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1 original contributions nature publishing group See reviewer commentary page 77 Upregulation of AT 1 R and inos in the Rostral Ventrolateral Medulla (RVLM) Is Essential for the Sympathetic Hyperactivity and Hypertension in the Wistar Rat Model Elizabeth Barbosa de Oliveira-Sales 1, Erika Emy Nishi 1, Mirian Aparecida Boim 2, Miriam Sterman Dolnikoff 1, Cássia T. Bergamaschi 3 and Ruy R. Campos 1 Background We hypothesized that upregulation of angiotensin type 1 receptor (AT 1 R) and inducible nitric oxide (NO) synthase (inos) within the rostral ventrolateral medulla (RVLM) could contribute to two-kidney, one-clip () hypertension. Methods The experiments were performed in male Wistar rats, 6 weeks after the renal surgery. The animals were divided into control (, n = 18) and hypertensive groups (, n = 18). Bilateral tissue punches were taken from sections containing the RVLM to perform inos gene expression analyses by the real-time PCR technique, and AT 1 R and inos protein expression analyses by western blotting. In addition, we injected losartan (1 nmol), an AT 1 R antagonist, and aminoguanidine (25 pmol), an inos inhibitor, bilaterally into the RVLM to analyze the mean arterial pressure (MAP) and renal sympathetic nerve activity (rsna). 1 Cardiovascular Division, Department of Physiology, Federal University of Sao Paulo, São Paulo, Brazil; 2 Nephrology Division, Department of Medicine, Federal University of Sao Paulo, São Paulo, Brazil; 3 Department of Bioscience, Federal University of Sao Paulo Baixada Santista, São Paulo, Brazil. Correspondence: Ruy R. Campos (campos@fcr.epm.br) Received 4 November 29; first decision 16 December 29; accepted 25 February 21; advance online publication 1 April 21. doi:1.138/ajh American Journal of Hypertension, Ltd. Results inos mrna expression levels were greater (P <.5) in the group compared to the group. Furthermore, the AT 1 R and inos protein expression were significantly increased in the RVLM of rats compared to rats. Injection of losartan into the RVLM reduced the MAP (11%) and rsna (18%) only in the rats, whereas injection of aminoguanidine in the same region decreased the MAP (31%) and rsna (34%) in hypertensive rats. Conclusions The present study suggests that upregulation of AT 1 R and inos in the RVLM is important in the maintenance of high blood pressure and renal sympathetic activation in hypertension. Keywords: angiotensin receptors; blood pressure; genes; Goldblatt hypertension; hypertension; nitric oxide; rats; RVLM; sympathetic nervous system Am J Hypertens 21; 23: American Journal of Hypertension, Ltd. There is evidence suggesting that an excess of angiotensin II (Ang II) plays an important role in the development and maintenance of two-kidney, one-clip () Goldblatt hypertension. 1 Previous studies have shown that Ang II can act at various sites within the brain to regulate cardiovascular function. 2,3 Furthermore, increasing evidence suggests that brain Ang II can contribute to increased sympathetic activity in hypertension. 4 However, the exact mechanism by which Ang II acts in sympathetic premotor neurons remains unclear. 5 One region of particular interest is the rostral ventrolateral medulla (RVLM), which contains a group of sympathoexcitatory neurons that are involved in the tonic and reflex control of blood pressure. These neurons project directly to sympathetic preganglionic neurons in the spinal cord. 6 Studies have demonstrated that this region contains a high density of angiotensin type 1 receptors (AT 1 Rs) and that the activation of these receptors results in an increase in blood pressure and sympathetic activity. 7 We recently identified that the mrna expression of AT 1 R was greater in compared to the control group in the RVLM. 8 These effects appear to be specific to sympathoexcitatory neurons in the RVLM because Ang II has no effect on respiratory neurons that are located close to the RVLM. Previous studies have shown that not only Ang II in the RVLM but also nitric oxide (NO) in this region is important for controlling sympathetic activity. In fact, NO in the central nervous system, including the brainstem, plays an important role in the regulation of blood pressure via the sympathetic nervous system. 9,1 There are three types of NOSs: the endothelial (enos), the neuronal (nnos), and the inducible (inos). enos and nnos are Downloaded from by guest on 13 January July 21 VOLUME 23 NUMBER AMERICAN JOURNAL OF HYPERTENSION

2 AT 1 R Receptor and inos in the RVLM original contributions constitutively expressed, but inos is expressed only during pathophysiological states such as hypertension, heart failure, endotoxin shock, and in aging. 11,12 Recent study suggested that NO, specifically nnos, mediates sympathetic cardiaccardiovascular responses through its action in the RVLM. 13 Interactions between NO and Ang II have been observed in the central nervous system; 14 however, the role of inos within the RVLM in the regulation of sympathetic outflow is not well established in hypertension. Therefore, the first aim of the present study was to examine whether AT 1 R and inos are upregulated within the RVLM the major region involved in the central control of sympathetic vasomotor tone in the hypertension model. For this purpose, inos mrna, and AT 1 R and inos protein expression were quantified in the RVLM of hypertensive rats. Furthermore, to test the functional significance of these molecular results, we performed injection of losartan (antagonist of the AT 1 R receptor) and aminoguanidine (specific inhibitor of inos) into the RVLM in renovascular hypertensive rats. Methods All experimental procedures were conducted according to the National Institutes of Health guidelines for the use and care of animals, and the study protocol was approved by the Ethics in Research Committee of the Federal University of Sao Paulo School of Medicine (process no. 662/4). Male Wistar rats (n = 36, g) were obtained from the animal care facility of the University. The animals were housed in cages in groups of 5 6 rats with respect to their group ( or ), with free access to rat chow and tap water. Animals were maintained in a temperature-controlled environment (23 C) on a 12-h light/dark cycle. The drugs (urethane (ethyl carbamate), hexamethonium, and amoniguanidine: Sigma Chemical, St Louis, MO and losartan: Fluka Analytical, Shanghai, China) were all dissolved in saline (ph 7 7.4). Experimental protocols. The study was divided into five independent series of experiments. In the first series, the inos mrna expression levels in the RVLM were quantified by real-time PCR. In the second series, western blotting was used to quantify the AT 1 R and inos protein expression levels in the RVLM. In these series, the groups were divided into control () (n = 5) and rats (n = 5). Series three to five were performed in urethane-anesthetized rats. In a third series of experiments, saline (1 nl) was bilaterally injected into the RVLM in two different groups of animals: (i) + saline (n = 5); and (ii) + saline (n = 5). This series served as a control for series four and five. In a fourth series of experiments, losartan (1 nmol/1 nl) 15 was bilaterally injected into the RVLM in two groups of rats: (i) + losartan (n = 4); (ii) + losartan (n = 5). Finally, in a fifth series of experiments, aminoguanidine (25 pmol/1 nl) 14 was bilaterally injected into the RVLM in two groups of rats: (i) + aminoguanidine (n = 4); (ii) + aminoguanidine (n = 3). The cardiovascular parameters analyzed in the last three series of experiments were the mean arterial pressure (MAP), heart rate, and renal sympathetic nerve activity (rsna). Hypertensive animals. To obtain hypertensive animals, male Wistar rats (15 18 g) were anesthetized with ketamine and xylazine (4 and 2 mg/kg, IP, respectively), and the left renal artery was partially obstructed with a.2 mm wide silver clip. 16 The control animals () were subjected to the same surgical procedure but without partial renal artery occlusion. All experiments were performed 6 weeks after the renal surgery. Extraction of RNA from RVLM. Rats were euthanized, the brainstem quickly removed, and bilateral punches of RVLM were obtained. 8 Histology was then performed to confirm the localization of the punches (Figure 1b). Quantitative real-time PCR. The RVLM region was homo genized in 1 µl of 1% Triton and RNasin (Invitrogen, Carlsbad, CA). To avoid contamination with genomic DNA, the RNA samples were treated with DNase (RQ1 RNase-free DNase; Promega, Madison, WI). Genomic contamination was excluded by performing PCR with samples that were not treated with reverse transcriptase. The following primer sequences were used: β-actin, forward (CCT-CTA TGC-CAA-CAC-AGT-GC), reverse (ACA-TCT- GCT-GGA AGG-TGG-AC); inos, forward (AGG-TGT-TCA- GCG-TGC-TCC-AC), reverse (AGT-TCA-GCT-TGG-CGG- CCA-CC). The sizes of the PCR products amplified with the primers were as follows: β-actin, 191 base pairs (bp); and inos, 114 bp. Real-time amplification was carried out using a Rotor- Gene 6 (Uniscience, Sydney, Australia). 8 Western blotting. The total protein present in the RVLM samples was subjected to electrophoresis in 8% SDS-PAGE gels (Bio-Rad, Hercules, CA). Separated proteins were transferred onto a nitrocellulose membrane (.2 mm). The blots were then blocked with 5% nonfat dry milk for 6 h, followed by incubation with anti-at1 Dy8 (1:1,), anti-inos (1:1,2), and anti-β-actin (1:1,) at 4 C for 18 h. Blots were visualized using the secondary antibody at a fluorescence emission of 7 or 8 nm. The band intensity obtained by western blotting was measured using the Odyssey system (Li-COR Biosciences, Lincoln, NE). The antibodies used were anti-at1 (Proteimax Biotecnologia, Cotia, Brazil), anti-inos (GeneTex, Irvine, CA), and anti-β-actin (Proteimax Biotecnologia). Indeed, the second antibody employed was anti-rabbit IgG with a fluorescence at 7 nm at a dilution of 1:1,. Surgical instrumentation for the acquisition of cardiovascular parameters. Six weeks after the renal surgery, rats were anesthetized with ketamine and xylazine (4 and 2 mg/kg, IP, respectively) and fitted with femoral venous and arterial catheters for drug injection and arterial pressure recording, respectively. After 1 day, the MAP and heart rate were first recorded in conscious rats, and then the rats were slowly anesthetized with urethane ( g/kg, IV). 8 These procedures were performed in the fifth to fourth series of experiments. Downloaded from by guest on 13 January 219 AMERICAN JOURNAL OF HYPERTENSION VOLUME 23 NUMBER 7 july 21 79

3 original contributions AT 1 R Receptor and inos in the RVLM Injection procedures. In the third, fourth, and fifth series of experiments, urethane-anesthetized rats were placed in a stereotaxic frame (David Kopf Instruments, Tujunga, CA). Saline (1 nl), losartan (1 nmol/1 nl), or aminoguanidine (25 pmol/1 nl) were bilaterally injected into the RVLM. 8 An interval of 1 2 s separated the injections between sides in the RVLM. At the end of the experiments, 1 nl of Evans blue dye (2%) was injected into the RVLM, and histology was subsequently performed to confirm injection sites. Figure 1a shows a representative histological coronal view of the dye distribution within the RVLM region. Analysis of the sympathetic nerve activity. For the rsna recording, the left renal nerve was retroperitoneally exposed, placed on bipolar silver electrodes, and covered with paraffin oil. The signal from the renal nerve was displayed on an oscilloscope (TDS 22; Tektronix, Beaverton, OR), and the nerve activity was amplified (gain 2K, NeuroLog; Digitimer, Welwyn Garden City, UK), filtered by a band-pass filter (5 1, Hz), and collected for display and later analysis using a PowerLab data acquisition system (ADInstruments, Bella Vista, Australia). At the end of the experiments, the background noise level was determined following hexamethonium bromide (3 mg/ kg, IV) administration. The rsna was rectified online, integrated from the raw data obtained for each heart period, and expressed as volts per second. Additionally, the neural activity was analyzed offline using the appropriate software (Spike Histogram; ADInstruments). The responses of rsna to the various stimuli are expressed as the percentage of change compared to the basal value obtained immediately before each test. For this purpose, the raw nerve signal was passed through a spike discriminator (PowerLab) to remove background noise, and then the total nerve activity expressed in spikes/s was computed from the time at which it changed from the basal value to when it returned to the basal value. The basal rsna is expressed as spikes/s over a period of 6 s. The mean value obtained was a b c compared to the mean value determined before each test. Only experiments in which the level of background noise was confirmed at the end of the experiments following hexamethonium and terminal anesthesia are included in this report. The clip and rsna were performed in the same side (left), however not in the same place. The clip was positioned in the left renal artery close to the kidney, and the electrode to record rsna was positioned at the junction of the aorta and the left renal artery. Considering that the surgery for the clip implantation was performed very carefully to avoid any damage in the renal nerve, therefore, we believe that the integrity of nerve was not affected after clipping. Statistical analyses. Results are presented as means ± s.e. Data were evaluated by one-way analysis of variance, followed by the Student Newman Keuls method and the paired Student s t-test when appropriate. The level of statistical significance was defined as P <.5. Statistical evaluation was carried out using GraphPad Prism 4 (GraphPad Software, San Diego, CA). Results Analysis of the gene and protein expression levels of AT 1 R and inos in the RVLM of and rats According to the western blotting results, AT 1 R protein expression was increased in the RVLM of the rats when compared to rats (, 133 ± 3 vs., 1 ± 7%, P <.5) (Figure 2b). This data corroborate with the levels of AT 1 R gene expression in the RVLM shown previously in our laboratory 8 (Figure 2a). Concerning inos, the gene expression was significantly higher in rats (58.3 ± 16.5 arbitrary units) compared to rats (1. ±.1 arbitrary units) (P <.2) in the RVLM, as shown in Figure 2c. The increase was also observed for the protein expression, with values of 29 ± 26% and 1 ± 7% in the and rats in the RVLM, respectively (P <.4) (Figure 2d). STN NA RVLM NTS NTS NA STN RVLM Downloaded from by guest on 13 January mm 1.25 mm ION CST ION CST Bregma-11.8 mm.7 mm.73 mm Figure 1 Representative histological coronal view of the brain stem. Typical injection site in the RVLM as evaluated by (a) 1 nl of Evans blue diffusion, (b) bilateral punches, and (c) a schematic representation. CST, corticospinal tract; ION, inferior olivary nucleus; NA, nucleus ambiguous; NTS, nucleus of the tractus solitarii; RVLM, rostral ventrolateral medulla; STN, spinal trigeminal nucleus. 71 july 21 VOLUME 23 NUMBER 7 AMERICAN JOURNAL OF HYPERTENSION

4 AT 1 R Receptor and inos in the RVLM original contributions a 1. c 75 mrna expression of AT 1 R (arbitrary units) mrna expression of inos (arbitrary units) b Protein expression of AT 1 R (%) 5 kda AT 1 R β-actin Effects of losartan injection into the RVLM of urethane-anesthetized rats Six weeks after the renal surgery, the group displayed a significant increase in MAP and rsna, as compared to the group (, 193 ± 8 mm Hg;, 115 ± 5 mm Hg and, 152 ± 4 spikes/s; 115 ± 5 spikes/s, respectively, P <.5). Heart rate and body weight did not differ between the groups. In hypertensive rats, losartan decreased MAP by 6 ± 4% (from 18 ± 8 to 168 ± 8 mm Hg) in 5 min, with a further gradual reduction until 3 min after the injection (11 ± 6%, from 18 ± 8 to 162 ± 8 mm Hg, P <.5), as shown in Figure 3a. The reduction in MAP was accompanied by a significant decrease in rsna, with a maximal reduction during the 3 min ( 18 ± 2%, P <.5) following losartan injection in the animals (Figure 3b). In contrast, MAP and rsna remained unchanged after injection in the animals. Figure 3c,d show the representative responses to bilateral losartan injection into the RVLM of the or rats. Furthermore, saline was injected in the RVLM in five experiments, and no changes in cardiovascular parameters or sympathetic nerve activity were observed. Effects of aminoguanidine injection into the RVLM of urethane-anesthetized rats In the group, a higher reduction in MAP was observed 45 min following aminoguanidine injection (from 188 ± 8 to 128 ± 15 mm Hg, P <.5), with a gradual return to the basal condition by ~6 min after the injection (146 ± 9 mm Hg) (Figure 4a). The aminoguanidine injected into the RVLM d Protein expression of inos (%) Figure 2 AT 1 R and inos mrna and protein expression. Relative amounts of (a) AT 1 R mrna (as published by Oliveira-Sales et al. 8 ), (b) AT 1 R protein expression, and (c,d) inos mrna and protein expression in the rostral ventrolateral medulla compared to β-actin mrna, in the and groups. P <.5 compared to the. AT 1 R, angiotensin type 1 receptor; inos, inducible nitric oxide synthase. 131 kda inos β-actin significantly reduced the rsna only at 45 min after injection (34 ± 8%). In contrast, MAP and rsna remained unchanged in rats (Figure 4b). Figure 4c,d show the representative responses to aminoguanidine injection into the RVLM of or rats. Discussion In the present study, we showed that AT 1 R protein levels and inos at both the gene and protein levels are upregulated in the RVLM of hypertensive animals. Additionally, we observed that losartan and aminoguanidine administration into the RVLM decreased MAP and rsna only in hypertensive rats. Taken together, the results suggest that Ang II (via AT 1 R) and NO (produced by inos) in the RVLM are involved in the maintenance of arterial hypertension and sympathoexcitation in the rats. It has been suggested that the increase in peripheral sympathetic activity present in model could be due to changes in the central nervous system triggered by an increase in circulating Ang II. Katholi et al., in 1982, 17 showed that renal denervation of the nonclipped kidney produced no change in systolic blood pressure, whereas renal denervation of the clipped kidney resulted in a significant decrease in systolic blood pressure. This data indicate that the depressor effect of clipped kidney denervation in the model is associated with a decrease in sympathetic nervous system activity in the absence of changes in renin angiotensin system activity. 17 One mechanism by which circulating Ang II exerts its effects centrally is by binding to its receptors on neurons in specialized brain regions that lack a blood brain barrier, known as Downloaded from by guest on 13 January 219 AMERICAN JOURNAL OF HYPERTENSION VOLUME 23 NUMBER 7 july

5 original contributions AT 1 R Receptor and inos in the RVLM a 1 b 5 5 % of MAP 5 1 % of rsna Time (min) Time (min) 3 c PAP HR (bpm) MAP rsna (AU) rsna (V/s) Losartan -RVLM Hexamethonium (IV) circumventricular organs. 2 Densely populated with fenestrated capillaries for high permeability, the circumventricular organs are localized around the third and fourth ventricles and are known to be pivotal in mediating the increases in blood pressure. Neurons in these regions, such as in the subfornical organ and organum vasculosum of the lamina terminalis, may send their signal to other downstream cardiovascular control regions of the brain, such as the RVLM, which additionally activates several neurohumoral pathways. 18 It is well known that Ang II plays a key role in the central cardiovascular control through the activation of AT 1 R in the RVLM. 15 In the present study, we discovered the protein upregulation of AT 1 R in the RVLM in the hypertension model. Similar results have been observed in other experimental models, such as in experimental myocardial infarction; 19 likewise, this model also presents a sympathetic hyperactivity. Accordingly, we observed a slow and gradual decrease in MAP following losartan administration into the RVLM of hypertensive animals only. The maximum response was observed 3 min later. This slow depressing response can be explained by the mechanism of action of Ang II in the RVLM. In vitro studies of the neurons present in the RVLM region demonstrated that C1 cells possess AT 1 R, whose activation leads to a reduction of the resting potassium conductance. This effect increases the discharge rate and excitability of these cells, which is likely to contribute to the sympathoexcitation observed when Ang II Losartan -RVLM. Baseline 1 min 15 min 3 min Baseline 1 min 15 min 3 min d PAP HR (bpm) MAP rsna (AU) rsna (V/s) Hexamethonium (IV) Figure 3 Losartan microinjection into the RVLM. Values of Δ% of (a) mean arterial pressure (MAP) and (b) renal sympathetic nerve activity (rsna) during bilateral injection of losartan (1 nmol) into the RVLM in the and groups. Representative traces of pulsatile arterial pressure (PAP), heart rate (HR), MAP, rsna, and integral rsna ( rsna) responses during the bilateral injection of losartan (1 nmol) into the RVLM in the (c) hypertensive and (d) animals. AU, arbitrary units; bpm, beats/min; IV, intravenous; RVLM, rostral ventrolateral medulla. P <.5 compared to. is injected into the RVLM in vivo. 2 Moreover, in the present study, blocking of the AT 1 R also promoted a gradual sympathetic inhibition in hypertensive animals. Different experimental models have demonstrated that Ang II may act through the AT 1 R pathway in the RVLM, promoting hypertension, as occurs in spontaneously hypertensive rats 21 and deoxycorticosterone acetate-salt 22 hypertension in transgenic rats harboring the mouse renin Ren-2 gene (TGR (mren2) 27). 23 Thus, our aim was to investigate whether a similar mechanism occurs in the hypertension model because it is an angiotensin renin-dependent model. In 21, Allen showed that the administration of candesartan (AT 1 R antagonist) into the RVLM produces hypotension and a 2% reduction in lumbar SNA in spontaneously hypertensive rat. 24 Additionally, further studies performed by the same author showed that transfection of the AT 1 R A adenovirus into the RVLM results in arterial hypertension, which is maintained for 3 4 days. This is an interesting finding, showing for the first time that a chronic increase in AT 1 R A activity in the RVLM promotes an increase in blood pressure over a period of days. 7 Our data suggest an important role of the activation of the central angiotensin renin system in the maintenance of hypertension in the model and corroborate previous reports from the literature. In 1992, Nishimura et al. showed increases of mrna angiotensinogen levels in several brain regions, such Downloaded from by guest on 13 January july 21 VOLUME 23 NUMBER 7 AMERICAN JOURNAL OF HYPERTENSION

6 AT 1 R Receptor and inos in the RVLM original contributions c a % of MAP PAP HR (bpm) MAP rsna (AU) rsna (V.s) d PAP HR (bpm) MAP rsna (AU) rsna (V.s) Time (min) Aminoguanidine Aminoguanidine RVLM Baseline 1 min 15 min 3 min 45 min 6 min -RVLM Baseline 1 min 15 min 3 min 45 min 6 min Time (min) Hexamethonium (IV) Hexamethonium (IV) Figure 4 Aminoguanidine microinjection into the RVLM. Values of Δ% of (a) mean arterial pressure (MAP) and (b) renal sympathetic nerve activity (rsna) during bilateral injection of aminoguanidine (25 pmol) into the RVLM in the and groups. Representative traces of pulsatile arterial pressure (PAP), heart rate (HR), MAP, rsna, and integral rsna ( rsna) responses during the bilateral injection of aminoguanidine (25 pmol) into the RVLM in the (c) hypertensive and (d) animals. AU, arbitrary units; bpm, beats/min; IV, intravenous; RVLM, rostral ventrolateral medulla. P <.5 compared to. as the medulla oblongata and hypothalamus, during the stable phase of hypertension. 25 Moreover, there is evidence that intracerebroventricular infusion of AT 1 R receptor antisense prevents hypertension by the inhibition of receptor synthesis. 26 It is possible that the described prevention of hypertension is related to the antisense actions on RVLM. It is known that losartan binds with high affinity and specificity to the AT 1 R with a slow dissociation rate, and it is b % of RSNA 6 3,-fold more selective for the AT 1 R than for the AT 2 R. Pharmacologically, AT 1 R antagonists block the pressor and functional responses of Ang II both in vitro and in vivo. 27 In contrast to AT 1 R, the functions of AT 2 R regarding the central regulation are not well understood. However, the patch clamp data from the cultured individual neurons of the hypothalamus and brainstem clearly demonstrated an increase in the potassium current induced by AT 2 R, 28 an effect contrary to that of the AT 1 R on neuronal channel function. 29 The functions and intracellular signaling pathways of the AT 2 R in most peripheral tissues and organs are also opposite to that of the AT 1 R. For example, stimulating AT 2 R induces vasodilation, stimulates NO production and hypotension. Taken together, these data led us to suggest that the blockade of AT 1 R leaves Ang II available for AT 2 R in the RVLM and this may be critical to maintain sympathetic tone in normal conditions. However, further integrative and molecular studies are necessary for a better understanding of the balance between AT 1 R and AT 2 R expression in the RVLM and its contribution to Ang II hypertension. Another important finding of the present study was that both gene and protein inos expression were increased in the RVLM of hypertensive animals. Furthermore, the inos inhibition in the RVLM promoted hypotension and sympathetic inhibition only in hypertensive animals. Thus, either the AT 1 R receptor and/or NO produced by inos plays a key role in the maintenance of hypertension and sympathoexcitation. However, we have thus far obtained no information regarding which cell types are involved in the upregulation of AT 1 R and inos within the RVLM. The role of glial cells and the phenotypes of neurons and endothelial cells must be addressed in future work. The inos can be induced by a variety of factors involved in inflammation such as LPS and cytokines in macrophages, endothelial cells, hepatocytes, and neutrophils. 3 Previous experiments of our laboratory showed that the gene expression of interleukin-1β is upregulated in the RVLM of animal (data not shown). However, further experiments are needed to explain this mechanism. The effects of NO in blood pressure regulation are controversial. Some studies showed that the NO in the RVLM reduces blood pressure by inhibiting the sympathetic nervous system; however, conflicting results have also been reported Moreover, there is evidence that NO can cause biphasic responses depending on the administered dose. 37 However, differences between these results may be related to different approaches; for example, most experiments were performed in anesthetized rats and evaluated the acute effects of NO donors, and sometimes the blockers administered were not specific for NO. In 25, Kimura et al. showed that adenovirus vectors encoding inos transfected into the RVLM in Wistar-Kyoto rats increase blood pressure via activation of the sympathetic nervous system. 14 Aminoguanidine administration into the RVLM after transfection resulted in a reduction in blood pressure and in sympathetic activity. Additionally, tempol (SOD mimetic) administration promoted hypotension and Downloaded from by guest on 13 January 219 AMERICAN JOURNAL OF HYPERTENSION VOLUME 23 NUMBER 7 july

7 original contributions AT 1 R Receptor and inos in the RVLM sympathetic inhibition, with a consequent reduction in the production of superoxide and lipid peroxidation in the same animals. The results thus showed that upregulation of inos in the RVLM elicits hypertension by activating the sympathetic nervous system, and these effects might be mediated by an increase in oxidative stress in the RVLM because NO may be inhibitory or excitatory for neurons in the RVLM region according to its bioavailability. Previous studies from our laboratory have shown that vitamin C (antioxidant) and tempol administration into the RVLM promote depressor effects, such as rsna reduction in hypertensive animals. 8,38 Thus, we cannot exclude the possibility that the increased production of NO resulting from inos activation promotes a greater interaction with superoxide. Superoxide anions react rapidly with NO, forming peroxy nitrite and decreasing the bioavailability of NO. Therefore, an increase in the superoxide anion levels in the RVLM might decrease the bioavailability of NO in these regions, leading to an increase in the neuronal firing. This mechanism might contribute to the increase in sympathetic nerve activity and hypertension. Our results corroborate a recent study in spontaneously hypertensive rat. The authors of that study demonstrated that upregulation of inos in the RVLM contributes to the increase in blood pressure in this model and that blocking of the inos enzyme promotes hypotension and bradycardia. 39 In conclusion, the present study suggests that the upregulation of AT 1 R and activation of inos in the RVLM may be important mechanisms in maintaining the high blood pressure and the sympathetic activation in the Ang II dependent model of hypertension. In summary, changes in the excitatory and/or inhibitory neuro transmission within the RVLM might constitute a mechanism that is essential for the development and maintenance of arterial hypertension. Taken together with other evidence, it is conceivable that the increase in the AT 1 R receptor and inos expression levels in the RVLM occurs under conditions such as arterial hypertension, thereby modulating central sympathetic outflow and resulting in blood pressure changes. An interesting recent work showed that an enrichment of AT 1 R receptors in the RVLM is associated with increased sympathetic drive in heart failure, 4 and a similar phenomenon is described in the present study in renovascular arterial hypertension. Therefore, the present study provides new insights regarding the central mechanism underlying the actions of Ang II and NO in conditions characterized by increased sympathetic activity, such as arterial hypertension and other chronic cardiovascular diseases. Acknowledgment: This study was funded by the Fundação de Amparo Pesquisa do Estado de São Paulo (5/6151-6). We are grateful to Andreia Sterman Heimann, PhD (Proteimax, Cotia, Brazil) for her assistance in the western blotting analyses. Disclosure: The authors declared no conflict of interest. 1. Navar LG, Zou L, Von Thun A, Tarng Wang C, Imig JD, Mitchell KD. Unraveling the mystery of Goldblatt hypertension. News Physiol Sci 1998; 13: Ku YH, Jia YF, Chang YZ. Mechanisms underlying pressor response of subfornical organ to angiotensin II. Peptides 1999; 2: Potts PD, Hirooka Y, Dampney RA. Activation of brain neurons by circulating angiotensin II: direct effects and baroreceptor-mediated secondary effects. Neuroscience 1999; 9: Dampney RA, Tan PS, Sheriff MJ, Fontes MA, Horiuchi J. Cardiovascular effects of angiotensin II in the rostral ventrolateral medulla: the push-pull hypothesis. Curr Hypertens Rep 27; 9: Campos RR, Bergamaschi CT. 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