Orexin A regulates cardiovascular responses in stress-induced hypertensive rats

Neuropharmacology 67 (2013) 16e24

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Orexin A regulates cardiovascular responses in stress-induced hypertensive rats Fen Xiao a,1, Meiyan Jiang a, b,1, Dongshu Du a, Chunmei Xia a, Jin Wang a, Yinxiang Cao a, Linlin Shen a, Danian Zhu a, * a b

Department of Physiology and Pathophysiology, Shanghai Medical College of Fudan University, 138 Yixueyuan Road, Shanghai 200032, PR China Oregon Hearing Research Center, Oregon Health & Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97239, USA

a r t i c l e i n f o

a b s t r a c t

Article history: Received 3 May 2012 Received in revised form 16 October 2012 Accepted 20 October 2012

Several pieces of evidence indicate that the rostral ventrolateral medulla (RVLM) is probably one of the key neural structures mediating the pressor effects of orexins in the brain. Nitric oxide synthase/nitric oxide (NOS/NO) system in the RVLM modulates cardiovascular activities. Our experiments were designed to test the hypothesis that orexin-A (OXA) is involved in the mechanism of stress-induced hypertension (SIH) by adjusting NOS/NO system in the RVLM. The stress-induced hypertensive rats (SIHR) model was established by electric foot-shocks and noises. Here we examined the expression of OXA immunoreactive (OXA-IR) cells in the lateral hypothalamus (LH) and the protein level of orexin 1 receptor (OX1R) in the RVLM of SIHR, and we found that the expressions of OXA-IR and OX1R were higher than those of the control group. The double-staining immunohistochemical evidence showed that OX1R immunoreactive (OX1R-IR) cells and neuronal nitric oxide synthase (nNOS) or inducible nitric oxide synthase (iNOS) immunoreactive (IR) cells were co-localizated in the RVLM. Microinjection of OXA (10, 50, 100 pmol/ 100 nl) into the unilateral (right) RVLM of control rats or SIHR produced pressor and tachycardiac effects in a dose-dependent manner. SB-408124 (100 pmol/100 nl, an antagonist of OX1R) or TCS OX2 29 (100 pmol/100 nl, an antagonist of OX2R) partly abolished the cardiovascular effects of exogenouslyadministrated OXA into the RVLM of control rats and SIHR, and lowered the increased systolic blood pressure (SBP) and heart rate (HR) of SIHR, with no difference in statistical significance between the two antagonists’ effects. Microinjection into the RVLM of both control and SIHR groups of 7-Ni (0.05 pmol/ 100 nl, nNOS inhibitor) or Methylene Blue [100 pmol/100 nl, an inhibitor of soluble guanylate cyclase (sGC)] suppressed the OXA-induced increase of SBP and HR, whereas microinjection of AG (1, 10, 100 pmol/100 nl) had no obvious effects on the OXA-induced increase of SBP and HR. Our results indicate that OXA in the RVLM may participate in the central regulation of cardiovascular activities in SIHR, and OX1R and OX2R both have important roles in it. The cardiovascular effects of OXA in the RVLM may be induced by nNOS-derived NO, which activated sGC-associated signaling pathway. Ó 2012 Elsevier Ltd. All rights reserved.

Keywords: Orexin A Rostral ventrolateral medulla Lateral hypothalamus Stress-induced hypertension Nitric oxide synthase/nitric oxide Systolic blood pressure Heart rate

1. Introduction Since the discovery as a hypothalamic peptide in 1998, orexin A and B (OXA/OXB), also known as hypocretin 1 and 2, have been recognized for their diverse functions in appetite, arousal (Fukuda et al., 2007; Furlong et al., 2009), metabolism (Yi et al., 2009), regulation of sleep-wakefulness (Chemelli et al., 1999) and neuroendocrine function (de Lecea et al., 1998; Sakurai et al., 1998; Shirasaka et al., 1999). These peptides form from the

* Corresponding author. Tel./fax: þ86 21 54237405. E-mail address: [email protected] (D. Zhu). 1 These authors contributed equally to this work. 0028-3908/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuropharm.2012.10.021

precursor prepro-orexin (PPO), and exert their effects by targeting two G protein-coupled receptors, the OX1 and OX2 receptors (OX1R and OX2R), with different affinities. OXA binds to both OX1R and OX2R with equal affinity, whereas OXB binds to OX2R with a 10-fold greater affinity than to OX1R (Sakurai et al., 1998). Immunohistochemical and in situ hybridization studies have revealed that orexins (OX)-containing cell bodies are restricted to the lateral hypothalamus (LH), perifornical area (PeF), and dorsomedial hypothalamus (DMH) (Shahid et al., 2011). OXcontaining nerve terminals and receptors, on the other hand, are widely distributed throughout the brain, including the brainstem area that includes the rostral ventrolateral medulla (RVLM) (Chen et al., 1999; Llewellyn-Smith et al., 2003; Peyron et al., 1998).

F. Xiao et al. / Neuropharmacology 67 (2013) 16e24

Ample evidence indicates a probable contribution of OXA to the central cardiovascular regulation. When given by an intracerebroventricular (Samson et al., 1999; Shirasaka et al., 1999), or intrathecal (Antunes et al., 2001) injection, OXA increases arterial pressure (AP), heart rate (HR), and sympathetic activity in rats and rabbits. However, some targeted structures may play a more important role than others. The RVLM is known as a critical cardiovascular nucleus that contains cardiac presympathetic neurons. Indeed, intracisternal- and RVLM-injected OX in anesthetized rats evoke rises in blood pressure and HR (Chen et al., 2000), with OXA being more effective. And injections of OXA in the vasopressor region of the RVLM depolarize vasopressor neurons (Huang et al., 2010). These data indicate that the RVLM is probably one of the key neural structures mediating the pressor effects of OXA in the brain. The neuropeptide OXA is involved in the control of arousal and has been implicated in many different states and behaviors, such as wakefulness, emotional stress, feeding, and addiction (de Lecea, 2010; Sakurai et al., 2010). OXA plays a key role in the stabilization of wakefulness and is thought to be an arousal-promoting peptide (Carter et al., 2009). Localization of orexinergic cell bodies in the PeF and DMH, which overlap the “defense area”, prompted us to determine the possible role of OXA in the defense response against stressors (Kuwaki and Zhang, 2010). Studies showed that blood pressure in ORX-KO (prepro-orexin knockout) and ORX-AB (orexin neuron-ablated) mice was significantly lower by about 20 mmHg than that of the wild-type controls in both anesthetized and conscious conditions (Kayaba et al., 2003; Zhang et al., 2006). Approximately 50% of hypothalamic neurons that innervate both the sympathetic efferent motor cortex and medial prefrontal cortex, which are implicated in mental stress, show OXlike immunoreactivity (Krout et al., 2003). Together these findings suggested that OXA took part in the defense and cardiovascular regulation. However, it is currently unknown whether OXA neurons and OXR would be activated by foot-shocks combined with noises for l4 consecutive days which could induce stress-induced hypertension (SIH) (Huang et al., 2005), and the underlying mechanisms would mediate the OXA-induced cardiovascular effects. It is reported that an injection of TCS OX2 29, a selective OX2R antagonist, into the cisterna magna could suppress intracisternal OXA-induced increases of AP and HR, whereas the effects of SB-334867, a selective OX1R antagonist, is not obvious. It is concluded that the cardiovascular functions of OX in the RVLM are more dependent on OX2R than on OX1R (Huang et al., 2010). Our experiments were designed to find out whether the same results would be presented in the SIHR. As a neurotransmitter, nitric oxide (NO) plays an important role in a variety of physiological and pathophysiological processes, such as in the regulation of vascular tone and central cardiovascular activities (Chan et al., 2003). NO is an important neurotransmitter for central cardiovascular regulation in the RVLM (Chan et al., 2003; Chang et al., 2003). Nitric oxide synthase (nNOS) or inducible nitric oxide synthase (iNOS) mRNA are present in the ventrolateral medulla (Chan et al., 2001). Our previous studies showed that after l4 consecutive days’ stimulation of foot-shocks combined with noises, the systolic blood pressure (SBP) of stress-induced hypertensive rats (SIHR) was increased significantly, accompanied with an increase in expression of nNOS and an decrease in expression of iNOS in the RVLM (Huang et al., 2005). NO derived only from nNOS exerts pressor effects (Chan et al., 2003), whereas NO derived from endothelial NO synthase (eNOS) or iNOS has depressor effects (Chan et al., 2003; Kishi et al., 2001). One of the mechanisms for NO-induced actions is through stimulation of sGC, which, in turn, catalyzes production of intracellular cyclic guanosine-30 , 50 -monophosphate (cGMP) (Arnold et al., 1977). This NO-sGC-cGMP cascade

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is involved in various NO-induced effects in the brain, including NO-mediated cardiovascular responses in the RVLM (Chan et al., 2003; Wu et al., 2001). OX-IR cells in the hypothalamus of the Long-Evans rats have co-expression with NOS-IR cells (Cheng et al., 2003). We investigated whether OX1R-IR cells and NOS-IR cells coexpressed in the RVLM and what was their relationship in the mechanism of SIH. 2. Methods 2.1. Experimental reagents The reagents and detection kits are described as following: L-glutamate, OXA, N(6, 8-difluoro-2-methyl-4-quinolinyl)-N0 -[4-(dimethylamino) phenyl] urea (SB408124), 7-nitroindazole (7-Ni), aminoguanidine (AG) and Methylene Blue, and rabbit polyclonal antibodies against OXA (1:1000) (Sigma-Aldrich, St Louis, Mo, USA); (2S)-1-(3,4-dihydro-6,7-dimethoxy-2(1H)-isoquinolinyl)-3,3-dimethyl-2-[(4pyridinylmethyl)amino]-1-butanone hydrochloride (TCS OX2 29) (Tocris, Minneapolis, MN, USA); 40 ,6-diamidino-2-phenylindole (DAPI), goat polyclonal anti-OX1R (1:300), rabbit polyclonal anti-Neurofilament (1:100), rabbit polyclonal anti-nNOS and rabbit polyclonal anti-iNOS antibody (1:200 separately) (Santa Cruz Biotechnology, CA, USA); cy3-labeled donkey anti-goat IgG, Fluorescein isothiocyanate (FITC)-labeled donkey anti-rabbit IgG, mouse polyclonal antibodies against b-actin and ECL-Plus detection kit (Beyotime Institute of Biotechnology, Haimen, Jiangsu, China); anti-rabbit IgG, avidin-biotin-peroxidase complex (ABC) and 3, 3diaminobenzidine (DAB) (Shanghai Shen-Hang Bio-Tech Co., Ltd., Shanghai, China). Chemicals microinjected into the RVLM include: L-glutamate (2 nmol), OXA (10, 50, 100 pmol), AG (1, 10, 100 pmol) and Methylene Blue (100 pmol), which were dissolved in the artificial cerebrospinal fluid (aCSF) (pH 7.4, composition in mM: NaCl 130, KCl 2.99, CaCl2 0.98, MgCl2$6H2O 0.80, NaHCO3 25, Na2HPO4$12H2O 0.039, NaH2PO4$2H2O 0.46); SB-408124 (100 pmol), TCS OX2 29 (100 pmol) and 7-Ni (0.05 pmol) were dissolved in 1% dimethyl sulfoxide (DMSO) in aCSF. All injections were made in a volume of 100 nl within 1 min. The doses of 7-Ni, AG or Methylene Blue were chosen based on separate experiments, which showed that unilateral microinjections of individual agents, at the doses used, had no significant effect on basel SBP and HR of control rats and SIHR. 2.2. Animal preparations Adult Sprague-Dawley rats (male, 250e300 g) were used in all experiments. They were housed on a 12-h light/dark cycle with food and water ad libitum and room temperature maintained at 23  Ce24  C. These studies were performed in accordance with the Guiding Principles for Research Involving Animals and Human Beings. The SIHR model was established as before (Xia et al., 2008). Briefly, animals were randomly divided into normotensive (control; n ¼ 10) and SIHR (n ¼ 10) groups. Animals in the SIHR group were placed in a cage (22 cm  22 cm  28 cm) with a grid floor and subjected to electric foot-shocks. The delivery of intermittent electric shocks (75e150 V, 0.5 ms duration) through the grid floor every 2e30 s was randomly controlled by a computer. Noises (88e98 dB) produced by a buzzer were given synchronously as a conditioned stimulus. The computer program was designed to give two types of stimulus: noises plus electric foot-shocks and noises only, which were delivered randomly. On the 6th day when SBP and HR of stressed group were increased to a stable level, the computer linked to the stress apparatus was adjusted to decrease the times of electric foot-shocks gradually and prolonged the interval between shocks until just noises remained. The control group of rats underwent sham stress; they were put into the same cage for the same period of time, but were not subjected to foot-shocks or noises. Rats were subjected to stress for 2 h twice daily for l4 consecutive days. Two hours after stress, SBP was measured in conscious condition by using the tail-cuffed method. Measurements were repeated three times and the average value was taken as the SBP. 2.3. Immunohistochemistry Animals were anesthetized with sodium pentobarbitone (100 mg/kg, i.p.) and perfused through the ascending aorta with 200 ml heparinized saline followed by 200 ml freshly prepared 4% paraformaldehyde in 0.1 M phosphate buffer saline (PBS, pH 7.4). After the perfusion, the brain tissues were removed from the skull and postfixed in 4% paraformaldehyde at 4  C for 2 h. Then the brain tissues were placed in 20% sucrose at 4  C until they sunk to the bottom. After that the brain tissues were placed in 30% sucrose at 4  C until they sunk to the bottom. The LH-PeF (1.0e5.0 mm behind the bregma) was cut according to the atlas of Paxinos and Watson (Paxinos and Watson, 2007). Coronal sections of the brain tissues (30 mm) were made with a cryostat microtome (Leica CM 1900, Germany). Immunohistochemical visualization was performed by using a conventional ABC procedure. All experiments were conducted at room temperature, unless stated otherwise. A commercially available antibody against OXA (1:1000) was used to stain one set of free-floating sections from each rat for OXA. Preparations were incubated at 37  C for one hour, and then

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incubated at 4  C overnight with the diluted primary antibody. After being rinsed with PBS, sections were subjected to anti-rabbit IgG and subsequently to ABC complex. Then, DAB was applied to visualize immunostaining. Sections were examined using light microscope to check OXA immunoreactive (OXA-IR) neurons in the LH-PeF. Regions of the LH-PeF was identified based on the rat brain stereotaxic atlas of Paxinos and Watson (Paxinos and Watson, 2007). The staining density of OXA-IR neurons in the LH-PeF was evaluated as absolute numbers, as well as the relative optical density (ROD), which was calculated at (ODOXA  ODbackground)/ODbackground as reported before (Liu et al., 2010). Image analysis of OXA-IR neurons number and its ROD were performed by using Image Measurement Version 1.00 (Department of Physiology and Pathophysiology, Shanghai Medical College of Fudan University, China). 2.4. Double immunofluorescence staining Animals were processed for double immunofluorescence staining to find out whether OX1R immunoreactive (OX1R-IR) cells were co-localizated with nNOS or iNOS or Neurofilament-IR cells in the RVLM. Rats were anesthetized with a mixture of urethane and chloralose (Liu et al., 2010) (140 g urethane, 7 g chloralose and 7 g borax per 1 L normal saline) in a dose of 7 ml/kg, i.p. In brief, free-floating sections of the medulla oblongata were incubated simultaneously with two primary antibodies. These included a goat polyclonal anti-OX1R (1:300), together with a rabbit polyclonal anti-Neurofilament (1:100), a rabbit polyclonal anti-nNOS or a rabbit polyclonal anti-iNOS antibody (1:200 separately). The same sections were subsequently incubated concurrently with two appropriate secondary antibodies (1:100). These included donkey anti-goat IgG conjugated with cy3 for OX1R and donkey anti-rabbit IgG conjugated with FITC for nNOS, iNOS or Neurofilament. Then the same sections were incubated in DAPI (0.1 mg/ml) for 10 min at room temperature. The processed sections were examined through a Leica DM IRB microscope equipped with Leica C-plan optics. Photomicrographs were taken with a Leica DC300F digital camera using IM50 software and saved as JPEG images. And the sections were viewed under the laser scanning confocal microscope (Leica Microsystems, Bensheim, Germany). Immunoreactivity for individual OX1R exhibited red fluorescence, NOS isoforms or Neurofilament immunoreactivity exhibited green fluorescence, DAPI exhibited blue fluorescence when viewed, and co-localization of OX1R immunoreactivity with NOS isoforms or Neurofilament immunoreactivity exhibited yellow fluorescence (Chan et al., 2004). 2.5. Intra-RVLM microinjection and hemodynamic measurements Intra-RVLM microinjection and hemodynamic measurements were performed as described previously (Jiang et al., 2011a). Briefly, the animal was anesthetized with a mixture of urethane and chloralose just mentioned above. The trachea was incubated with a polyethylene tube, and the animal breathed in room air spontaneously. Then, the head of the rat was fixed on a stereotaxic apparatus flexed to an angle of about 45 . The occipital bone was carefully removed to expose the fourth ventricle, and its floor was kept at horizontal level. A stainless steel cannula (the outward diameter of 200 mm) was inserted into the RVLM (1.5e1.9 mm ahead of the obex, 1.5e 2.0 mm right to the midline and 6.6e7.0 mm deep from the dorsal surface of the cerebellum) according to the atlas of Paxinos and Watson (Paxinos and Watson, 2007). SBP was measured via a femoral-artery cannula by a pressure transducer and a polygraph (Model SMUP-A, Department of Physiology and Pathophysiology, Shanghai Medical College of Fudan University). HR was derived automatically from the phasic wave of the arterial blood pressure by the computer. L-glutamate (2 nmol/ 100 nl) was microinjected into the RVLM to preliminarily judge whether the needle tip was located precisely in the RVLM by an elevation of SBP (DSBP  20 mmHg) and HR (DHR  30 bpm). In all animals, a period of 30 min was allowed at the beginning of the experiment for stabilization of SBP and HR at baseline before drug or aCSF administration. During the experiment, body temperature was measured with a rectal thermometer and maintained at 37.5  0.5  C by using a temperature controller (H-KWDY, Quanshui Experimental Instrument, China). Arterial blood PaO2 and PaCO2 were periodically monitored by means of a blood gas analyzer (Medica Easy Blood, Medica, USA) and were maintained within normal limits. Arterial blood pH was maintained between 7.35 and 7.45. At the end of the experiment, 2% Pontamine Blue Dye (100 nl) was microinjected to confirm the accurate injection sites within the RVLM. The animal was scarified by injection (i.p.) of an overdose of composite anesthetic agent and the brain was removed. After the rat brain was fixed in 10% formalin for 7 days, frozen brain cross-sections (30 mm) were made and stained with 1% Neutral Red to identify the sites of microinjection (Jiang et al., 2011a). The location of each site studied was identified and mapped on diagrams of the rat brain according to the atlas of Paxinos and Watson (Paxinos and Watson, 2007). 2.6. Western blot analysis Tissue samples of the RVLM were subject to western blot analysis as before (Jiang et al., 2011a). Briefly, a total protein extract was prepared by homogenizing RVLM tissues in lysis buffer with protease inhibitor. The concentration of the total proteins extracted from the tissue samples was determined using the bicinchoninic acid (BCA) assay. Protein samples (60 mg) were separated by 10% sodium dodecyl sulfateepolyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride

membranes (Millipore Corporation, Billerica MA, USA), blocked with 5% non-fat milk and then incubated at 4  C overnight with rabbit polyclonal antibodies against OX1R (1:3000; Santa Cruz, CA, USA) in 5% non-fat milk. Mouse polyclonal antibodies against anti-GAPDH (1:10,000; Kangcheng Biotechnology, Shanghai, China) were incubated as an internal standard. Peroxidase-conjugated anti-rabbit (1:5000) or anti-mouse (1:5000) antibodies were used as the secondary antibody. Membranes were probed using an ECL-Plus detection kit on film. Films were scanned using a photoscanner and analyzed with gel-pro Analyzer. 2.7. Statistical analysis Data are expressed as the mean  SE. Statistical analyses were performed using unpaired Student’s t-test (for the control group and the SIHR group comparisons) or one-way ANOVA with Dunnett’s test (for the control group and the drug groups comparisons). P < 0.05 was considered statistical significant.

3. Results 3.1. Effects of chronic stress on SBP Rats were subjected to stress for 2 h twice daily for l4 consecutive days. Two hours after every stress, tail SBP was measured in conscious rats. After experiencing the first 2e3 days of stress, rats in stressed group had obvious changes in performance and behavior, such as being alert and restless, which meant the conditioned reflex to noises was established. On the 4th day, SBP and HR have increased significantly, and the peak of HR for stress was around the 6th day (from 375  19 bpm to 445  23 bpm), and then HR decreased gradually to a stable condition (420  23 bpm; Fig. 1B). After the 6th day, SBP for stressed group kept rising gradually but less significantly until staying in a stable condition around the 14th day (from 110  5 mmHg to 142  8 mmHg; Fig. 1A). So the 6th day was a very important day, when the times of electric foot-shocks should be decreased till noises were used only. Compared with the control group, the stressed group experienced a significant increase in SBP and HR when given stress for 14 consecutive days. 3.2. Expression of OXA in the LH-PeF and OX1R in the RVLM Using immunohistochemistry, we measured OXA expression in the LH-PeF of the control group and the SIHR group. The number of OXA-IR neurons in the LH-PeF of the SIHR group was significantly greater than that of the control group (27  3.2 vs. 13  1.5; Fig. 2). The ROD of OXA expression was greater in SIHR than in control rats (3.53  0.39 vs. 2.1  0.26; Fig. 2). Furthermore, Western immunoblotting analysis showed that the protein level of OX1R in the RVLM of the SIHR group was higher than that of the control group (9.5  0.8 vs. 6.1  0.5; Fig. 3). 3.3. Co-localization of OX1R with nNOS, iNOS or Neurofilament in the RVLM of control rats In order to study the extent of co-localization of OX1R immunoreactivity with nNOS, iNOS or Neurofilament immunoreactivity in the RVLM, double-staining immunohistochemistry was performed in our experiments. OX1R-IR cells were seen as red fluorescence; nNOS, iNOS and Neurofilament-IR cells as green fluorescence; DAPI-IR cells as blue fluorescence, which stains all cell nuclei, and double labeling as yellow fluorescence (Fig. 4). We observed that the presence of OX1R-IR cells throughout the confines of the RVLM were extensive, and about 70% of OX1R-IR cells were co-localized with nNOS-IR cells while about 40% of OX1R with iNOS-IR cells. About 60% OX1R-IR cells were co-localized with Neurofilament-IR cells, a type of intermediate filament that served as major elements of the cytoskeleton supporting the axon cytoplasm, which indicated OX1R mainly in the neurons, with others in the neuroglia cells. DAPI was used in our experiments to

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bradycardiac responses compared with DMSO of SIHR group (128  7 mmHg vs. 140  6 mmHg; 406  25 bpm vs. 421  24 bpm, Fig. 5). However, microinjections of SB-408124 alone into the RVLM did not alter SBP and HR in normotensive rats. Microinjection of SB408124 (100 pmol) followed by OXA (100 pmol) 5 min later into the unilateral RVLM of control rats or SIHR mostly blocked hypertension and tachycardia induced by OXA alone (the SIHR group: 143  6.3 mmHg vs. 172  10 mmHg, 423  17.4 bpm vs. 462  31 bpm, Fig. 6). To investigate whether the OXA-induced cardiovascular responses were also mediated by OX2R, we coinjected OXA with TCS OX2 29, an OX2R antagonist. Interestingly, TCS OX2 29 (100 pmol), which induced no changes in normotensive rats, partly reduced the SBP and HR in stressed rats (136  5 mmHg vs. 172  10 mmHg; 412  29.7 bpm vs. 462  31 bpm, Fig. 6) and partly abolished the increased SBP and HR evoked by OXA (the SIHR group: 153  8.1 mmHg vs. 172  10 mmHg, 448  26 bpm vs. 462  31 bpm, Figs. 5 and 6). 3.5. Effect of NOS inhibitors on cardiovascular responses to OXA

Fig. 1. Difference of SBP and HR during the observed days (0e14 days) between the control group and the SIHR groups. *P < 0.05, **P < 0.01 vs. the control group. Values are means  SE, n ¼ 10.

This series of experiments evaluated whether or not the OXAinduced pressor response was mediated via the activation of the NO/NOS system. 7-Ni and AG were selected as the nNOS and iNOS inhibitors. 7-Ni (0.05 pmol, an inhibitor of nNOS) pretreatment 5 min prior to OXA (100 pmol) (7-Ni þ OXA group) microinjection into the unilateral RVLM significantly attenuated the OXA-induced cardiovascular responses both in the control group and the SIHR group. In SIHR group, SBP decreased from 172  10 mmHg to 141  6.1 mmHg and HR decreased from 462  31 bpm to 422  22.5 bpm (OXA group vs. 7-Ni þ OXA group, Fig. 6). Microinjection of AG (10 pmol/100 nl, an inhibitor of iNOS) into the unilateral RVLM followed by OXA (100 pmol) 5 min later did not cause significant cardiovascular responses compared with the effect of OXA alone in control rats and SIHR (175  11.5 mmHg vs. 172  10 mmHg, 472  29 bpm vs. 462  31 bpm, Fig. 6). To detect whether the dose of AG would influence the results, we employed two other doses (1 pmol and 100 pmol). Our experiments showed that they both failed to change the OXA-induced cardiovascular responses (Fig. 7). 3.6. Effect of sGC inhibitor on cardiovascular responses to OXA

mark the nucleolus of the RVLM cells, and we found OX1R-IR was absent from the nucleolus. 3.4. Effect of OXA, SB-408124 and TCS OX2 29 on SBP and HR Microinjection of aCSF or DMSO into the unilateral RVLM of both control rats and SIHR produced little change in both SBP and HR. Microinjection of OXA (10, 50, 100 pmol) into the unilateral RVLM of both groups produced increases in SBP and HR in a dosedependent and site-specific manner, with in SIHR being more effective. In the lowest dose (10 pmol), OXA caused no significant changes in SBP and HR in the control group compared with aCSF of the control group (114  5.3 mmHg vs. 110  4.5 mmHg; 380  15.4 bpm vs. 375  14.4 bpm), but caused statistical increase in SBP and HR in the SIHR group compared with aCSF of the SIHR group (151  8.7 mmHg vs. 142  5.4 mmHg; 434  27 bpm 420  vs. 23.5 bpm). In the highest dose (100 pmol) in SIHR, OXA caused a significant increase in SBP (172  10 mmHg vs. 142  5.4 mmHg,) and HR (462  31 bpm vs. 420  23.5 bpm, Fig. 5) compared with aCSF of SIHR group. Cardiovascular responses to OXA fully recovered after 40 min in the control group and after 1e1.5 h in stressed group. Microinjection of SB-408124 (100 pmol), selective antagonist of OX1R, into the unilateral RVLM of SIHR produced depressor and

Microinjection of Methylene Blue (100 pmol, an inhibitor of sGC) into the unilateral RVLM followed by OXA (Methylene Blue þ OXA group) 5 min later significantly attenuated the OXA-induced cardiovascular responses both in the control group and the SIHR group. In SIHR group, the SBP decreased from 172  10 mmHg to 143  6.8 mmHg, and HR decreased from 462  31 bpm to 425  18 bpm (OXA group vs. Methylene Blue þ OXA group, Fig. 6). 4. Discussion In this study, we investigated the effects of OXA on cardiovascular regulation in SIHR, and the role of medullary NOS/NO signaling in the mechanism. The primary findings of the present study are: 1) the expressions of both OXA-IR in the LH-PeF and OX1R in the RVLM of SIHR are up-regulated, compared with that of control rats; 2) in the RVLM, OX1R-IR cells are partly co-localizated with nNOS-IR or iNOS-IR cells; 3) microinjection of OXA into the RVLM evokes vasopressor effects in control rats and SIHR in a dosedependent and site-specific manner; 4) both SB-408124, a selective OX1R antagonist, and TCS OX2 29, a selective OX2R antagonist, decrease SBP and HR in SIHR, and partly abolish the cardiovascular response evoked by exogenously-administrated OXA, with no difference in statistical significance between the two antagonists’

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Fig. 2. Expressions of OXA-IR neurons in the lateral hypothalamic-perifornical area (LH-PeF) of the control group and the SIHR groups. a, b, c: the control group; d, e, f: the SIHR group; Scale bars ¼ 100 mm in a, d; 50 mm in b, e; 20 mm in c, f. The number and the relative optical density (ROD) of OXA-IR neurons in SIHR increase markedly compared with those of the control rats. Bars represent means  SE. **P < 0.01 vs. the control group, n ¼ 6.

Fig. 3. Changes of OX1R expressions of protein level in the RVLM of the control group and the SIHR group by Western Blot. The percentage of OX1R to GADPH in the RVLM of SIHR group increase markedly compared with that of the control group. Values are means  SE. **P < 0.01 vs. the control group, n ¼ 4.

effects; 5) 7-Ni, an nNOS inhibitor, and Methylene Blue, an sGC inhibitor, block the hypertensive response activated by OXA, while AG, an iNOS inhibitor, has no significant effects. Hypertension affects approximately 1 billion individuals worldwide (Al Ghatrif et al., 2011). Chronic and excessive exposure to psychological stress can lead to hypertension development. The speedy life and fierce competition seen in modern society can result in a growing occurrence of stress-induced hypertension, which is inflicted on more and more people. The recent quantitative data in hypertensives suggest that stress may be related to exaggerated hypertension (Kucukler et al., 2011). So it is very important to understand the mechanism of stress-induced hypertension. The stress-induced hypertensive model is well established by stimulus of foot-shocks combined with noises. We used the model in the present study because it may mimic the present environment in which humans live: fast paced and with high work pressures (Herd, 1991; Xia et al., 2008). Our previous (Huang et al., 2005; Xia et al., 2008) and present studies demonstrate that chronic stress on rats result in a long-term increase in SBP, which indicates success of a stress-induced hypertensive model. OXA has been found to be involved in cardiovascular regulation via effect on the brainstem neurons, including neurons in the RVLM, the nucleus of tractus solitarii (NTS), nucleus ambiguous (NA), pre-Bötzinger complex, et al. (Ciriello and de Oliveira, 2003; Ciriello et al., 2003; Cluderay et al., 2002; Marcus et al., 2001; Sunter et al., 2001; Trivedi et al., 1998). Microinjection of OXA into the RVLM resulted in hypertension and tachycardia for 25e30 min in anesthetized rats and for about 7 min in conscious rats (Machado

Fig. 4. Laser scanning confocal microscope images of the RVLM of normotensive rats showing individual immunofluorescence staining for OX1R, nNOS, iNOS and Neurofilament or double immunofluorescence. Note that OX1R was seen as red (A(b), B(b), C(b)); nNOS (A(a)), iNOS (B(a)) and Neurofilament (C(a)) as green; DAPI stained all cell nuclei as blue (A(c), B(c), C(c)), and double label was displayed yellow fluorescence (A(d), B(d), C(d)). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 5. Changes of SBP and HR in control rats and SIHR that received unilateral microinjections into the RVLM of OXA (10, 50, 100 pmol), SB-408124 (100 pmol) and TCS OX2 29 (100 pmol). *P < 0.05, **P < 0.01 vs. the control group with aCSF; yP < 0.05, yy P < 0.01 vs. the SIHR group with aCSF; #P < 0.05, ##P < 0.01 vs. the SIHR group with DMSO. Values are means  SE, n ¼ 8.

Fig. 6. Changes of SBP and HR in control rats and SIHR that received unilateral microinjections into the RVLM of SB-408124 (100 pmol), TCS OX2 29 (100 pmol), 7-Ni (0.05 pmol), Methylene Blue (100 pmol) or AG (10 pmol), which are all followed by OXA (100 pmol) 5 min later respectively. *P < 0.05, **P < 0.01 vs. the control group with OXA (100 pmol); #P < 0.05, ##P < 0.05 vs. SIHR group with OXA (100 pmol). 7-Ni þ OXA group, n ¼ 10; other groups, n ¼ 7.

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Fig. 7. Changes of SBP and HR in control rats and SIHR that received unilateral microinjections of AG (1, 10, 100 pmol) into the RVLM, which are all followed by OXA (100 pmol) 5 min later respectively. n ¼ 7.

et al., 2002). It was reported in previous studies that the hypertension and tachycardia caused by central administration of OXA (Chen et al., 2000; Machado et al., 2002) were likely due to an increase in sympathetic vasomotor tone mediated by the RVLM. In our present study, the expression of OXA-IR neurons in the LH-PeF of SIHR was higher than that of the control group. Moreover, unilateral microinjection of OXA (10, 50, 100 pmol) into the RVLM caused hypertension and tachycardia in SIHR in a dose-dependent manner. The effects were much more obvious in SIHR than in control rats. The reason may, as we discovered, be that the protein level of OX1R in the RVLM of SIHR was higher than that of the control group. When OXA was exogenously-administrated into the RVLM, increased OX1R could be combined with OXA, leading to hypertension and tachycardia. It is worth noting that unilateral microinjection of SB-408124 alone into the RVLM of control rats has no obvious influence on SBP and HR. However, unilateral microinjection of SB-408124 into the RVLM could almost totally abolish the cardiovascular responses evoked by OXA exogenouslyadministrated into the RVLM of control rats and SIHR. Similarly, Kazuyoshi Hirota et al. reported that SB-334867 could reverse the responses evoked by OXA both in vivo and in vitro, and when

administrated SB-334867 alone did not significantly change baseline hemodynamic variables (Hirota et al., 2003). On the other hand, unilateral microinjection of SB-408124 into the RVLM of SIHR decreased both SBP and HR, but did not return them to the normal level, which indicated SB-408124 only partly abolished the effects of the intrinsic OXA increased during stress. So we want to know whether OX2R has some potential effects on the mechanism. TCS OX2 29, a selective OX2R antagonist which was used in the experiment, partly abolished the cardiovascular response evoked by OXA exogenously-administrated into the RVLM of control rats and SIHR, and lowered the increased SBP and HR of SIHR to some degree. There was no difference in statistical significance between the effects of SB-408124 and those of TCS OX2 29. The results suggested that OXA may be involved in the mechanism of SIH, and both OX1R and OX2R play important roles in it. Huang et al. demonstrated OX directly excited RVLM neurons mainly via OX2R, with a smaller contribution from the OX1R (Huang et al., 2010). They did their experiments by using in vitro recordings of neuronal activity of the RVLM and intracisternal administration of OX1R and OX2R antagonists. The difference between our results and Huang’s may be caused by the different in vivo methods we tookdours is RVLM-microinjection while Huang’s is intracisternal administrationdand Huang’s additional in vitro method. SHIH et al. investigated the cardiovascular effects of OX in the NTS and the effects of NOS/NO on OX-induced cardiovascular responses. The authors observed that the physiological effects of OXA in the NTS were mediated mainly through the OX1R and the vasodepressor effects of OXA were induced by the nNOSderived NO and activation of sGC-associated signaling pathway (Shih and Chuang, 2007). Many reports suggested that OX fibers and OX receptors, especially OX2R, were found extensively in the paraventricular nucleus (PVN) (Kannan et al., 2007). The PNV was thought to be one of the critical sites for OX in regulating cardiovascular and autonomic functions. Chang et al. demonstrated increases in ACTH levels after administration of OXA or swimming stress were attenuated by prior administration of OX2R into PVN, which suggested that swimming stress facilitates ACTH release, at least in part via activation of OX2R (Chang et al., 2007). Another study in our laboratory related to OXA indicates microinjection of TCS OX2 29 into PVN could abolish the cardiovascular response evoked by OXA exogenously-administrated in control rats and SIHR (Data has not been published). All the studies mentioned above showed OX2R may be more effective in PVN than in the RVLM. It is worth further investigation. NO in the RVLM decreases sympathetic activities in cardiovascular responses (Jiang et al., 2011b). Our previous study has proved that the number and optical density (OD) of nNOS immunoreactive neurons in the RVLM of SIHR increased, while the number and OD of iNOS immunoreactive neurons decreased (Huang et al., 2005). Microiontophoresis of 7-Ni in the RVLM of SIHR inhibited the activities of Glutamate-sensitive neurons, while microiontophoresis of AG increased the activities of Glutamate-sensitive neurons (Data has not been published). All these studies suggested it was possible for NO in the RVLM to mediate the cardiovascular effect of OXA. Nevertheless, NO derived from nNOS induced sympathoexcitation and NO derived from iNOS elicited sympathoinhibition (Chan et al., 2003). We also delineate the role of NO, which is involved in central cardiovascular regulation via an action in the RVLM (Martins-Pinge et al., 2007), in the OX-induced cardiovascular responses. In the double-labeling studies, we observed that the OX1R-IR cells were co-localized with NOS isoforms immunoreactive cells (both nNOS and iNOS) in the RVLM, about 70% of OX1R-IR cells were co-localized with nNOS-IR cells, and about 40% of OX1R-IR cells were colocalized with iNOS-IR cells. The results suggested that NO may

F. Xiao et al. / Neuropharmacology 67 (2013) 16e24

play a role in the mechanism of cardiovascular regulation evoked by OXA microinjected into the RVLM. So we also investigated the effect of 7-Ni on the pressor response evoked by centrally-administered OXA. 7-Ni administered alone had no appreciable effects on SBP and HR, whereas 7-Ni microinjected before OXA significantly diminished the pressor effect of OXA. An unanticipated finding of this study was that AG (1, 10, 100 pmol), an inhibitor of iNOS, did not strengthen the pressor and tachycardiac effects of centrallyadministered OXA. All these results supported our hypothesis that OXA in the RVLM increases SBP and HR by increasing nNOS-derived NO, while iNOS-derived NO had no significant effect. Guo et al. observed that iontophoresis of 7-Ni (0.1 mM) reduced the increased discharge of cardiovascular sympathoexcitatory of the RVLM neurons in response to cardiac stimulation by bradykinin (BK), while unilateral microinjection of the iNOS inhibitor into the RVLM failed to evoke any change. The authors suggested NO, specifically nNOS, mediated sympathetic cardiovascular responses through its action in the RVLM (Guo et al., 2009). NO, which mediates various biological responses by diffusion into the target cells, exerts its effects through activation of sGC, which increases the concentration of intracellular cGMP, achieving further phosphodiesterase signal transduction and downstream cellular effects (Chan et al., 2005; Hamid et al., 2010). The NO-sGCcGMP signaling pathway is involved in NO-mediated central nervous system, including the RVLM, the important area of cardiovascular regulation (Chan et al., 2005). So we wanted to know whether the NO-sGC-cGMP signaling pathway was involved in OXA-dependent hypertensive effects in the RVLM of SIHR. We observed that the cardiovascular effects of OXA were abolished by Methylene Blue, the sGC inhibitor which was administrated prior to OXA, in both the control group and the SIHR group. Therefore, we speculated that pressor effect of OXA in the RVLM of SIHR was mediated by NO derived from nNOS, followed by the activation of NO-sGC-cGMP signaling pathway. In summary, results in the present study support the hypothesis that OXA in the RVLM participated in the central regulation of cardiovascular activities in SIHR. OXA administrated into the RVLM evoked the pressor response via OX receptor-dependent mechanism, on which both OX1R and OX2R have important effects. nNOS and sCG, but not iNOS, inhibitors abolished the OXA-induced cardiovascular responses, which indicated that NO derived from nNOS and activation of sGC-associated signaling pathway may play an important role in the OXA-induced cardiovascular effects in the RVLM. Acknowledgments The study was supported by National Key Basic Research Development Program (973) of China (No. 2006CB504509 and 2007CB512502973) and Shanghai Leading Academic Discipline Project (Project Number: B112). References Al Ghatrif, M., Kuo, Y.F., Al Snih, S., Raji, M.A., Ray, L.A., Markides, K.S., 2011. Trends in hypertension prevalence, awareness, treatment and control in older Mexican Americans, 1993e2005. Ann. Epidemiol. 21, 15e25. Antunes, V.R., Brailoiu, G.C., Kwok, E.H., Scruggs, P., Dun, N.J., 2001. Orexins/hypocretins excite rat sympathetic preganglionic neurons in vivo and in vitro. Am. J. Physiol. Regul. Integr. Comp. Physiol. 281, R1801eR1807. Arnold, W.P., Mittal, C.K., Katsuki, S., Murad, F., 1977. Nitric oxide activates guanylate cyclase and increases guanosine 30 :50 -cyclic monophosphate levels in various tissue preparations. Proc. Natl. Acad. Sci. U. S. A. 74, 3203e3207. Carter, M.E., Borg, J.S., de Lecea, L., 2009. The brain hypocretins and their receptors: mediators of allostatic arousal. Curr. Opin. Pharmacol. 9, 39e45. Chan, J.Y., Chan, S.H., Chang, A.Y., 2004. Differential contributions of NOS isoforms in the rostral ventrolateral medulla to cardiovascular responses associated with mevinphos intoxication in the rat. Neuropharmacology 46, 1184e1194.

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