Increased superoxide anion in rostral ventrolateral medulla contributes to hypertension in spontaneously hypertensive rats via interactions with nitric oxide

Free Radical Biology & Medicine 38 (2005) 450 – 462 www.elsevier.com/locate/freeradbiomed

Original Contribution

Increased superoxide anion in rostral ventrolateral medulla contributes to hypertension in spontaneously hypertensive rats via interactions with nitric oxide Ming-Hong Tai, Ling-Lin Wang, Kay L.H. Wu, Julie Y.H. Chan* Department of Medical Education and Research, Kaohsiung Veterans General Hospital, 386, Ta-chung 1st Road, Kaohsiung, 813, Taiwan, Republic of China Received 10 October 2004; revised 9 November 2004; accepted 9 November 2004 Available online 2 December 2004

Abstract Oxidative stress because of an excessive production of superoxide anion (O2S ) is associated with hypertension. The present study evaluated the hypothesis that in the rostral ventrolateral medulla (RVLM), where the premotor neurons for the maintenance of vascular vasomotor activity are located, increased O2S contributes to hypertension in spontaneously hypertensive rats (SHR) by modulating the cardiovascular depressive actions of nitric oxide (NO). Compared with normotensive Wistar-Kyoto (WKY) rats, SHR manifested significantly increased basal O2S production, along with reduced manganese superoxide dismutase (MnSOD) expression and activity, in the RVLM. The magnitude of hypotension, bradycardia, or suppression of sympathetic neurogenic vasomotor tone elicited by microinjection bilaterally into the RVLM of a membrane-permeable SOD mimetic, Mn(III)-tetrakis-(4-benzoic acid) porphyrin (MnTBAP), was also significantly larger in SHR. Transfection bilaterally into the RVLM of adenoviral vectors encoding endothelial nitric oxide synthase resulted in suppression of arterial pressure, heart rate, and sympathetic neurogenic vasomotor tone in both WKY rats and SHR. Microinjection of MnTBAP into the RVLM of SHR further normalized those cardiovascular parameters to the levels of WKY rats. We conclude that an elevated level of O2S in the RVLM is associated with hypertension in SHR. More importantly, this elevated O2S may contribute to hypertension by reducing the NO-promoted cardiovascular depression. D 2004 Elsevier Inc. All rights reserved. Keywords: Superoxide anion; Nitric oxide; Oxidative stress; Hypertension; Rostral ventrolateral medulla; Superoxide dismutase; Free radicals

Introduction Abbreviations: aCSF, artificial cerebrospinal fluid; AdGFP, adenovirus encoding green fluorescence protein; AdeNOS, adenovirus encoding endothelial nitric oxide synthase; cGMP, guanosine 3V,5V-cyclic monophosphate; Cu/ZnSOD, cupper/zinc superoxide dismutase; HEt, hydroethidine; HR, heart rate; MAP, mean arterial pressure; MSAP, mean systemic arterial pressure; MnSOD, manganese superoxide dismutase; MnTBAP, Mn(III)-tetrakis-(4-benzoic acid) porphyrin; NO, nitric oxide; eNOS, endothelial nitric oxide synthase; nNOS, neuronal nitric oxide synthase; iNOS, inducible nitric oxide synthase; O2 , superoxide anion; ODQ, 1H-[1,2,4]oxadiazole[4,3- a]quinoxalin-1-one; RVLM, rostral ventrolateral medulla; SAP, systemic arterial pressure; SHR, spontaneously hypertensive rat; sGC, soluble guanylate cyclase; WKY, Wistar-Kyoto; pfu, plaque-forming unit. * Corresponding author. Fax: +886 7 3468056. E-mail address: [email protected] (J.Y.H. Chan).

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0891-5849/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2004.11.015

Accumulated evidence suggests an active role for nitric oxide (NO) in central regulation of sympathetic vasomotor tone and systemic arterial pressure (SAP) ([1–3], for reviews). In the rostral ventrolateral medulla (RVLM), where sympathetic premotor neurons for the maintenance of basal vasomotor tone are located [4], microinjection of NO donor [5] or overexpression of endothelial nitric oxide synthase (eNOS) [6] results invariably in greater depressor and sympathoinhibitory responses in spontaneously hypertensive rats (SHR) or stroke-prone SHR than normotensive Wistar-Kyoto (WKY) rats. Accordingly, a defect in bioavailability of NO at the vasculature already suggested for

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the pathogenesis of hypertension [7] may also be a contributing factor in the RVLM of SHR [8]. Diminished bioavailability of NO may result from reduction in production or augmentation in inactivation of the gaseous molecule. In the peripheral vasculature of SHR, the reduced NO production is the result of inefficient utilization of L-arginine [9] or impaired activity of eNOS [10]. On the other hand, an augmented oxidative inactivation of NO by superoxide anions (O2S ) also accounts for the decreased NO availability in the vasculature of hypertensive animals [11,12]. Oxidative stress because of an enhanced O2S production has been demonstrated in peripheral blood vessels during hypertension [13,14]. Moreover, activity of superoxide dismutase (SOD), which dismutates O2S to H2O2, is decreased in experimental hypertension [15]. Administration of SOD mimetics [16,17] or in vivo overexpression of SOD [18–20] improves endothelial function and normalizes arterial pressure in animal models of hypertension. Our laboratory reported previously [21,22] that an innate reduction in NO activity at the RVLM as a result of downregulation of both molecular synthesis and functional expression of the inducible NOS (iNOS) underlies the augmented sympathetic vasomotor outflow in SHR. We investigated in the present study that an enhanced production of O2S in the RVLM may also play a role in the manifestation of hypertension. We found that an elevated O2S production in the RVLM is associated with augmented sympathetic neurogenic vasomotor tone and hypertension in SHR. Moreover, the increased O2S may contribute to hypertension in SHR by reducing the cardiovascular depression induced by NO in the RVLM.

Methods Animals Experiments were carried out in accordance to the guidelines for animal experimentation endorsed by our institutional animal care committee. Male adult (10–12 weeks old) SHR (200–220 g, n = 136) or WKY rats (200– 230 g, n = 128), purchased from the Experimental Animal Center of the National Applied Research Laboratories, Taiwan, were used. They were housed in an animal room under temperature control (24 F 0.58C) and 12-h light–dark (08:00–20:00) cycle. Standard laboratory rat chow (Purina) and tap water were available ad libitum. All animals were allowed to acclimatize for at least 7 days prior to experimental manipulations. In situ detection of superoxide anion The oxidation-dependent fluorescent dye hydroethidine (HEt, Molecular Probes, Eugene, OR) was used to evaluate in situ production of O2S [23,24]. HEt is taken up by living

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cells and oxidizes to a red fluorescent ethidium bromide in the presence of O2S , but not by other reactive oxygen species such as hydroxyl radical and NO/O2S -derived oxidants in the cells [23,25]. Unfixed frozen caudal brain stem was cut on a Cryostat into 20-Am-thick section at the level of the RVLM, and was mounted on glass slides. The entire procedure was completed within 30 min. HEt (40 AM) and NADPH (1 mM) were applied to the surface of each section, and the slides were incubated in a light-protected humid chamber at 378C for 30 min. Slides were then examined under an Olympus Fluoview 300 laser confocal microscope equipped with a krypton/argon laser. Fluorescent ethidium bromide was detected with a 585 nm long-pass filter. Brain sections from SHR and WKY rats were processed and imaged in parallel. Specificity for O2S image was determined by adding diphenylene iodonium chloride (10 AM), an inhibitor of flavoprotein, into the incubation medium. Measurement of O2S chemiluminescence

by lucigenin-enhanced

O2S production was also determined by lucigeninenhanced chemiluminescence according to previously described and validated methods [24,26]. The ventrolateral medulla was homogenized in a 20 mM sodium phosphate buffer, pH 7.4, that contains 0.01 mM EDTA by a glassto-glass homogenizer. The homogenate was subjected to low speed centrifugation at 1000g for 10 min at 48C to remove nuclei and unbroken cell debris. The pellet was discarded and the supernatant was obtained immediately for O2S measurement. Background chemiluminescence in buffer (2 ml) containing lucigenin (5 Amol/L) was measured for 5 min. An aliquot of 100 AL of supernatant was then added, and the chemiluminescence measured for 30 min at room temperature (Sirius Luminometer, Berthold, Germany). O2S production was calculated and expressed as counts per minute per milligram protein. Specificity for O2S was determined by adding SOD (350 U/mL) into the incubation medium. This method was also used to determine the effectiveness of the membranepermeable superoxide dismutase mimetic, MnTBAP, to scavenge O2S in the RVLM. Isolation of cytosolic and mitochondrial fractions Tissues samples from both sides of ventrolateral medulla collected from SHR or WKY rats were immediately placed in ice-cold buffer containing 10 mM Tris–HCl (pH 7.4), 1 mM EDTA, and 250 mM sucrose. Aprotinin (10 Ag/ml), phenylmethylsulfonyl fluoride (20 Ag/ml), and trypsin inhibitor (10 Ag/ml) were included in the isolation buffer to prevent protein degradation. Isolation of rat mitochondria was carried out according to the procedures previously described [27]. The entire procedure was performed at 48C and completed within 2 h. Tissue samples were gently

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homogenized with a glass–glass homogenizer. Homogenates were centrifuged at 1400 g (258C) for 5 min, and supernatants were collected and centrifuged at 11,000 g (48C) for 20 min to pellet the mitochondria. The supernatants were further centrifuged at 12,000 g (48C) for 10 min to yield the cytosolic fraction. The purity of mitochondrial-rich fraction was verified by the expression of the mitochondrial inner membrane-specific protein, prohibitin [28]. Superoxide dismutase assay Activity of SOD from the mitochondrial or cytosolic fraction was measured using a SOD assay kit (Calbiochem, San Diego, CA). This assay kit utilizes 5,6,6a,11btetrahydro-3,9,10-trihydroxybenso[c]fluorine as the substrate. This reagent undergoes alkaline autoxidation, which is accelerated by SOD. Autoxidation of the reagent yields a chromophore, which absorbs maximally at 525 nm. The activity of manganese SOD (MnSOD) or copper/ zinc SOD (Cu/ZnSOD) was measured respectively in the mitochondrial or cytosolic fraction according to the manufacturer’s instructions. A 50% inhibition is defined as 1 unit of SOD and the specific activity was expressed as units per milligram of mitochondrial or cytosolic protein. Construction and purification of adenovirus vectors Adenoviral vectors were constructed and purified as detailed previously [29]. In brief, a full-length green fluorescence protein (GFP) or human eNOS cDNA was inserted into multiple cloning sites of pCA13 shuttle vector (Microbix). Recombinant adenovirus was generated by cotransfection of pCA13-GFP or pCA13-eNOS with pJM17 parental vector into 293 cells, followed by further amplification and purification. Transfection efficacy of the adenoviral vectors and their effects to enhance NO production were characterized in the GH3 neuronal cells [29]. In vivo gene transfer into the RVLM Microinjection bilaterally of adenoviral vectors encoding either the GFP (AdGFP) or the eNOS (AdeNOS) gene was carried out stereotaxically and sequentially into RVLM sites [29]. An adenoviral suspension containing 0.5  108 or 1  108 plaque-forming units (pfu)/100 nl was administered into each injection site over 10–15 min using a glass micropipette. A total of eight injections (four on each side) were made at stereotaxic coordinates of 4.5–5 mm posterior to lambda, 1.8–2.1 mm lateral to the midline, and 8.0–8.5 mm below the dorsal surface of cerebellum. These coordinates cover the RVLM in which sympathetic premotor neurons reside [4]. Animals were allowed to recover in their home cages with free access to food and water.

Histology and analysis of transferred gene At the end of each experiment, the animal was killed by an overdose of pentobarbital sodium, and the brain stem, except those for isolation of cytosolic and mitochondrial fractions, was removed from animals and fixed in 30% sucrose in 10% formaldehyde–saline solution for z72 h. Histological verification of the microinjection sites was carried out on 20-Am frozen sections stained with neutral red. The expression of transferred GFP was viewed under a Fluoroview 300 laser scanning confocal microscope (Olympus, Japan). Expression of transfected eNOS was confirmed by immunohistochemical staining or Western blot analysis [29]. Measurement of arterial pressure and heart rate We routinely measured mean arterial pressure (MAP) and heart rate (HR) at 13:00–15:00 in conscious rats using a noninvasive tail-cuff method. Rats were handled repeatedly and allowed to adapt to the restraint chamber for 3 days before the actual measurements commenced. MAP and HR were measured using an electrosphygmomanometer (MK-2000, Momuroki Kikai Co., Japan) [22,29]. These measurements were considered valid only when five consecutive readings did not differ by more than 5 mm Hg. The mean of the five readings was recorded as the individual MAP. To confirm the validity of measurements obtained by tail-cuff plethysmography, systemic arterial pressure and HR were measured directly in a separate group of animals with implanted cannula inserted into the carotid artery of the rats under pentobarbital sodium (50 mg/kg, ip) anesthesia. The insertion point was sealed with cyanoacrylate adhesive, and the catheter was externalized at the back of the neck through a subcutaneous tunnel. The rats were allowed to recover from surgery for at least 2 days. On the date of blood pressure measurement, the rats were connected via the catheter to a polygraph (Gould RS3400, Valley View, OH, USA) under unrestrained conditions. The pulsatile and mean SAP (MSAP) and HR of each rat were recorded for 30 min, and 1-min tracings were taken every 5–6 min. These values were averaged to provide a single MSAP and HR [30]. Evaluation of sympathetic vasomotor tone In experiments that evaluated sympathetic vasomotor activity, animals were anesthetized initially with pentobarbital sodium (50 mg/kg, ip) to perform intubation of the trachea and cannulation of the femoral artery and vein. Animals received thereafter continuous infusion of propofol (Zeneca, Macclesfield, UK) at 25–30 mg/kg/h. This scheme provided satisfactory anesthetic maintenance while preserving the capacity of central cardiovascular regulation [31]. Pulsatile and MSAP from the femoral artery, as

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well as HR, were recorded on a polygraph (Gould RS3400), and the SAP signals were simultaneously subject to on-line power spectral analysis [21,22]. We were particularly interested in the very low-frequency (0– 0.25 Hz) and low-frequency (0.25–0.8 Hz) components in the SAP spectrum. Our laboratory demonstrated previously [32] that these spectral components of SAP signals take origin from the RVLM, and their power density reflects the prevailing sympathetic neurogenic vasomotor tone. Microinjections of test agents into the RVLM Microinjection bilaterally of a membrane-permeable SOD mimetic, Mn(III)-tetrakis-(4-benzoic acid) porphyrin (MnTBAP; Calbiochem) or a soluble guanylyl cyclase inhibitor, 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ; Tocris Cookson, Bristol, UK), was carried out stereotaxically and sequentially into functionally identified RVLM sites, using a glass micropipette and at a injection volume of 50 nL [21,22]. The coordinates used were: 4.5 to 5 mm posterior to lambda, 1.8 to 2.1 mm lateral to midline, and 8.1 to 8.4 mm below the dorsal surface of cerebellum. Functional location of RVLM neurons on either side was carried out at the beginning of each experiment by monitoring a transient increase in SAP (20–25 mm Hg) after microinjection of L-glutamate (2 nmol). Subsequent microinjections of test agents were delivered to the identified pressor loci. The doses used were the same as in our recent study [33] in which MnTBAP or ODQ was used for the same purpose as in the present study. Protein extraction and Western blot analysis At the end of experiments, the brain stem was rapidly removed and placed on dry ice. Tissues on both sides of the ventrolateral part of medulla oblongata, at the level of RVLM (0.5 to 1.5 mm rostral to the obex) that contained the injection sites, were collected by micropunches made with a stainless-steel bore (1 mm ID) and frozen in liquid nitrogen. Medullary samples thus obtained from 4 to 6 rats under the same experimental treatment were stored at 808C and were pooled to provide sufficient tissue for protein extraction. Extraction of total protein from the ventrolateral part of the medulla was carried out as detailed previously [21,22] at Day 7 or 10 after gene transfer. Western blot analysis was carried out using a rabbit polyclonal antiserum against eNOS, nNOS, Cu/ ZnSOD, MnSOD, soluble guanylate cyclase (sGC) subunit a1 and h1 (Calbiochem), or h-tubulin (Sigma, St. Louis, MO), or a mouse monoclonal antiserum against the mitochondrial inner membrane protein, prohibitin (Calbiochem), as the primary antiserum. This was followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG (Jackson Immunoresearch Laboratories,

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Inc., West Grove, PA). Specific antibody–antigen complex was detected using an enhanced chemiluminescence Western blot detection system (Perkin-Elmer Life Sciences, Boston, MA). The amount of detected protein was quantified by Photo-Print Plus software (ETS VilberLourmat, France), and was expressed as the ratio to htubulin protein. Statistical analysis All values are expressed as mean F SE. One-way or twoway analysis of variance with repeated measures was used to assess group means, as appropriate, to be followed by the Scheffe´ or Dunnett multiple-range test for post hoc assessment of individual means. P b 0.05 was considered statistically significant.

Results Differential production of superoxide anion in RVLM of SHR or WKY rats Compared to age-matched normotensive WKY rats, both the intensity and the number of cells in the RVLM that showed red fluorescent ethidium bromide, which reflects O2S level [23–25], were discernibly greater in SHR (n = 4) (Fig. 1A). Similarly, lucigenin-enhanced chemiluminescence assay indicated that the production of O2S was significantly greater in the ventrolateral medulla of SHR (1903 F 196 versus 1321 F 113 cpm/mg protein, P b 0.05, n = 6). Differential expression and activity of MnSOD or Cu/ZnSOD in ventrolateral medulla Western blot analysis revealed that protein expression of MnSOD, but not Cu/ZnSOD (Fig. 1B), in the ventrolateral medulla was significantly reduced in SHR. Such a reduction in MnSOD expression was accompanied by a significant decrease in SOD activity in the mitochondrial (2.6 F 0.4 versus 4.7 F 0.6 U/mg protein, P b 0.05, n = 5), but not cytosolic (5.6 F 0.3 versus 4.5 F 0.9 U/mg protein, n = 5) fraction of protein extracts from the ventrolateral medulla. Differential effects of SOD mimetic in RVLM on cardiovascular parameters or O2S production Compared to aCSF, microinjection bilaterally of a membrane-permeable SOD mimetic, MnTBAP (1, 10, or 20 pmol), into the RVLM of anesthetized SHR or WKY rats resulted in a dose-related decrease in MSAP, HR, or power density of the vasomotor components of SAP signals (Fig. 1C). Given at 20 pmol, the magnitude of hypotension, bradycardia, or suppression of sympathetic

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Fig. 1. Representative photomicrographs showing expression of red fluorescent ethidium bromide in the RVLM of SHR or WKY rats (A), and representative gels (inset) or amount of MnSOD or Cu/ZnSOD (B) protein relative to h-tubulin detected from the ventrolateral medulla of SHR or WKY rats. Also shown are time-course changes in MSAP, HR, or total power density of vasomotor components (0–0.8 Hz) in SAP spectrum in SHR or WKY rats that received microinjection bilaterally of MnTBAP or aCSF into the RVLM (C). Photomicrographs shown on (A) are typical results obtained from 4 animals. Values are mean F SE of quadruplicate analyses on samples pooled from 4 to 5 animals in each group (B) or n = 6 to 7 animals in each group (C). *P b 0.05 vs WKY rats (B) or corresponding aCSF group (C) in the Scheffe´ multiple-range test. Scale bar in A: 200 Am.

neurogenic vasomotor tone induced by MnTBAP was significantly greater, and the duration of these actions significantly longer in SHR. On normalization against basal values, the percentage decrease in MSAP ( 27 F 3% versus 17 F 4%), HR ( 18 F 4% versus 11 F 3%), or power density of vasomotor components of SAP signals ( 38 F 4% versus 27 F 3%) after MnTBAP (20 pmol) treatment was still significantly greater ( P b 0.05, n = 6) in SHR than WKY rats. On the other hand, microinjection of the same dose of MnTBAP into ventrolateral medullary areas adjacent to, but outside the confine of, the RVLM (e.g., spinal trigeminal nucleus, lateral

paragigantocellular nucleus) elicited no discernible changes in those hemodynamic parameters in both rat strains (data not shown). Compared to aCSF, microinjection bilaterally into the RVLM of MnTBAP (20 pmol) also significantly decreased O2S production in the ventrolateral medulla of SHR (1893 F 149 versus 1249 F 118 cpm/mg protein, P b 0.05, n = 5) or WKY rats (1433 F 132 versus 1052 F 89 cpm/mg protein, P b 0.05, n = 5), detected by lucigenin-enhanced chemiluminescence assay 30 min posttreatment. Of note was the magnitude of reduction which was again larger in SHR ( 36 F 6% versus 25 F 4%, P b 0.05, n = 5) than WKY rats.

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Expression of green fluorescence protein or eNOS protein in RVLM after gene transfer Microinjection bilaterally of AdGFP or AdeNOS into the RVLM of SHR or WKY rats resulted in the expression of GFP (Fig. 2A) or eNOS-like immunoreactivity (Fig. 2B) in this medullary site. Those eNOS-positive cells in the RVLM appeared at Day 3, peaked at Day 7 to Day 10, and returned to basal level at Day 21 after AdeNOS application. Western blot analysis further revealed that whereas AdGFP was ineffective, gene transfer of eNOS (a total of 4  108 or 8  108 pfu delivered over 8 injections) into the RVLM resulted in a significant and titer-related increase in eNOS protein expression in the ventrolateral medulla of SHR or WKY rats (Fig. 2C). As a control, application of AdGFP or AdeNOS into the RVLM did not affect nNOS expression (Fig. 2D). We noted that the basal expression of eNOS in the ventrolateral medulla was comparable between these two strains of rats. The magnitude of AdeNOSpromoted upregulation of eNOS expression was also similar between SHR and WKY rats. Gene transfer of eNOS (a total of 8  108 pfu delivered over 8 injections) into the RVLM, on the other hand, elicited

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an insignificant effect on O2S production in the ventrolateral medulla of SHR (1754 F 206 cpm/mg protein, n = 5) or WKY rats (1356 F 124 cpm/mg protein, n = 5), measured 10 days after treatment. The level of O2S in the ventrolateral medulla of SHR after Ad-eNOS transfection was still significantly greater than WKY rats ( P b 0.05, n = 5). Differential effects of eNOS gene transfer into RVLM on cardiovascular parameters Based on indirect tail-cuff measurements under conscious conditions, microinjection bilaterally of AdeNOS (a total of 4  108 or 8  108 pfu delivered over 8 injections) into the RVLM resulted in significant titerrelated decreases in MAP (Fig. 3A) or HR (Fig. 3B) from Day 3 to Day 14 in SHR or WKY rats. Treatment with AdGFP or aCSF was ineffective (Figs. 3A and 3B). Given at the high titer (a total of 8  108 pfu delivered over 8 injections), the magnitude of hypotension or bradycardia was significantly larger in SHR at Day 10 after AdeNOS administration. We noted that at the peak of cardiovascular depression, the level of MAP or sympathetic vasomotor activity was significantly higher in SHR than WKY rats. Comparable results were obtained at 10 days after

Fig. 2. Representative photomicrographs showing expression of green fluorescence protein (GFP; A) or eNOS-like immunoreactivity (black arrows; B) in the RVLM 7 days after microinjection bilaterally into the RVLM of adenovirus vector encoding GFP (AdGFP) or eNOS (AdeNOS). Scale bars, 100 Am in left panel, and 25 Am in right panels. Also shown are representative gels (inset) or amount of eNOS (C) or nNOS (D) protein relative to h-tubulin, detected from the ventrolateral medulla of SHR or WKY rats 7 days after receiving gene transfer or aCSF. Values are mean F SE of quadruplicate analyses on samples pooled from 4 to 5 animals in each group. *P b 0.05 vs corresponding aCSF group in one-way ANOVA.

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Fig. 3. Time-course changes in mean arterial pressure (MAP) (A), HR (B), or total power density of vasomotor components in SAP spectrum (C) in SHR or WKY rats that received microinjection bilaterally into the RVLM of AdGFP, AdeNOS, or aCSF. Values are mean F SE, n = 6 to 8 animals in each group. *P b 0.05 vs corresponding aCSF group, and #P b 0.05 vs corresponding WKY rats in the Scheffe´ multiple-range test.

AdeNOS treatment in conscious SHR or WKY rats implanted with catheters for direct SAP and HR measurements (data not shown). We also found that hypotension in AdeNOS-treated SHR, but not WKY rats, was followed by a rebound pressor response that became significant from Day 28 to Day 35 after gene transfer into the RVLM (Fig. 3A). No rebound tachycardia, however, was detected in both strains of rats (Fig. 3B). In a separate series of experiments on anesthetized animals, we found that SHR exhibited significantly higher basal sympathetic neurogenic vasomotor tone that was not affected 10 days after receiving AdGFP (Fig. 3C). Superimposed on the maximal decreases in MAP and HR already

observed (Figs. 3A and 3B), the power density of the vasomotor components of SAP signals also underwent significant reduction in SHR or WKY rats 10 days after microinjection bilaterally of AdeNOS (a total of 8  108 pfu delivered over 8 injections) into the RVLM. The magnitude (Fig. 3C) of this decrease in sympathetic vasomotor tone was again larger in the SHR, although the power density of the vasomotor components of SAP signals remained significantly higher in SHR. We also found that hypotension (Fig. 4A) and bradycardia (Fig. 4B) detected at Day 7 or 10 after transfecting eNOS gene to the RVLM were significantly reversed by microinjection bilaterally into the RVLM of

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Fig. 4. Time-course changes in mean systemic arterial pressure (MSAP) (A) or HR (B) in anesthetized SHR or WKY rats that received microinjection bilaterally of ODQ into the RVLM 7 or 10 days after gene transfer with AdGFP or AdeNOS into the RVLM. For clarity, data on AdGFP + ODQ at Day 7 after gene delivery are not shown because they essentially duplicated those at Day 10. Also illustrated are representative gels (inset) or amount of sGCa1 or sGCh1 (C) protein relative to h-tubulin, detected from the ventrolateral medulla of SHR or WKY rats 10 days after receiving AdeNOS bilaterally into the RVLM. Values are mean F SE, n = 5 to 6 animals in each group (A and B) or of quadruplicate analyses on samples pooled from 4 to 5 animals in each group (C). *P b 0.05 vs corresponding aCSF group in the Scheffe´ multiple-range test, and #P b 0.05 vs preinjection baseline (Time 0) in the Dunnett multiple-range test. No significant difference was detected by one-way ANOVA in C.

the soluble guanylate cyclase inhibitor, 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (500 pmol). Western blot analysis further revealed that the expression of a1 or h1 (Fig. 4C) subunit of sGC protein in the ventrolateral medulla of SHR or WKY rats was comparable at Day 10 after eNOS gene transfection.

Effects of SOD mimetic in RVLM on biphasic cardiovascular responses to eNOS gene transfer Microinjection of MnTBAP (10 pmol) bilaterally into the RVLM of anesthetized SHR 10 days after transfection of eNOS promoted further decreases in the already reduced

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MSAP (Fig. 5A) or HR (Fig. 5B) to the level of WKY rats. At Day 10, the percentage change of MnTBAP-induced hypotension (Fig. 5C) or bradycardia (Fig. 5D) was significantly greater in the AdeNOS-treated SHR. In addition, the maximal decrease in MSAP or HR induced by MnTBAP in the AdeNOS-treated SHR (MSAP, 76.3 F 4.3 mm Hg; HR, 134 F 7 bpm; n = 6 ), but not WKY rats, was significantly greater than the sum of cardiovascular

depression induced by MnTBAP (MSAP, 20.4 F 3.8 mm Hg; HR, 32 F 7 bpm) or AdeNOS (MSAP, 38.9 F 5.1 mm Hg; HR, 72 F 11 bpm; n = 6) treatment alone. Augmentation of cardiovascular depression by MnTBAP in the AdeNOS-treated SHR or WKY rats endured at least 90 min, and returned to preinjection level by 120 min. Rebound pressor response detected in the SHR at Day 28 or Day 35 after eNOS gene transfer was also reversed to

Fig. 5. Time-course changes in MSAP (A) or HR (B) in anesthetized SHR or WKY rats that received microinjection bilaterally of MnTBAP into the RVLM 10 days after gene transfer with AdGFP or AdeNOS into the RVLM. Also illustrated are percentage changes in MSAP (C) or HR (D) 10 days after rats received gene transfer or aCSF. Values are mean F SE, n = 6 to 7 animals in each group. In A and B, *P b 0.05 vs corresponding aCSF group in the Scheffe´ multiple range test and #P b 0.05 vs preinjection baseline (Time 0) in the Dunnett multiple-range test. In C and D, *P b 0.05 vs corresponding aCSF group and #P b 0.05 vs corresponding WKY rats in the Scheffe´ multiple-range test.

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pretransfection levels (Fig. 6) by microinjection bilaterally into the RVLM of MnTBAP (10 pmol). The same MnTBAP treatment, on the other hand, exerted a minimal effect on MSAP of AdeNOS-treated WKY rats.

Discussion The present study provides novel evidence to suggest that an elevated O2S level in the RVLM is associated with augmented sympathetic neurogenic vasomotor tone and hypertension in SHR. Our observations also support the notion that an interaction between O2S and NO in the RVLM is an important determinant in the manifestation of neurogenic hypertension. An augmented O2S level in peripheral vasculature is associated with dysfunction of endothelium-dependent vasodilation [11,12] and elevated peripheral resistance

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[16] in SHR. An increase in O2S production in brain reportedly plays a key role in sympathoexcitation after myocardial infarction [34,35]. Our results extend these observations to indicate that O2S in the RVLM is involved in tonic regulation of sympathetic vasomotor outflow under both normotensive and hypertensive conditions. More importantly, we identified an elevated basal production of O2S in the ventrolateral medulla of SHR. An increased basal level of O2S in the RVLM was reported recently to contribute to the neural mechanism underlying hypertension in stroke-prone SHR [20]. Similar to a native SOD [36], we also found that microinjection of a SOD mimetic into the RVLM elicited greater magnitudes of hypotension and bradycardia or decrease in sympathetic neurogenic vasomotor tone in SHR. Together these results suggest that an abundance of O2S in the RVLM is related to the augmented sympathetic neurogenic vasomotor tone and hypertension in SHR.

Fig. 6. Time-course changes in MSAP (A) or HR (B) in anesthetized SHR or WKY rats that received microinjection bilaterally of MnTBAP into the RVLM 28 or 35 days after gene transfer with AdGFP or AdeNOS into the RVLM. For clarity, data on AdGFP + MnTBAP at Day 35 after gene delivery are not shown because they essentially duplicated those at Day 28. Values are mean F SE, n = 6 to 7 animals in each groups. *P b 0.05 vs aCSF group in the Scheffe´ multiple-range test.

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The increase in O2S in the RVLM of SHR may result from an exaggerated production due to increased oxidase activity or a decrease in activity of antioxidant enzymes such as SOD. Whereas the present study did not explore the candidate oxidases in the RVLM, we noted that an overactivity of NAD(P)H oxidase [12,37] or xanthine oxidase [38] in vascular cells plays a major role in the enhanced vascular O2S production in hypertension. Our results, on the other hand, indicate that a reduction in MnSOD expression and activity may contribute to the heightened O2S level in the RVLM of SHR. Abnormal mitochondrial metabolism of reactive oxygen species is observed selectively in the brain during hypertension [39]. As the enzyme responsible for dismutating O2S generated by the mitochondrial respiratory enzyme complexes [40], a reduction in MnSOD activity would reasonably lead to the augmented level of O2S in the RVLM of SHR because of retarded inactivation. In this regard, Kishi et al. [20] reported recently that overexpression of MnSOD in the RVLM of stroke-prone SHR increases MnSOD expression and activity, decreases oxidative stress, and evokes hypotension and bradycardia. Our results also indicated that both MnSOD and Cu/ZnSOD are present in the RVLM. Whereas the expression and activity of cytosolic Cu/ZnSOD in the RVLM were comparable between SHR and WKY rats, we are aware that a marked decrease in Cu/ZnSOD is associated with renal oxidative stress and renal damage in Dahl saltsensitive hypertensive rats [15]. We postulated recently [21,22] that a reduction in NO availability because of innate down-regulation of synthesis and activity of inducible NOS in the RVLM is associated with the augmented sympathetic vasomotor activity and hypertension in SHR. The present study points to the importance of interactions between O2S and NO at the RVLM in central cardiovascular regulation during hypertension. In agreement with recent observations [6,41], we found that overexpression of eNOS in the RVLM promoted a greater extent of decrease in SAP, HR, or sympathetic vasomotor activity in SHR. We noted, however, that at the peak of cardiovascular depression seen after AdeNOS (Figs. 3A and C) treatment, the level of SAP or sympathetic vasomotor tone in SHR was still significantly higher than that in WKY rats. These results suggest that despite a reduction in NO activity in SHR [21,22], the sympathetic premotor neurons in the RVLM remain responsive to exogenously generated NO. More importantly, they denote that the heightened O2S level in the RVLM of SHR discernibly reduces the cardiovascular depressive actions of NO. This notion is substantiated by four additional observations. First, O2S levels in the RVLM after eNOS gene transfer were still higher in SHR than WKY rats. Second, microinjection of MnTBAP bilaterally into the RVLM at Day 10 after AdeNOS gene transfer further normalized the lingering elevations in SAP to the level of WKY rats (Fig. 5A). Third, there were synergistic actions between MnTBAP and AdeNOS in promoting hypotension

and bradycardia in SHR but not WKY rats. We found that the maximal decrease in SAP or HR induced by MnTBAP in AdeNOS-treated SHR was significantly greater than the mathematical sum of cardiovascular depression induced by MnTBAP or AdeNOS treatments alone. Fourth, this SOD mimetic also reversed the rebound hypertension seen in SHR 28–35 days after AdeNOS treatment (Fig. 6). It follows that reducing the cardiovascular depressive actions of NO by the elevated production of O2S in the RVLM may underlie the elevated blood pressure and the augmented sympathetic neurogenic vasomotor tone seen in SHR. Glutamatergic neurotransmission plays a crucial role in mediating the tonic and phasic excitation of sympathetic preganglionic neurons by the sympathetic premotor pathway from the RVLM [42]. It is intriguing that, at the cellular level, a recent study [43] reported that high levels of NO elicit synaptic depression on rat RVLM neurons through an inhibition of presynaptic N-type Ca2+-channel activity via peroxynitrite formation, leading to reduced presynaptic release of the excitatory neurotransmitter, glutamate. Excessive O2S may thus decrease the bioavailability of NO through the reactions with NO to form peroxynitrite [44]. Whether the observed interplay between O2S and NO in central regulation of sympathetic neurogenic vasomotor activity and blood pressure is restricted to the RVLM of SHR, however, remains to be delineated. Kishi and co-workers [6,20] reported that increased O2S production and overexpression of eNOS in the RVLM contribute to the neural mechanisms of hypertension in stroke-prone SHR. Whether the increased O2S in the RVLM contributes to hypertension via an inhibition of NOpromoted cardiovascular depression and how these two important blood pressure-regulating signals interact in the RVLM under hypertensive condition are hereunto undefined. As such, the present study provides novel observations indicating that a suppression of NO-promoted cardiovascular depression by an increase of O2S level in the RVLM contributes to the neural mechanism of hypertension in SHR. Our findings also support the notion that a balance between NO and O2S in the RVLM is more important than their individual levels in neural regulation of cardiovascular functions. That an increase in O2S level in the RVLM contributes to sympathetic overexcitation and hypertension in both SHR and stroke-prone SHR [20] further suggests that oxidative stress in the RVLM may serve as a common mechanism in the brain stem during the pathogenesis of hypertension. Two methods were used in the present study to detect the production of O2S in the RVLM. The oxidation-dependent fluorescent dye, HEt, was used to qualitatively evaluate in situ production of O2S , and the lucigenin-chemiluminescence reaction was used to compare quantitatively the O2S levels between SHR and WKY rats. Whereas results obtained from both measurements revealed an elevated O2S production in the RVLM of SHR, we are aware that there are suggested limitations to these methods [24]. We

M.-H. Tai et al. / Free Radical Biology & Medicine 38 (2005) 450–462

also recognize that even under a low concentration (5 AM), the potential for lucigenin to measure an undefined reductase activity that may only be tangentially related to O2S cannot be overlooked. To reduce these potentially confounding factors, the specificity of these two methods was verified in control experiments when the addition of SOD or diphenylene iodonium chloride, an inhibitor of NAD(P)H oxidase [45], significantly diminished the measured level of O2S . We are aware that expression of nNOS or iNOS in the brain stem and hypothalamus is higher in stroke-prone SHR than in WKY rats [46]. SHR also manifest a reduction in iNOS expression and activity in the RVLM [21,22]. We thus chose to minimize the confounding influence of nNOS or iNOS by transfection of AdeNOS, which behaves as constitutive NOS, into the RVLM [41]. Basal expression of eNOS, which is present in the RVLM [47], is comparable between SHR and WKY rats (cf. Fig. 2C). That the resultant cardiovascular depression was attributed mainly to NO production in the RVLM is confirmed by the reversing action of a sGC inhibitor (Fig. 4). Within the brain stem, the cardiovascular actions of NO are mediated by activation of sGC, resulting in accumulation of guanosine 3V,5V-cyclic monophosphate (cGMP) in target cells [8]. The closely correlated temporal profile among transgene expression of eNOS in the ventrolateral medulla and hemodynamic suppression in AdeNOS-treated SHR or WKY rats or its reversal by OQD suggest that activation of the sGC/cGMP signaling pathway by NO may underlie our observed cardiovascular consequences of eNOS gene transfer in the RVLM. Reduction in the sGC/ cGMP signaling pathway has been demonstrated to contribute to the development of hypertension [48]. Endothelium-independent vasorelaxation in response to the sGC activator, expression of sGC, and the basal content of cGMP are all reduced significantly in SHR aorta [48]. Such alterations, however, may not take place in the RVLM of SHR. We found similar expression of the a1 or h1 subunit of heterodimeric sGC in the ventrolateral medulla of SHR and WKY rats (Fig. 4C). In addition, the extent of reversal of AdeNOS-promoted cardiovascular depression by ODQ was comparable between these two strains of rats (Figs. 4A and 4B). In conclusion, the present study demonstrates that an elevated level of O2S in the RVLM may be associated with the heightened sympathetic neurogenic vasomotor tone and hypertension in SHR. We further propose that the enhanced level of O2S may contribute to hypertension by reducing the cardiovascular depressive actions of NO in the RVLM.

Acknowledgments This study was supported by the Academic Excellence Program (89-B-FA08-1-4) from the Ministry of Education, and Research Grants VGHKS93-19 and 93-16 from

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Kaohsiung Veterans General Hospital (M.H.T. and J.Y.H.C.), Taiwan, Republic of China.

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