plotted against SBP for linear regression analysis with 2–8 shifts; the slope of SBP-HP was expressed as baroreflex sensitivity (BRSHP) (ms/mmHg). The data were recorded using a computer at three time points (preadministration, and 5 and 30 min after medication). Histological Analysis At the end of the experiment, the animal was perfused transcardially with 0.9 % NaCl and 10 % phosphate-buffered formalin. The brain tissue was stored overnight in 10 % phosphate-buffered formalin and then transferred to fixative containing 30 % sucrose. The frozen brain tissue was sectioned coronally (50 lm) and stained with neutral red. Rats with microinjection sites outside the NTS or RVLM were excluded from data analysis. Experimental Protocol Experiment 1 The immunoreactivity and protein levels of salusin b were investigated in the PVN, NTS and RVLM in WKY rats and SHR (four sections for each rat and six rats for each group). Experiment 2 Sixty-eight anesthetized and paralyzed SHR, which were randomized into 8 groups (n = 7–9 rats/group), bilateral or unilateral received application of salusin b (0.4, 4, and 40 pmol) or aCSF (100 nl) to test the dose-dependent effects of exogenous salusin b within the NTS. Experiment 3 Sixteen anesthetized SHR were randomly divided into two groups (n = 7–9 rats/group), which were subjected to the NTS microinjection of aCSF (100 nl) or salusin b (4 pmol)
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Fig. 1 Salusin b expression in the PVN, NTS and RVLM in WKY rats and SHR. a Immunoreactivity for salusin b-positive neurons in the PVN, NTS and RVLM in WKY rats and SHR. b Bar graph showing the number of neurons with salusin b immunoreactivity per section in the PVN, NTS and RVLM in WKY rats and SHR. n = 6 for each group. Values are mean ± SE. *P \ 0.05 versus WKY
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Fig. 2 Representative western blot (a) and densitometric (b) analysis of salusin b in the NTS, PVN and RVLM in WKY rats and SHR. AU arbitrary unit. n = 6 in each group. Values are mean ± SE. *P \ 0.05 versus WKY
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Fig. 3 a Representative original tracings showing mean arterial pressure (MAP) and heart rate (HR) responses to bilateral microinjection of vehicle (aCSF) or salusin b (0.4, 4, 40 pmol) into the NTS in anaesthetized and paralyzed SHR. b, c Line graph showing the
effects of bilateral microinjection of aCSF or salusin b (0.4, 4, 40 pmol) into the NTS on the MAP and HR in SHR. n = 7 or 9 for each group. Values are mean ± SE. *P \ 0.05 versus aCSF
to observe the effects of exogenous salusin b within NTS on the baroreflex sensitivity functions.
Experiment 6
Experiment 4 Sixteen anesthetized SHR were randomly divided into two groups (n = 7–9 rats/group), which were subjected to the NTS microinjection of aCSF (100 nl), glutamate receptors antagonist KYN (5 nmol), salusin b (4 pmol) pretreated with aCSF or salusin b pretreated with KYN. Salusin b was administered 10 min after the pretreatment. Experiment 5 Sixteen anesthetized SHR were successively prior applied bilateral vagus dissection or sham operation before intraNTS salusin b (4 pmol) in SHR (n = 7–9 rats/group) to identify whether the cardiovascular responses to intra-NTS salusin b were mediated by the cholinergic mechanism.
Sixteen anesthetized SHR were randomly divided into two groups (n = 7–9 rats/group), which were subjected to the RVLM microinjection of aCSF (100 nl) or a specific GABAA receptors agonist muscimol (100 pmol) before intra-NTS salusin b (4 pmol) in SHR. Salusin b was administered 10 min after the pretreatment.
Statistical Analysis Data were shown as mean ± SE and analyzed using Student’s t test and one-way analysis of variance (ANOVA). Regression analysis was made to determine the correlation between the doses and responses. Statistical comparison was performed using Prism 5 (GraphPad Software). Differences were considered to be significant at P \ 0.05.
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Fig. 4 a Representative original tracings showing mean arterial pressure (MAP) and heart rate (HR) responses to unilateral microinjection of vehicle (aCSF), Glu or salusin b (0.4, 4, 40 pmol) into the NTS in anaesthetized and paralyzed SHR. b, c Line graph showing
the effects of unilateral microinjection of aCSF, Glu or salusin b (0.4, 4, 40 pmol) into the NTS on the MAP and HR in SHR. n = 7 or 9 for each group. Values are mean ± SE. *P \ 0.05 versus aCSF
Results
The Cardiovascular Effects of Bilateral Microinjection of Salusin b (0.4–40 pmol) into the NTS
The Immunoreactivity of Salusin b in the PVN, NTS and RVLM As shown in Fig. 1, the number of salusin b positive neurons in the PVN, NTS and RVLM was significantly increased in SHR compared with WKY rats. The Protein Expression of Salusin b in the PVN, NTS and RVLM Western blot also further demonstrated that SHR had higher expression of salusin b in the PVN, NTS and RVLM than in WKY rats (Fig. 2).
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Intra-NTS in SHR injection of aCSF did not alter the basal MAP (157 ± 8 vs. 155 ± 7 mmHg, P [ 0.05) and HR (369 ± 18 vs. 367 ± 19 bpm, P [ 0.05). However, microinjection of salusin b (0.4–40 pmol) into the NTS decreased the MAP (0.4 pmol: -10 ± 2 mmHg; 4 pmol: -16 ± 5 mmHg; 40 pmol: -21 ± 7 mmHg vs. aCSF: -2 ± 1 mmHg, P \ 0.05) and HR (0.4 pmol: -9 ± 3 bpm; 4 pmol: -13 ± 6 bpm; 40 pmol: -19 ± 7 bpm vs. aCSF: -2 ± 2 bpm, P \ 0.05) in a dose-related manner in SHR. There was a significant linear correlation between the dose of salusin b and MAP or HR change in SHR (Fig. 3).
Cardiovasc Toxicol
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Fig. 5 Responses of baroreflex sensitivity (BRSHP) before and after microinjection of salusin b (4 pmol) or vehicle (aCSF) into the NTS in SHR. Microinjection of salusin b into the NTS produces a significant increase in the BRSHP of SHR compared with pretreatment
with vehicle (aCSF). n = 7 or 9 for each group. Values are mean ± SE. *P \ 0.05 versus aCSF. SBP indicates systolic blood pressure; HP heart period; values of slope are the values of baroreflex sensitivity
The Cardiovascular Effects of Unilateral Microinjection of Salusin b (0.4–40 pmol) into the NTS
The Effects of Intra-NTS Salusin b (4 pmol) on the Baroreflex Sensitivity (BRSHP)
Intra-NTS injection of aCSF produced no significant influences on the basal MAP (164 ± 4 vs. 161 ± 3 mmHg, P [ 0.05) or HR (374 ± 10 vs. 372 ± 19 bpm, P [ 0.05) of SHR. Unilateral microinjection of salusin b into the NTS decreased the MAP (0.4 pmol: -14 ± 2 mmHg; 4 pmol: -20 ± 3 mmHg; 40 pmol: -24 ± 8 mmHg vs. aCSF: -2 ± 1 mmHg, P \ 0.05) and HR (0.4 pmol: -13 ± 3 bpm; 4 pmol: -19 ± 4 bpm; 40 pmol: -22 ± 9 bpm vs. aCSF: -2 ± 2 bpm, P \ 0.05) in a dose-related manner in SHR. There was a significant linear correlation between the dose of salusin b and MAP or HR change in SHR (Fig. 4).
Bilateral microinjection of salusin b (4 pmol) improved the BRSHP of SHR (before microinjection: 0.312 ± 0.02 ms/ mmHg vs. 5 min after microinjection: 0.457 ± 0.06 ms/ mmHg; 30 min after microinjection: 0.494 ± 0.06 ms/ mmHg, P \ 0.05; Fig. 5). The Effects of Pretreatment with Glutamate Receptors Antagonist KYN on the Cardiovascular Responses of Intra-NTS Salusin b (4 pmol) Prior unilateral microinjection of aCSF in SHR did not alter the MAP and HR responses of intra-NTS salusin b. Unilateral injection of glutamate receptors antagonist KYN
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(5 nmol) into the NTS in SHR significantly raised the basal MAP (154 ± 5 vs. 167 ± 7 mmHg, P \ 0.05) and HR (354 ± 15 vs. 371 ± 17 bpm, P \ 0.05). However, pretreatment with KYN did not affect MAP (-18 ± 5 mmHg with KYN vs. -16 ± 4 mmHg with vehicle, P [ 0.05) and HR (-25 ± 5 bpm with KYN vs. -22 ± 3 bpm with vehicle, P [ 0.05) responses of salusin b within the NTS of SHR (Fig. 6).
The Effects of Bilateral Vagotomy on the Cardiovascular Functions of Intra-NTS Salusin b (4 pmol) Sham operation had no significant effects on the MAP and HR responses of intra-NTS salusin b in SHR. Bilateral vagatomy increased in the basal MAP (151 ± 8 vs. 164 ± 5 mmHg, P \ 0.05) and HR (342 ± 13 vs. 354 ± 11 bpm, P \ 0.05) of SHR. However, bilateral vagatomy did not alter the MAP (-13 ± 4 mmHg with vagatomy vs. -15 ± 5 mmHg with sham, P [ 0.05) and HR (-17 ± 3 bpm with vagatomy vs. -20 ± 3 bpm with sham, P [ 0.05) responses induced by bilateral microinjection of salusin b (4 pmol) within the NTS of SHR (Fig. 7).
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Fig. 6 a Representative original tracings showing the effects of prior application of vehicle (aCSF) or glutamate receptor antagonist kynurenic acid (KYN, 5 nmol) into NTS on the mean arterial pressure (MAP) and heart rate (HR) responses to intra-NTS salusin b (4 pmol) in SHR. b, c The effects of pretreatments with aCSF or glutamate receptor antagonist kynurenic acid (KYN, 5 nmol) on the MAP and HR responses of intra-NTS salusin b (4 pmol) in SHR. n = 7 or 9 for each group. Values are mean ± SE
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The Effect of Prior Microinjection of GABAA Receptors Agonist Muscimol into the RVLM on the Cardiovascular Responses to Microinjection of Salusin b (4 pmol) into the NTS Prior unilateral microinjection of aCSF in SHR did not alter the MAP and HR responses of salusin b within the NTS of SHR. Unilateral microinjection of GABAA receptors agonist muscimol into the RVLM produced a significant decrease in basal MAP (155 ± 6 vs. 132 ± 5 mmHg, P \ 0.05) and HR (371 ± 15 vs. 354 ± 10 bpm, P \ 0.05) of SHR. Notable, pretreatment with muscimol within the RVLM of SHR completely abolished the MAP (-2 ± 2 mmHg with muscimol vs. -18 ± 2 mmHg with vehicle, P \ 0.05) and HR (-4 ± 2 bpm with muscimol vs. -28 ± 4 bpm with vehicle, P \ 0.05) responses induced by application of salusin b into the NTS of SHR (Fig. 8).
Discussion The NTS is a key region for central control of BP and plays a pivotal role in maintaining ABR. In the present study, we investigated the effects and its possible mechanism of
Cardiovasc Toxicol Fig. 7 a Representative original tracings showing the effects of prior application sham operation or bilateral vagotomy on the mean arterial pressure (MAP) and heart rate (HR) responses to intra-NTS salusin b (4 pmol) in the NTS in SHR. b, c The effects of pretreatments with sham operation or bilateral vagotomy on the MAP and HR responses of intra-NTS salusin b (4 pmol) in SHR. n = 7 or 9 for each group. Values are mean ± SE
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salusin b within NTS in essential hypertension. The novel findings of the present study are: (1) salusin b expressed was increased in the PVN, NTS and RVLM in SHR compared with WKY rats; (2) intra-NTS application of salusin b decreased the MAP and HR in a dose-dependent manner in SHR; (3) intra-NTS microinjection of salusin b also improved baroreflex sensitivity of SHR; and (4) pretreatment with muscimol within the RVLM abolished the cardiovascular effects of salusin b in the NTS in SHR. These results indicate that increased salusin b in the NTS in SHR may be beneficial to decrease hypertension and improve baroreflex sensitivity functions possibly through suppression of presympathetic neurons in the RVLM. Salusins are detected strongly in a variety of human, rat and mouse tissues, serum and urine [1, 29]. Salusins are confirmed to exist in human plasma in their originally predicted forms [29, 30] indicating their possible role as peptide hormones in humans. Our study also found that the immunoreactivity and protein levels of salusin b were increased in the PVN, NTS and RVLM in SHR compared with in WKY rats. Furthermore, salusin b in the NTS reduced the MAP and HR in a dose-dependent manner in SHR. These results suggest that increased salusin b in the NTS of SHR may participate in hypertension. ABR plays a crucial role in
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the regulation of cardiovascular activities. ABR function was found impaired in hypertension, heart failure and diabetes [26]. The impairment of ABR function is related to the poor prognosis in patients with acute myocardial infarction (MI) or with heart failure [26, 31]. Baroreflex sensitivity is enhanced when the BP level is lowered by an antihypertensive drug. It is well known that the NTS plays a crucial role in mediating baroreflex pathway, because neuronal inhibition in the NTS decreased ABR function [26, 31, 32]. Our present work showed that bilateral microinjection of salusin b into NTS improved baroreflex sensitivity functions in SHR. Therefore, we conclude that salusin b may be beneficial for the treatment of hypertension. Salusin b stimulates the release of arginine vasopressin (AVP) and oxytocin from the rat neurohypophysis in vitro and also coexists with AVP in the hypothalamo-neurohypophyseal system of the rat under normal [1, 11, 33]. Previous study showed that human salusin b is a surrogate ligand of the mouse MrgA1 (mas-related G protein-coupled receptors); however, it could not activate human MrgA1 [34]. Because the exact receptor and post-receptor signaling pathways are unclear, it is difficult to elucidate the mechanisms of cardiovascular roles of salusin b. It is well established that the NTS is considered as the site of the first
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synapse of the visceral sensory inputs in the brainstem, including those related to baro-, chemo- and cardiopulmonary reflexes [15, 35]. Hence, it is reasonable for us to hypothesize that salusin b probably produce regulative actions in the NTS. In our study, we found that unilateral or bilateral microinjection of salusin b (0.4–40 pmol) into the NTS decreased the MAP and HR in SHR, very similar to those of glutamate within the NTS. The wide distribution of glutamate receptors in medulla [36] provides the potential that the cardiovascular functions of salusin b within NTS are probably mediated by glutamate receptors. However, pretreatment with a non-selective glutamate receptors antagonist KYN, which has been reported to effectively abolish the cardiovascular functions of glutamate within the NTS, could not attenuate the cardiovascular functions of intra-NTS salusin b in SHR in present study. These mentions shown above strongly struggle against the cardiovascular effects of salusin b within the NTS are mediated by glutamate receptor mechanisms. Shichiri et al. [1] indicates that the hypotension, bradycardia and cardiac dysfunction by an bolus intravenous injection of salusin b in rats are probably mediated by the cholinergic mechanism, and the cardiovascular functions of salusin b within the NTS in SHR might not be mediated by peripheral
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Fig. 8 a Representative original tracings showing the effects of pretreatments with vehicle (aCSF) or GABAA receptors agonist muscimol (100 pmol) into RVLM on the mean arterial pressure (MAP) and heart rate (HR) responses to intra-NTS salusin b (4 pmol) in SHR. b, c The effects of prior application of aCSF or muscimol (100 pmol) into RVLM on the MAP and HR responses to intra-NTS salusin b (4 pmol) in SHR. n = 7 or 9 for each group. Values are mean ± SE. *P \ 0.05 versus aCSF ? Salusin b
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cholinergic mechanisms because our present study showed that bilateral vagatomy had no significant effects on the MAP or HR of intra-NTS salusin b. However, the studies above could not explain the exact mechanism of the effects of salusin b in the NTS on the MAP and HR. The activation of excitatory amino acid receptors within the NTS are involved in the inhibition of presympathetic neurons in the RVLM [37]. In turn, presympathetic neurons in the RVLM project to sympathetic preganglionic neurons and provide the major drive for sympathetic vasomotor tone [38, 39]. We supposed that the cardiovascular effects of intra-NTS salusin b are also probably the results of inhibition of presympathetic neurons in the RVLM in SHR. To test this hypothesis, we applied muscimol into RVLM to selectively activate GABAA receptors in the RVLM in SHR. It has been reported that microinjection of muscimol into RVLM could silence most activities of presympathetic neurons [40]. Our study showed that the cardiovascular effects of intra-NTS salusin b was completely abolished by prior application of muscimol within the RVLM in SHR, indicating that the cardiovascular effects of intra-NTS salusin b is due to the result of inhibition of the presympathetic neurons within the RVLM in SHR.
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In conclusion, the data from this study demonstrate that salusin b excites neurons within the NTS, which might directly, or might, by suppressing the activities of presympathetic neurons in the RVLM produce inhibitory effects on the cardiovascular functions in SHR. The present study provides new information to expand our knowledge of the salusin b adjustment of cardiovascular activity. Acknowledgments This work was supported by National Basic Research Program of China (No. 2012CB517805) and National Natural Science Foundation of China (Nos. 30700266, 91439120, 81170248, 31171095, 81370356). Conflict of interest
None.
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Salusin b Within the Nucleus Tractus Solitarii Suppresses Blood Pressure Via Inhibiting the Activities of Presympathetic Neurons in the Rostral Ventrolateral Medulla in Spontaneously Hypertensive Rats Hong-Bao Li1 • Yan Lu2 • Jin-Jun Liu1 • Yu-Wang Miao1 • Tian-Zhen Zheng3 • Qing Su1 • Jie Qi1 • Hong Tan1 • Zu-Yi Yuan4 • Guo-Qing Zhu5 • Yu-Ming Kang1
Ó Springer Science+Business Media New York 2015
Abstract Salusin b is a newly identified bioactive peptide, which shows peripheral hypotensive, mitogenic and proatherosclerotic effects. The present study was undertaken to investigate the role of salusin b within the nucleus tractus solitarii (NTS) and the underlying mechanism in regulating blood pressure and heart rate (HR) in spontaneously hypertensive rats (SHR). Our results showed that bilateral or unilateral microinjection of salusin b (0.4–40 pmol) into the NTS in SHR decreased mean arterial pressure and HR in a dose-dependent manner. Bilateral microinjection of salusin b (4 pmol) within NTS improved baroreflex sensitivity functions in SHR. Pretreatment with glutamate receptors antagonist kynurenic acid (5 nmol) into the NTS in SHR did not alter the salusin b (4 pmol)
induced hypotension and bradycardia. Likewise, bilateral vagotomy also did not alter the salusin b (4 pmol) induced hypotension and bradycardia. However, pretreatment with GABAA receptors agonist muscimol (100 pmol) within the rostral ventrolateral medulla (RVLM) in SHR almost completely abolished the hypotension and bradycardia evoked by intra-NTS salusin b (4 pmol). Our findings suggested that microinjection of salusin b into the NTS produced hypotension and bradycardia, as well as improved baroreflex sensitivity functions, via inhibiting the activities of presympathetic neurons in the RVLM in SHR.
Keywords Salusin Nucleus tractus solitarii Rostral ventrolateral medulla Baroreflex Presympathetic neuron & Yan Lu [email protected] & Yu-Ming Kang [email protected] 1
Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Xi’an Jiaotong University Health Science Center, Xi’an 710061, China
2
Department of Clinical Laboratory, SanAiTang Hospital, Lanzhou 730030, China
3
Department of Physiology and Psychology, School of Basic Medical Sciences, Lanzhou University, Lanzhou 730000, China
4
Department of Cardiovascular Medicine, First Affiliated Hospital of Medical College of Xi’an Jiaotong University, Xi’an 710061, China
5
Key Laboratory of Cardiovascular Disease and Molecular Intervention, Department of Physiology, Nanjing Medical University, Nanjing 210029, China
Introduction Salusins derive from the prosalusin precursor molecule and regulate hemodynamics, mitogenesis and atherogenesis. Salusins are considered to be the source of two bioactive peptides, salusin a and salusin b, which comprise 28 and 20 amino acid residues, respectively. Salusins induce immediate early response genes and stimulate proliferation of human vascular smooth muscle cells (VSMCs) [1]. They can be concomitantly biosynthesized from their precursor, prosalusin, after alternative splicing and subsequent processing of the human torsion dystonia-related gene (TOR2A) [1]. Preprosalusin and its mRNA are widely expressed in both human tissues and rat tissues, including vasculature, central nervous system (CNS), lung and kidney [1–3]. Salusin b was also abundantly expressed in the CNS in both human and rats [3, 4].
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Previous studies show that salusins exert multiple functions in the cardiovascular, endocrine and immune systems [1, 5, 6]. Bolus intravenous administration of salusin b in rats produces hypotension, bradycardia and cardiac dysfunction, which is mainly mediated by the cholinergic mechanism [7]. In additional, salusins also stimulate the proliferation of VSMCs, cardiomyocytes and fibroblasts, participate in the formation of atherosclerosis, modulate the intracellular signaling pathways and may be a potential survival factor against serum deprivation-induced myocardial cell death in cardiomyocytes [2, 8, 9]. Preprosalusin and mRNA have been reported to exist abundantly in the hypothalamus and pituitary [3, 4]. Salusin b in the paraventricular nucleus (PVN) increases blood pressure (BP) and sympathetic outflow via vasopressin in hypertensive rats [10]. Salusin b causes potent hypotensive/ bradycardic effects [1], stimulates arginine vasopressin secretion from the neurohypophysis [1, 11] and exerts antiapoptotic and growth-promoting effects on cardiovascular cells [8]. Hypertension is characterized by elevated levels of BP and sympathetic tone, which are closely related to a poor prognosis in this disease [12]. It is well known that the nucleus tractus solitarii (NTS), which is situated at the dorsomedial part of medulla oblongata, serves as a putative gateway to various visceral sensory [13, 14] and plays a pivotal role in maintaining arterial baroreflex (ABR) and resting BP in cardiovascular system [14, 15]. Stimulation of baroreceptor afferents is generally thought to activate excitatory amino acid receptors within the NTS. The NTS at first integrates afferent signals and sends an excitatory transmitter, glutamic acid, to the caudal ventrolateral medulla (CVLM) and stimulates a GABAergic inhibitory pathway to the rostral ventrolateral medulla (RVLM) [16– 18]. However, it is unknown whether salusin b in the NTS is involved in regulating BP and baroreflex sensitivity functions. The present study was designed to determine whether salusin b in the NTS attenuates BP and improves baroreflex sensitivity functions, and the mechanisms of exogenous salusin b regulating cardiovascular activity in spontaneously hypertensive rats.
National Institutes of Health Guide for the Care and Use of Laboratory Animals. Salusin b and Anti-salusin b Serum Human salusin b was obtained from Phoenix Pharmaceuticals (CA, USA). Rabbit anti-salusin b (human) serum was obtained from Bachem (Bubendorf, Switzerland). The function of salusin b in rats was investigated by human salusin b and rabbit anti-salusin b (human) serum because human salusin b has been proved to be highly homologous to the rat salusin b [10, 19]. Rabbit anti-salusin b (human) serum was diluted in 0.01 M PBS (1:400) for immunohistochemistry or 5 % goat serum (1:1000) for western blot. Immunofluorescence Studies Immunofluorescence labeling was performed in floating sections as described previously [20–22] to identify salusin b expressions. Briefly, rats (n = 6 per group) were anaesthetized by a ketamine (80 mg/kg) and xylazine (10 mg/ kg) mixture (ip). The transcardiac perfusion was performed with 200 ml of 1 % phosphate buffer saline (PBS), followed by 400 ml of 4 % buffered paraformaldehyde (pH 7.4). Brains were removed and fixed in 4 % buffered paraformaldehyde for 48 h. Free-floating sections (18 lm) were incubated with 0.3 % Triton-X for 20 min and with 5 % goat serum for 30 min. The brain sections were then incubated with salusin b serum diluted in 0.01 M PBS at 4 °C overnight followed by incubation with Alexa 488-labeled anti-rabbit secondary antibody (1:200 green fluorescence) or Alexa 594-labeled anti-rabbit secondary antibody (1:200 red fluorescence) (Invitrogen, CA) for 60 min at 37 °C. The salusin b positive neurons in the RVLM, NTS and PVN were observed using a fluorescence microscope (Nikon, Tokyo, Japan). The number of neurons with salusin b immunoreactivity in the RVLM, NTS and PVN was determined by averaging the numbers of four sections for each animal. Western Blot Analysis
Materials and Methods Animals One hundred and fifty-six ten-week-old male normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) were used in this study. All animal and experimental procedures in this study were approved by the Animal Care and Use Committees of Xi’an Jiaotong University and were conducted in accordance with the US
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Proteins extracted from the PVN, NTS and RVLM were used for the measurement of salusin b expression by western blot. The procedures of western blot were described previously [23, 24]. The protein concentration was measured and loaded onto a SDS-PAGE gel and then transferred to a polyvinylidene fluoride membrane. The membrane was probed with salusin b serum and secondary antibody (1:2000, Santa Cruz Biotechnology). The protein bands were visually detected and analyzed. Protein loading was controlled by probing all blots with b-actin antibody
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(Santa Cruz Biotechnology) and normalizing salusin b protein intensity to that of b-actin. The bands were quantified using NIH Image J software.
solution was injected into the NTS and RVLM to mark the site. Baroreflex Sensitivity (BRSHP)
General Experimental Procedure The methods for animal preparation and microinjection were similar to those described previously [25, 26]. Briefly, rats were anesthetized with a a-chloralose (80 mg/kg) and urethane (500 mg/kg) mixture (ip). Then, the right femoral artery and vein were cannulated with polyethylene catheters for the measurement of arterial BP and drug administration, respectively. The catheters were prior filled with heparinized saline (50 U/ml) or 0.9 % NaCl. BP and (heart rate) HR were measured with an analysis software (RM6240, China) by a computer, and heart period (HP) was monitored by the differential signal of BP pulse. A trachea was cannulated and connected to animal ventilator (WJ-2000, China). a-Chloralose (25 mg/kg) was intravenously supplemented when necessary. Adequacy of anesthesia was assessed by monitoring the stability of BP and HP. The anesthetized rats were fixed in the stereotaxic apparatus (MP-8003, China). The dorsal surface of medulla oblongata was exposed by removing the partial occipital and cerebellum. In this process, an infrared heating lamp was utilized to keep rat body temperature at 37 °C. Microinjection Procedure According to stereotaxic coordinates, the multi-barreled micropipette (tip diameter 20–30 lm) was inserted into the NTS (0.4–0.5 mm rostral to the calamus scriptorius, 0.5–0.6 mm lateral to the midline and 0.4–0.5 mm below the dorsal surface of the medulla) or RVLM (2.0–2.5 mm rostral to the calamus scriptorius, 1.8–2.0 mm lateral to the midline and 2.8–3.2 mm below the dorsal surface of the medulla). The micropipette was filled with L-glutamate, salusin b, glutamate receptors antagonist kynurenic acid (KYN) or GABAA receptors agonist muscimol. KYN was initially dissolved in 10 % sodium hydroxide (NaOH) and finally diluted with artificial cerebrospinal fluid (aCSF, in mM: 133.3 NaCl, 3.4 KCl, 1.3 CaCl2, 1.2 MgCl2, 0.6 NaH2PO4, 32.0 NaHCO3 and 3.4 glucose, pH 7.4). The other drugs were dissolved into aCSF. The dose of salusin b, KYN, and muscimol were based on previous studies [10, 27]. The bilateral or unilateral microinjections were completed within 10–30 s, and the microinjection volume was 100 nl for each microinjection site. Functional location of NTS or RVLM on either side was carried out at the beginning of each experiment by eliciting a transient decrease or increase in mean arterial pressure (MAP) (15–25 mmHg) by microinjection of glutamate (2 nmol), respectively [26]. Finally, 20 nl of 2 % pontamine sky blue
Rats were anesthetized with a-chloralose (80 mg/kg, ip) and urethane (500 mg/kg, ip). The bradycardiac response to phenylephrine was measured by using previously described method [28]. A rapid bolus injection of phenylephrine was used to induce a BP elevation and then baroreceptor activation. The dose of phenylephrine (2–10 lg/kg) was adjusted to elevate systolic blood pressure (SBP) between 20 and 40 mmHg. There exists a delay between the elevation of BP (stimulus) and the prolongation of heart period HP (response) for ABR. In rat, HR is about 7 per second. HP was plotted against SBP for linear regression analysis with 2–8 shifts; the slope of SBP-HP was expressed as baroreflex sensitivity (BRSHP) (ms/mmHg). The data were recorded using a computer at three time points (preadministration, and 5 and 30 min after medication). Histological Analysis At the end of the experiment, the animal was perfused transcardially with 0.9 % NaCl and 10 % phosphate-buffered formalin. The brain tissue was stored overnight in 10 % phosphate-buffered formalin and then transferred to fixative containing 30 % sucrose. The frozen brain tissue was sectioned coronally (50 lm) and stained with neutral red. Rats with microinjection sites outside the NTS or RVLM were excluded from data analysis. Experimental Protocol Experiment 1 The immunoreactivity and protein levels of salusin b were investigated in the PVN, NTS and RVLM in WKY rats and SHR (four sections for each rat and six rats for each group). Experiment 2 Sixty-eight anesthetized and paralyzed SHR, which were randomized into 8 groups (n = 7–9 rats/group), bilateral or unilateral received application of salusin b (0.4, 4, and 40 pmol) or aCSF (100 nl) to test the dose-dependent effects of exogenous salusin b within the NTS. Experiment 3 Sixteen anesthetized SHR were randomly divided into two groups (n = 7–9 rats/group), which were subjected to the NTS microinjection of aCSF (100 nl) or salusin b (4 pmol)
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Fig. 3 a Representative original tracings showing mean arterial pressure (MAP) and heart rate (HR) responses to bilateral microinjection of vehicle (aCSF) or salusin b (0.4, 4, 40 pmol) into the NTS in anaesthetized and paralyzed SHR. b, c Line graph showing the
effects of bilateral microinjection of aCSF or salusin b (0.4, 4, 40 pmol) into the NTS on the MAP and HR in SHR. n = 7 or 9 for each group. Values are mean ± SE. *P \ 0.05 versus aCSF
to observe the effects of exogenous salusin b within NTS on the baroreflex sensitivity functions.
Experiment 6
Experiment 4 Sixteen anesthetized SHR were randomly divided into two groups (n = 7–9 rats/group), which were subjected to the NTS microinjection of aCSF (100 nl), glutamate receptors antagonist KYN (5 nmol), salusin b (4 pmol) pretreated with aCSF or salusin b pretreated with KYN. Salusin b was administered 10 min after the pretreatment. Experiment 5 Sixteen anesthetized SHR were successively prior applied bilateral vagus dissection or sham operation before intraNTS salusin b (4 pmol) in SHR (n = 7–9 rats/group) to identify whether the cardiovascular responses to intra-NTS salusin b were mediated by the cholinergic mechanism.
Sixteen anesthetized SHR were randomly divided into two groups (n = 7–9 rats/group), which were subjected to the RVLM microinjection of aCSF (100 nl) or a specific GABAA receptors agonist muscimol (100 pmol) before intra-NTS salusin b (4 pmol) in SHR. Salusin b was administered 10 min after the pretreatment.
Statistical Analysis Data were shown as mean ± SE and analyzed using Student’s t test and one-way analysis of variance (ANOVA). Regression analysis was made to determine the correlation between the doses and responses. Statistical comparison was performed using Prism 5 (GraphPad Software). Differences were considered to be significant at P \ 0.05.
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Fig. 4 a Representative original tracings showing mean arterial pressure (MAP) and heart rate (HR) responses to unilateral microinjection of vehicle (aCSF), Glu or salusin b (0.4, 4, 40 pmol) into the NTS in anaesthetized and paralyzed SHR. b, c Line graph showing
the effects of unilateral microinjection of aCSF, Glu or salusin b (0.4, 4, 40 pmol) into the NTS on the MAP and HR in SHR. n = 7 or 9 for each group. Values are mean ± SE. *P \ 0.05 versus aCSF
Results
The Cardiovascular Effects of Bilateral Microinjection of Salusin b (0.4–40 pmol) into the NTS
The Immunoreactivity of Salusin b in the PVN, NTS and RVLM As shown in Fig. 1, the number of salusin b positive neurons in the PVN, NTS and RVLM was significantly increased in SHR compared with WKY rats. The Protein Expression of Salusin b in the PVN, NTS and RVLM Western blot also further demonstrated that SHR had higher expression of salusin b in the PVN, NTS and RVLM than in WKY rats (Fig. 2).
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Intra-NTS in SHR injection of aCSF did not alter the basal MAP (157 ± 8 vs. 155 ± 7 mmHg, P [ 0.05) and HR (369 ± 18 vs. 367 ± 19 bpm, P [ 0.05). However, microinjection of salusin b (0.4–40 pmol) into the NTS decreased the MAP (0.4 pmol: -10 ± 2 mmHg; 4 pmol: -16 ± 5 mmHg; 40 pmol: -21 ± 7 mmHg vs. aCSF: -2 ± 1 mmHg, P \ 0.05) and HR (0.4 pmol: -9 ± 3 bpm; 4 pmol: -13 ± 6 bpm; 40 pmol: -19 ± 7 bpm vs. aCSF: -2 ± 2 bpm, P \ 0.05) in a dose-related manner in SHR. There was a significant linear correlation between the dose of salusin b and MAP or HR change in SHR (Fig. 3).
Cardiovasc Toxicol
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slope = 0.312 r = 0.405
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Fig. 5 Responses of baroreflex sensitivity (BRSHP) before and after microinjection of salusin b (4 pmol) or vehicle (aCSF) into the NTS in SHR. Microinjection of salusin b into the NTS produces a significant increase in the BRSHP of SHR compared with pretreatment
with vehicle (aCSF). n = 7 or 9 for each group. Values are mean ± SE. *P \ 0.05 versus aCSF. SBP indicates systolic blood pressure; HP heart period; values of slope are the values of baroreflex sensitivity
The Cardiovascular Effects of Unilateral Microinjection of Salusin b (0.4–40 pmol) into the NTS
The Effects of Intra-NTS Salusin b (4 pmol) on the Baroreflex Sensitivity (BRSHP)
Intra-NTS injection of aCSF produced no significant influences on the basal MAP (164 ± 4 vs. 161 ± 3 mmHg, P [ 0.05) or HR (374 ± 10 vs. 372 ± 19 bpm, P [ 0.05) of SHR. Unilateral microinjection of salusin b into the NTS decreased the MAP (0.4 pmol: -14 ± 2 mmHg; 4 pmol: -20 ± 3 mmHg; 40 pmol: -24 ± 8 mmHg vs. aCSF: -2 ± 1 mmHg, P \ 0.05) and HR (0.4 pmol: -13 ± 3 bpm; 4 pmol: -19 ± 4 bpm; 40 pmol: -22 ± 9 bpm vs. aCSF: -2 ± 2 bpm, P \ 0.05) in a dose-related manner in SHR. There was a significant linear correlation between the dose of salusin b and MAP or HR change in SHR (Fig. 4).
Bilateral microinjection of salusin b (4 pmol) improved the BRSHP of SHR (before microinjection: 0.312 ± 0.02 ms/ mmHg vs. 5 min after microinjection: 0.457 ± 0.06 ms/ mmHg; 30 min after microinjection: 0.494 ± 0.06 ms/ mmHg, P \ 0.05; Fig. 5). The Effects of Pretreatment with Glutamate Receptors Antagonist KYN on the Cardiovascular Responses of Intra-NTS Salusin b (4 pmol) Prior unilateral microinjection of aCSF in SHR did not alter the MAP and HR responses of intra-NTS salusin b. Unilateral injection of glutamate receptors antagonist KYN
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(5 nmol) into the NTS in SHR significantly raised the basal MAP (154 ± 5 vs. 167 ± 7 mmHg, P \ 0.05) and HR (354 ± 15 vs. 371 ± 17 bpm, P \ 0.05). However, pretreatment with KYN did not affect MAP (-18 ± 5 mmHg with KYN vs. -16 ± 4 mmHg with vehicle, P [ 0.05) and HR (-25 ± 5 bpm with KYN vs. -22 ± 3 bpm with vehicle, P [ 0.05) responses of salusin b within the NTS of SHR (Fig. 6).
The Effects of Bilateral Vagotomy on the Cardiovascular Functions of Intra-NTS Salusin b (4 pmol) Sham operation had no significant effects on the MAP and HR responses of intra-NTS salusin b in SHR. Bilateral vagatomy increased in the basal MAP (151 ± 8 vs. 164 ± 5 mmHg, P \ 0.05) and HR (342 ± 13 vs. 354 ± 11 bpm, P \ 0.05) of SHR. However, bilateral vagatomy did not alter the MAP (-13 ± 4 mmHg with vagatomy vs. -15 ± 5 mmHg with sham, P [ 0.05) and HR (-17 ± 3 bpm with vagatomy vs. -20 ± 3 bpm with sham, P [ 0.05) responses induced by bilateral microinjection of salusin b (4 pmol) within the NTS of SHR (Fig. 7).
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KYN + salusin (n=9)
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Fig. 6 a Representative original tracings showing the effects of prior application of vehicle (aCSF) or glutamate receptor antagonist kynurenic acid (KYN, 5 nmol) into NTS on the mean arterial pressure (MAP) and heart rate (HR) responses to intra-NTS salusin b (4 pmol) in SHR. b, c The effects of pretreatments with aCSF or glutamate receptor antagonist kynurenic acid (KYN, 5 nmol) on the MAP and HR responses of intra-NTS salusin b (4 pmol) in SHR. n = 7 or 9 for each group. Values are mean ± SE
KYN + salusin (n=9)
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The Effect of Prior Microinjection of GABAA Receptors Agonist Muscimol into the RVLM on the Cardiovascular Responses to Microinjection of Salusin b (4 pmol) into the NTS Prior unilateral microinjection of aCSF in SHR did not alter the MAP and HR responses of salusin b within the NTS of SHR. Unilateral microinjection of GABAA receptors agonist muscimol into the RVLM produced a significant decrease in basal MAP (155 ± 6 vs. 132 ± 5 mmHg, P \ 0.05) and HR (371 ± 15 vs. 354 ± 10 bpm, P \ 0.05) of SHR. Notable, pretreatment with muscimol within the RVLM of SHR completely abolished the MAP (-2 ± 2 mmHg with muscimol vs. -18 ± 2 mmHg with vehicle, P \ 0.05) and HR (-4 ± 2 bpm with muscimol vs. -28 ± 4 bpm with vehicle, P \ 0.05) responses induced by application of salusin b into the NTS of SHR (Fig. 8).
Discussion The NTS is a key region for central control of BP and plays a pivotal role in maintaining ABR. In the present study, we investigated the effects and its possible mechanism of
Cardiovasc Toxicol Fig. 7 a Representative original tracings showing the effects of prior application sham operation or bilateral vagotomy on the mean arterial pressure (MAP) and heart rate (HR) responses to intra-NTS salusin b (4 pmol) in the NTS in SHR. b, c The effects of pretreatments with sham operation or bilateral vagotomy on the MAP and HR responses of intra-NTS salusin b (4 pmol) in SHR. n = 7 or 9 for each group. Values are mean ± SE
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salusin b within NTS in essential hypertension. The novel findings of the present study are: (1) salusin b expressed was increased in the PVN, NTS and RVLM in SHR compared with WKY rats; (2) intra-NTS application of salusin b decreased the MAP and HR in a dose-dependent manner in SHR; (3) intra-NTS microinjection of salusin b also improved baroreflex sensitivity of SHR; and (4) pretreatment with muscimol within the RVLM abolished the cardiovascular effects of salusin b in the NTS in SHR. These results indicate that increased salusin b in the NTS in SHR may be beneficial to decrease hypertension and improve baroreflex sensitivity functions possibly through suppression of presympathetic neurons in the RVLM. Salusins are detected strongly in a variety of human, rat and mouse tissues, serum and urine [1, 29]. Salusins are confirmed to exist in human plasma in their originally predicted forms [29, 30] indicating their possible role as peptide hormones in humans. Our study also found that the immunoreactivity and protein levels of salusin b were increased in the PVN, NTS and RVLM in SHR compared with in WKY rats. Furthermore, salusin b in the NTS reduced the MAP and HR in a dose-dependent manner in SHR. These results suggest that increased salusin b in the NTS of SHR may participate in hypertension. ABR plays a crucial role in
Changes in HR (bpm)
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the regulation of cardiovascular activities. ABR function was found impaired in hypertension, heart failure and diabetes [26]. The impairment of ABR function is related to the poor prognosis in patients with acute myocardial infarction (MI) or with heart failure [26, 31]. Baroreflex sensitivity is enhanced when the BP level is lowered by an antihypertensive drug. It is well known that the NTS plays a crucial role in mediating baroreflex pathway, because neuronal inhibition in the NTS decreased ABR function [26, 31, 32]. Our present work showed that bilateral microinjection of salusin b into NTS improved baroreflex sensitivity functions in SHR. Therefore, we conclude that salusin b may be beneficial for the treatment of hypertension. Salusin b stimulates the release of arginine vasopressin (AVP) and oxytocin from the rat neurohypophysis in vitro and also coexists with AVP in the hypothalamo-neurohypophyseal system of the rat under normal [1, 11, 33]. Previous study showed that human salusin b is a surrogate ligand of the mouse MrgA1 (mas-related G protein-coupled receptors); however, it could not activate human MrgA1 [34]. Because the exact receptor and post-receptor signaling pathways are unclear, it is difficult to elucidate the mechanisms of cardiovascular roles of salusin b. It is well established that the NTS is considered as the site of the first
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synapse of the visceral sensory inputs in the brainstem, including those related to baro-, chemo- and cardiopulmonary reflexes [15, 35]. Hence, it is reasonable for us to hypothesize that salusin b probably produce regulative actions in the NTS. In our study, we found that unilateral or bilateral microinjection of salusin b (0.4–40 pmol) into the NTS decreased the MAP and HR in SHR, very similar to those of glutamate within the NTS. The wide distribution of glutamate receptors in medulla [36] provides the potential that the cardiovascular functions of salusin b within NTS are probably mediated by glutamate receptors. However, pretreatment with a non-selective glutamate receptors antagonist KYN, which has been reported to effectively abolish the cardiovascular functions of glutamate within the NTS, could not attenuate the cardiovascular functions of intra-NTS salusin b in SHR in present study. These mentions shown above strongly struggle against the cardiovascular effects of salusin b within the NTS are mediated by glutamate receptor mechanisms. Shichiri et al. [1] indicates that the hypotension, bradycardia and cardiac dysfunction by an bolus intravenous injection of salusin b in rats are probably mediated by the cholinergic mechanism, and the cardiovascular functions of salusin b within the NTS in SHR might not be mediated by peripheral
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Salusin
aCSF
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Changes in HR (bpm)
Fig. 8 a Representative original tracings showing the effects of pretreatments with vehicle (aCSF) or GABAA receptors agonist muscimol (100 pmol) into RVLM on the mean arterial pressure (MAP) and heart rate (HR) responses to intra-NTS salusin b (4 pmol) in SHR. b, c The effects of prior application of aCSF or muscimol (100 pmol) into RVLM on the MAP and HR responses to intra-NTS salusin b (4 pmol) in SHR. n = 7 or 9 for each group. Values are mean ± SE. *P \ 0.05 versus aCSF ? Salusin b
-15
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*
cholinergic mechanisms because our present study showed that bilateral vagatomy had no significant effects on the MAP or HR of intra-NTS salusin b. However, the studies above could not explain the exact mechanism of the effects of salusin b in the NTS on the MAP and HR. The activation of excitatory amino acid receptors within the NTS are involved in the inhibition of presympathetic neurons in the RVLM [37]. In turn, presympathetic neurons in the RVLM project to sympathetic preganglionic neurons and provide the major drive for sympathetic vasomotor tone [38, 39]. We supposed that the cardiovascular effects of intra-NTS salusin b are also probably the results of inhibition of presympathetic neurons in the RVLM in SHR. To test this hypothesis, we applied muscimol into RVLM to selectively activate GABAA receptors in the RVLM in SHR. It has been reported that microinjection of muscimol into RVLM could silence most activities of presympathetic neurons [40]. Our study showed that the cardiovascular effects of intra-NTS salusin b was completely abolished by prior application of muscimol within the RVLM in SHR, indicating that the cardiovascular effects of intra-NTS salusin b is due to the result of inhibition of the presympathetic neurons within the RVLM in SHR.
Cardiovasc Toxicol
In conclusion, the data from this study demonstrate that salusin b excites neurons within the NTS, which might directly, or might, by suppressing the activities of presympathetic neurons in the RVLM produce inhibitory effects on the cardiovascular functions in SHR. The present study provides new information to expand our knowledge of the salusin b adjustment of cardiovascular activity. Acknowledgments This work was supported by National Basic Research Program of China (No. 2012CB517805) and National Natural Science Foundation of China (Nos. 30700266, 91439120, 81170248, 31171095, 81370356). Conflict of interest
None.
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