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1.  Angiotensin Type 2 Receptors in the Intermediolateral Cell Column of the Spinal Cord: Negative Regulation of Sympathetic Nerve Activity and Blood Pressure 
International journal of cardiology  2013;168(4):10.1016/j.ijcard.2013.06.051.
BACKGROUND
Our previous study demonstrated that AT2R in brainstem nuclei participated in the regulation of sympathetic outflow and cardiovascular function. However, the functional significance of AT2R in the intermediolateral cell column (IML) of the thoracic spinal cord in normal rats remains elusive. We hypothesized that AT2R activation in the IML exert a sympatho-inhibitory effect.
METHODS and RESULTS
Using Western-blot analysis, immunohistochemical staining and quantitative Real-Time PCR, both AT1R and AT2R expression were detected in the spinal cord. The highest AT2R protein expression was found in the IML, while AT1R expression didn’t display regional differences within the gray matter. Microinjection of AngII into the IML dose-dependently elevated mean blood pressure (MAP, employing a transducer-tipped catheter) and renal sympathetic nerve activity (RSNA, using a pair of platinum-iridium recording electrodes), which was completely abolished by Losartan, and attenuated by TEMPOL and apocynin. Activation of AT2R in the IML with CGP42112 evoked hypotension (ΔMAP: −21 ± 4 mmHg) and sympatho-inhibition (RSNA: 73 ± 3% of baseline), which were completely abolished by PD123319 and L-NAME. Blockade of AT2R in the IML with PD123319 significantly increased MAP (11 ± 1 mmHg) and sympathetic nerve activity (RSNA: 133 ± 13 % of baseline). Moreover, PD123319 significantly enhanced the AngII induced pressor response. Furthermore, in isolated IML neurons, CGP42112 treatment augmented potassium current and decreased resting membrane potential by employing whole-cell patch clamp.
Conclusion
In the normal condition, AT2R in the IML tonically inhibit sympathetic activity through an NO/NOS dependent pathway and subsequent potassium channel activation.
doi:10.1016/j.ijcard.2013.06.051
PMCID: PMC3821163  PMID: 23871345
Intermediolateral cell column; Angiotensin type 1 receptor; Angiotensin type 2 receptor; arterial blood pressure; renal sympathetic nerve activity
2.  CENTRAL RHO KINASE INHIBITION RESTORES BAROREFLEX SENSITIVITY AND AT1R PROTEIN IMBALANCE IN CONSCIOUS RABBITS WITH CHRONIC HEART FAILURE 
Hypertension  2013;61(3):723-729.
The small GTPase RhoA and its associated kinase ROCKII are involved in vascular smooth muscle cell contraction and endothelial nitric oxide synthase (eNOS) mRNA destabilization. Overactivation of the RhoA/ROCKII pathway is implicated in a number of pathologies including chronic heart failure (CHF) and may contribute to the enhanced sympathetic outflow seen in CHF due to decreased nitric oxide (NO) availability. Thus, we hypothesized that central ROCKII blockade would improve the sympatho-vagal imbalance in a pacing rabbit model of CHF in an NO-dependent manner. CHF was induced by rapid ventricular pacing and characterized by an ejection fraction of ≤45%. Animals were implanted with an ICV cannula and osmotic minipump (rate: 1 µL/h) containing sterile saline, 1.5 µg/kg/day fasudil (Fas, a ROCKII inhibitor) for 4 days or Fas + 100 µg/kg/day L-NAME, a NOS inhibitor. Arterial baroreflex control was assessed by IV infusion of sodium nitroprusside and phenylephrine. Fas infusion significantly lowered resting HR by decreasing sympathetic and increasing vagal tone. Furthermore, Fas improved baroreflex gain in CHF in an NO-dependent manner. In CHF Fas animals, the decrease in HR in response to IV metoprolol was similar to sham and was reversed by L-NAME. Fas decreased AT1R and phospho-RhoA protein expression and increased eNOS expression in the brainstem of CHF animals. These data strongly suggest that central ROCKII activation contributes to cardiac sympatho-excitation in the setting of CHF and that central Fas restores vagal and sympathetic tone in an NO-dependent manner. ROCKII may be a new central therapeutic target in the setting of CHF.
doi:10.1161/HYPERTENSIONAHA.111.00396
PMCID: PMC3573242  PMID: 23283363
heart failure; rho-associated kinase; nitric oxide synthase; autonomic nervous system; Angiotensin AT1 receptor
3.  Angiotensin II Increased Neuronal Stem Cell Proliferation: Role of AT2R 
PLoS ONE  2013;8(5):e63488.
Angiotensin II (Ang II), known a potent vasoactive substance in the renin-angiotensin system in the brain, plays a critical role in systemic blood pressure control. However, increasing evidence indicated that the physiological role of Ang II go beyond its vasoactive effect. In the present study, we demonstrated that Ang II type-1 receptor (AT1R) and type-2 receptor (AT2R) were expressed in primary rat hippocampal neuronal stem cells (NSCs). Treatment of rat hippocampal NSCs with Ang II increased cell proliferation. Pretreatment of NSCs with specific AT2R, but not AT1R, antagonist significantly suppressed Ang II-induced cell proliferation. Furthermore, Ang II stimulated ERK and Akt phosphorylation in NSCs. Pretreatment of MEK inhibitor, but not PI3K inhibitor, inhibited Ang II-induced ERK phosphorylation as well as cell proliferation. In addition, stimulation of NSCs with Ang II decreased expression of KV 1.2/KV 3.1 channels and blocked K+ currents which lie downstream of ERK activation. Taken together, these findings underpin the role of AT2R as a novel target that regulates cell proliferation mediated by Ang II with implications for therapeutic intervention for regulation of neurogenesis.
doi:10.1371/journal.pone.0063488
PMCID: PMC3655161  PMID: 23691054
4.  Abnormal Baroreflex Function is Dissociated from Central Angiotensin II Receptor Expression in Chronic Heart Failure 
Shock (Augusta, Ga.)  2012;37(3):319-324.
Neurohumoral disturbances characterize chronic heart failure (CHF) and are reflected, in part, as impairment of baroreflex sensitivity (BRS) and sympathetic function. However the mechanisms that trigger these neurohumoral abnormalities in CHF are not clear. We hypothesized that the BRS is blunted early in CHF and that the humoral effects occur later and contribute to progressive loss of cardiovascular control in CHF. We assessed the BRS (bpm/mmHg) and recorded renal sympathetic nerve activity (RSNA) in four groups of conscious rabbits at varying time intervals; control, one week CHF, two week CHF and three week CHF. CHF was induced by ventricular pacing at 360 bpm and was assessed by echocardiography. Arterial blood pressure and heart rate were recorded by an implanted telemetric device and RSNA through an implanted electrode. A significant fall in the ejection fraction, fractional shortening and an increase in LVESD & LVEDD was observed in all CHF groups. The BRS was significantly reduced in all the CHF groups with no significant change in the basal RSNA(% of maximum) after 1 week of pacing, a small but insignificant rise in RSNA was seen at 2 weeks and a significant rise in RSNA was observed at 3 weeks. AT1 receptor protein (Western Blot) and mRNA (RT-PCR) expression in the rostral ventrolateral medulla (RVLM) exhibited a progressive increase with the duration of CHF, reaching significance after 3 weeks, the same time point in which RSNA was significantly elevated. These data are the first to examine early changes in central AT1 receptors in CHF and suggest that the fall in BRS and hemodynamic changes occur early in the development of CHF followed by sympatho-excitation and over-expression of AT1 receptors with the progression of CHF causing further impairment of cardiovascular control.
doi:10.1097/SHK.0b013e31824581e8
PMCID: PMC3281308  PMID: 22258229
Baroreflex; heart failure; sympathetic activity; angiotensin II receptor
5.  Tonic Glutamatergic Input in The Rostral Ventrolateral Medulla is Increased in Rats With Chronic Heart Failure 
Hypertension  2008;53(2):370-374.
Chronic heart failure (CHF) is characterized by increased sympathetic tone. The glutamatergic input in the rostral ventrolateral medulla (RVLM), which is a key region involved in sympathetic outflow, seems not to be involved in the generation of sympathetic tone in the normal state. The aim of this study was to determine the role of the RVLM glutamate receptors in generation of sympathetic tone in CHF. CHF was produced by left coronary artery ligation. Bilateral microinjection of the glutamate receptor antagonist kynurenic acid (KYN), the N-methyl-D-aspartate (NMDA) receptor antagonist D-AP5, or the non-NMDA receptor antagonist CNQX into the RVLM dose-dependently reduced resting blood pressure and renal sympathetic nerve activity in CHF but not in sham rats. Picoinjection of KYN (100 pmol in 5 nl) significantly decreased the basal discharge by 47% in 25 RVLM presympathetic neurons in CHF rats, In contrast, KYN had no effect on the discharge in all 22 RVLM presympathetic neurons tested in sham rats. These data suggest that upregulated glutamate receptors, including NMDA and non-NMDA, in the RVLM are involved in tonic control of elevated sympathetic tone in CHF.
doi:10.1161/HYPERTENSIONAHA.108.122598
PMCID: PMC3574554  PMID: 19029485
sympathoexcitation; glutamate receptors; micro/picoinjection; extracellular recording; presympathetic neuron
6.  Brain-Selective Overexpression of Angiotensin Converting Enzyme 2 Attenuates Sympathetic Nerve Activity and Enhances Baroreflex Function in Chronic Heart Failure 
Hypertension  2011;58(6):1057-1065.
Angiotensin-converting enzyme 2 (ACE2) has been suggested to be involved in the central regulation of autonomic function. During chronic heart failure (CHF), elevated central angiotensin II signaling contributes to the sustained increase of sympathetic outflow. This is accompanied by a downregulation of ACE2 in the brain. We hypothesized that central overexpression of ACE2 decreases sympathetic outflow and enhances baroreflex function in CHF. Transgenic mice overexpressing human ACE2 selectively in the brain (SYN-hACE2) and wild type littermates (WT) were used. CHF was induced by permanent coronary artery ligation (CAL). Four weeks after CAL, both WT and SYN-hACE2 mice exhibited a significant decrease in left ventricular ejection fraction (<40%). A slight decrease in MAP was found only in SYN-hACE2 mice. Compared with WT mice with CHF, brain-selective ACE2 overexpression attenuated left ventricular end-diastolic pressure; decreased urinary norepinephrine excretion; baseline RSNA (WT CHF: 71.6±7.6% Max vs. SYN-hACE2 CHF: 49.3±6.1% Max); and enhanced baroreflex sensitivity (Maximum Slope: WT Sham: 1.61±0.16 vs. SYN-hACE2 CHF: 1.51±0.17%/mmHg). Chronic subcutaneous blockade of mas receptor increased RSNA in SYN-hACE2 mice with CHF (A779: 67.3±5.8% vs. vehicle: 46.4±3.6% of Max). An up-regulation in angiotensin II type 1 receptor (AT1R) expression was detected in medullary nuclei in WT CHF mice, which was significantly attenuated in SYN-hACE2 mice with CHF. These data suggest that central ACE2 overexpression exerts a potential protective effect in CHF through attenuating sympathetic outflow. The mechanism for this effect involves angiotensin (1-7) mas signaling as well as a decrease in AT1R signaling in the medulla.
doi:10.1161/HYPERTENSIONAHA.111.176636
PMCID: PMC3224814  PMID: 22025374
heart failure; angiotensin converting enzyme 2; angiotensin II; angiotensin I (1-7); autonomic function; baroreflex
7.  Ontogeny of Angiotensin Type 2 and Type 1 Receptor Expression in Mice 
In the current experiment, we determined AT2R and AT1R protein expression by Western blot analysis in developing normal mice. The results indicate that, (1) in all detected brain regions and in the spinal cord, adult mice exhibited significantly higher AT2R expression and lower AT1R expression in total protein extracts compared to fetuses and neonates; (2) other major organs, including heart, lung, liver, and kidney, exhibited the same expression pattern as the brain and spinal cord; (3) reciprocal changes in AT2R and AT1R expression were found in the total protein extracts from the brainstems of mice from one day prenatal to six weeks of age. There was a negative correlation between AT2R and AT1R protein expression; (4) in both membrane and cytosolic fractions from the brainstem, adult mice exhibited higher AT2R and lower AT1R expression than did fetuses and neonates; (5) in the brainstem, there were no significant differences in AT2R and AT1R mRNA levels among fetal, neonatal, and adult mice. The above results reconfirmed our previous finding in rats that adult animals have higher AT2R and lower AT1R expression compared to fetuses and neonates. These data imply an involvement of AT1R in fetal development and of AT2R in adult function.
doi:10.1177/1470320312443720
PMCID: PMC3463952  PMID: 22526820
Angiotensin receptors; development; protein expression; mRNA expression
8.  CENTRAL ANG-(1–7) ENHANCES BAROREFLEX GAIN IN CONSCIOUS RABBITS WITH HEART FAILURE 
Hypertension  2011;58(4):627-634.
In chronic heart failure (CHF), arterial baroreflex function is impaired, in part, by activation of the central renin-angiotensin system. A metabolite of Angiotensin II (Ang II), Ang-(1–7), has been shown to exhibit cardiovascular effects that are in opposition to that of Ang II. However, the action of Ang-(1–7) on sympathetic outflow and baroreflex function is not well understood, especially in CHF. The aim of this study was to determine the effect of intracerebroventricular infusion of Ang-(1–7) on baroreflex control of heart rate (HR) and renal sympathetic nerve activity (RSNA) in conscious rabbits with CHF. We hypothesized that central Ang-(1–7) would improve baroreflex function in CHF. Ang-(1–7) (2 nmol/1 μl/hour) or artificial cerebrospinal fluid (1 μl/hour) was infused by an osmotic mini-pump for 4 days in sham and pacing-induced CHF rabbits (n=3–6/group). Ang-(1–7) treatment had no effects in sham rabbits but reduced HR and increased baroreflex gain (7.4±1.5 bpm/mm Hg vs. 2.5±0.4 bpm/mm Hg, P<0.05) in CHF rabbits. The Ang-(1–7) antagonist A779 (8 nmol/1 μl/hr) blocked the improvement in baroreflex gain in CHF. Baroreflex gain increased in CHF+Ang-(1–7) animals when only the vagus was allowed to modulate baroreflex control by acute treatment with the β-1 antagonist metoprolol, indicating increased vagal tone. Baseline RSNA was significantly lower and baroreflex control of RSNA was enhanced in CHF rabbits receiving Ang-(1–7). These data suggest that augmentation of central Ang-(1–7) inhibits sympathetic outflow and increases vagal outflow in CHF thus contributing to enhanced baroreflex gain in this disease state.
doi:10.1161/HYPERTENSIONAHA.111.177600
PMCID: PMC3187859  PMID: 21844487
angiotensin-(1–7); heart failure; sympathetic nervous system; baroreflex; vagus nerve; blood pressure; heart rate
9.  AT2 Receptor Signaling and Sympathetic Regulation 
Current opinion in pharmacology  2010;11(2):124-130.
There is a growing consensus that the balance between Angiotensin Type 1 (AT1R) and Angiotensin Type 2 (AT2R) signaling in many tissues may determine the magnitude and, in some cases the direction, of the biological response. Sympatho-excitation in cardiovascular diseases is mediated by a variety of factors and is, in part, dependent on Angiotensin II signaling in the central nervous system. Recent data have provided evidence that the AT2R can modulate sympatho-excitation in animals with hypertension and heart failure. The evidence for this concept is reviewed and a model is put forward to support the rationale that therapeutic targeting of the central AT2R may be beneficial in the setting of chronic heart failure.
doi:10.1016/j.coph.2010.11.004
PMCID: PMC3075409  PMID: 21159555
10.  Activation of Central Angiotensin Type 2 Receptors Suppresses Norepinephrine Excretion and Blood Pressure in Conscious Rats 
American Journal of Hypertension  2011;24(6):724-730.
Background
We have previously documented that central Angiotensin type 2 receptors (AT2R) negatively modulate sympathetic outflow and arterial blood pressure (BP). In the current study, we determined the effects of intracerebroventricular (icv) infusion of Compound 21 (C21), the first selective non-peptide AT2R agonist, on norepinephrine (NE) excretion and BP in rats.
Methods
C21 was icv infused by a Micro-osmotic pump for 7 days. Urinary NE concentration was measured using the Norepinephrine Enzyme Immunoassay kit. BP was recorded by radiotelemetry. After 7 days, the rats were euthanized and three sympathetic relevant brain regions and cerebral cortex were micro-punched to measure nNOS protein expression by Western Blot. In addition, the influence of C21 on neuronal potassium current (IKv) was determined by whole cell patch-clamp in a neuron cell-line, CATH.a.
Results
(1) icv treatment of C21 significantly decreased NE concentration and amount in nighttime urine but had no effect in daytime urine. (2) C21 treated rats exhibited a slight but significant decrease in BP. (3) The effects of C21 on NE excretion and BP were abolished by AT2R antagonist, PD123319, and nitric oxide synthase (NOS) inhibitor, L-NAME. (4) C21 treatment significantly up regulated nNOS expression in the PVN and RVLM, but not in the NTS and cerebral cortex. (5) In CATH.a neurons, C21 treatment significantly increased IKv, which was completely abolished by PD123319 and L-NAME.
Conclusion
These results demonstrate a central inhibitory influence of C21 on sympathetic outflow via a nNOS dependent mechanism, which might be mediated by facilitating neuronal potassium channel.
doi:10.1038/ajh.2011.33
PMCID: PMC3286515  PMID: 21394088
Angiotensin type 2 receptor; central nervous system; norepinephrine; blood pressure; potassium current
11.  The Attenuation of Central Angiotensin II-dependent Pressor Response and Intra-neuronal Signaling by Intracarotid Injection of Nanoformulated Copper/Zinc Superoxide Dismutase 
Biomaterials  2010;31(19):5218-5226.
Adenoviral-mediated overexpression of the intracellular superoxide (O2•−) scavenging enzyme copper/zinc superoxide dismutase (CuZnSOD) in the brain attenuates central angiotensin II (AngII)-induced cardiovascular responses. However, the therapeutic potential for adenoviral vectors is weakened by toxicity and the inability of adenoviral vectors to target the brain following peripheral administration. Therefore, we developed a non-viral delivery system in which CuZnSOD protein is electrostatically bound to a synthetic poly(ethyleneimine)-poly(ethyleneglycol) (PEI-PEG) polymer to form a polyion complex (CuZnSOD nanozyme). We hypothesized that PEI-PEG polymer increases transport of functional CuZnSOD to neurons, which inhibits AngII intra-neuronal signaling. The AngII-induced increase in O2•−, as measured by dihydroethidium fluorescence and electron paramagnetic resonance spectroscopy, was significantly inhibited in CuZnSOD nanozyme-treated neurons compared to free CuZnSOD- and non-treated neurons. CuZnSOD nanozyme also attenuated the AngII-induced inhibition of K+ current in neurons. Intracarotid injection of CuZnSOD nanozyme into rabbits significantly inhibited the pressor response of intracerebroventricular-delivered AngII; however, intracarotid injection of free CuZnSOD or PEI-PEG polymer alone failed to inhibit this response. Importantly, neither the PEI-PEG polymer alone nor the CuZnSOD nanozyme induced neuronal toxicity. These findings indicate that CuZnSOD nanozyme inhibits AngII intra-neuronal signaling in vitro and in vivo.
doi:10.1016/j.biomaterials.2010.03.026
PMCID: PMC2860066  PMID: 20378166
brain; superoxide dismutase; nanotechnology; drug delivery; copolymer; potassium current
12.  Developmental changes in AT1 and AT2 receptor-protein expression in rats 
It has long been known that angiotensin type-1 receptors (AT1R) play a critical role in sympathetic regulation, cardiovascular activity, and hormone secretion under physiological and pathological states. On the other hand, the functional significance of angiotensin type-2 receptors (AT2R) is poorly understood. In a recent study we demonstrated that, in rats with chronic heart failure, AT1R protein expression was increased but AT2R expression was decreased in the rostral ventrolateral medulla (RVLM). This imbalance of angiotensin receptors contributed to sympatho-excitation in the heart failure state. In the current experiment, we measured AT1R and AT2R protein expressions in the brainstem, kidney and liver from male foetuses (3 days before birth), male neonates (3 days after birth), male and female adults (8 weeks) and male aged (28 months) rats by Western blot analysis. In the brainstem, we found that the foetuses and neonates exhibited a significantly lower AT2R protein expression compared with adult rats (foetus 0.08 ± 0.01, neonate 0.12 ± 0.01, male adult 0.25 ± 0.01, female adult 0.22 ± 0.02; n = 4 per group, p < 0.001 foetus and neonate compared with male or female adults). In contrast, the foetuses and neonates expressed significantly higher AT1R protein than that of the adults (foetus 0.64 ± 0.09, neonate 0.56 ± 0.01, male adult 0.13 ± 0.02, female adult 0.08 ± 0.02; n = 4 each group, p < 0.001 foetus and neonate compared with male and female adults). In the liver, the AT2R protein was also higher in foetus and neonate, than in adult rats. Interestingly, the foetal liver expressed higher AT1R protein compared with that of the neonate. In the kidney, AT2R expression was significantly increased with age (foetus 0.08 ± 0.01, neonate 0.19 ± 0.02, male adult 0.49 ± 0.04, female adult 0.90 ± 0.10; n = 4 per group, p < 0.01–0.001). AT1R expression, on the other hand, was higher in the foetuses than that in both neonate and male adults. This study provides data contrary to existing dogma that AT2R expression is higher in foetal life and low in adults, suggesting an involvement of a potentially important functional role for AT2R in adult animals and AT1R in foetal development and/or physiology.
doi:10.1177/1470320310379065
PMCID: PMC3035052  PMID: 20807798
angiotensin II receptors; brainstem; development; kidney; liver; protein expression
13.  IMBALANCE OF AT1R AND AT2R IN THE RVLM: POTENTIAL MECHANISM FOR SYMPATHETIC OVERACTIVITY IN HEART FAILURE 
Hypertension  2008;52(4):708-714.
Up-regulation of Angiotensin type 1 receptors (AT1R) in the rostral ventrolateral medulla (RVLM) contributes to the sympatho-excitation in the chronic heart failure (CHF). However, the role of AT2R is not clear. In this study, we measured AT1R and AT2R protein expression in the RVLM and determined their effects on renal sympathetic nerve activity (RSNA), blood pressure (BP), and heart rate (HR) in anaesthetized sham and CHF rats. We found that: (1) while AT1R expression in the RVLM was up-regulated, the AT2R was significantly down-regulated (CHF: 0.06 ± 0.02 vs sham: 0.15 ± 0.02, P < 0.05); (2) simultaneously stimulating RVLM AT1R and AT2R by Ang II evoked sympatho-excitation, hypertension, and tachycardia in both sham and CHF rats, with greater responses in CHF; (3) stimulating RVLM AT1R with Ang II plus the specific AT2R antagonist PD123319 induced a larger sympatho-excitatory response than simultaneously stimulating AT1R and AT2R in sham rats, but not in CHF; (4) activating RVLM AT2R with CGP42112 induced a sympatho-inhibition, hypotension, and bradycardia only in sham rats (RSNA: 36.4 ± 5.1 % of baseline vs 102 ± 3.9 % of baseline in aCSF, P < 0.05); (5) pretreatment with ETYA, a general inhibitor of AA metabolism, into the RVLM attenuates the CGP42112 induced sympatho-inhibition. These results suggest that AT2R in the RVLM exhibits an inhibitory effect on sympathetic outflow, which is, at least partially, mediated by an AA metabolic pathway. These data implicate a down regulation in the AT2R as a contributory factor in the sympatho-excitation in CHF.
doi:10.1161/HYPERTENSIONAHA.108.116228
PMCID: PMC2760297  PMID: 18768398
Angiotensin II type 2 receptor; Angiotensin II type 1 receptor; rostral ventrolateral medulla; sympathetic outflow
14.  SIMVASTATIN INHIBITS CENTRAL SYMPATHETIC OUTFLOW IN HEART FAILURE BY A NOS MECHANISM 
Our previous study demonstrated that oral treatment with simvastatin (SIM) suppressed renal sympathetic nerve activity (RSNA) in the rabbits with chronic heart failure (CHF). The purpose of this experiment was to determine the effects of direct application of SIM to the central nervous system on RSNA and its relevant mechanisms. Experiments were carried out on 21 male New Zealand White rabbits with pacing induced CHF. The CHF rabbits received infusion of vehicle, SIM, or SIM + L-NAME into the lateral cerebral ventricle via osmotic minipump for 7 days. We found that, (1) In CHF rabbits, icv infusion of SIM significantly suppressed basal RSNA (1st day 69.5 ± 8.9 % of Max; 7th day 26.0 ± 6.0 % of Max. P < 0.05, n = 7) and enhanced arterial baroreflex function starting from the 2nd day and lasting through the following 5 days; (2) Statin treatment significantly upregulated nNOS protein expression in the rostral ventrolateral medulla (RVLM) (Control, n = 6, 0.12 ± 0.04; SIM treated, n = 7, 0.31 ± 0.05. P < 0.05); (3) In CATH.a neurons, incubation with SIM significantly upregulated the nNOS mRNA expression, which was blocked by co-incubation with Mevalonate, farnesyl-pyrophosphate, or geranylgeranyl-pyrophosphate; (4) Incubation with Y-27632 significantly upregulated nNOS mRNA expression in these neurons. These results suggest that central treatment with SIM decreased sympathetic outflow in CHF rabbits via up regulation of nNOS expression in RVLM, which may be due to the inhibition of HMG-CoA reductase and a decrease in Rho Kinase by SIM.
doi:10.1124/jpet.107.136028
PMCID: PMC2575087  PMID: 18441251
15.  The Role of Oxidant Stress on AT1 Receptor Expression in Neurons of Rabbits with Heart Failure and in Cultured Neurons 
Circulation research  2008;103(2):186-193.
We have previously reported that the expression of Angiotensin II (Ang II) type 1 receptors (AT1R) was increased in the rostral ventrolateral medulla (RVLM) of rabbits with chronic heart failure (CHF) and in the RVLM of normal rabbits infused with intracerebroventricular (ICV) Ang II. The present study investigated if oxidant stress plays a role in Ang II induced AT1R up-regulation and its relationship to the transcription factor activator protein 1 (AP1) in CHF rabbits and in the CATHa neuronal cell line. In CATHa cells, Ang II significantly increased AT1R mRNA by 123 ± 11%, P<0.01; c-Jun mRNA by 90 ± 20%, P<0.01; c-fos mRNA by 148 ± 49%, P<0.01; NADPH oxidase activity by 126 ± 43%, P<0.01 versus untreated cells. Tempol and Apocynin reversed the increased expression of AT1R mRNA, c-Jun mRNA, c-fos mRNA, and superoxide production induced by Ang II. We also examined the effect of ICV Tempol on the RVLM of CHF rabbits. Compared to vehicle treated CHF rabbits, Tempol significantly decreased AT1R protein expression (1.6±0.29 vs 0.88±0.16, P<0.05), phosphorylated Jnk protein (0.4 ± 0.05 vs 0.2 ± 0.04, P<0.05), cytosolic phosphorylated c-Jun (0.56 ± 0.1 vs 0.36 ± 0.05, P<0.05), and nuclear phosphorylated c-Jun (0.67±0.1 vs 0.3±0.08, P<0.01). Tempol also significantly decreased the AP-1-DNA binding activity in the RVLM of CHF rabbits compared to the vehicle group (9.14 × 103 vs 41.95 × 103 grey level P<0.01). These data suggest that Ang II induces AT1R up-regulation at the transcriptional level by induction of oxidant stress and activation of AP1 in both cultured neuronal cells and in intact brain of rabbits. Antioxidant agents may be beneficial in CHF and other states where brain Ang II is elevated by decreasing AT1R expression through the Jnk and AP1 pathway.
doi:10.1161/CIRCRESAHA.108.179408
PMCID: PMC2574821  PMID: 18566341
16.  NADPH oxidase-derived superoxide anion mediates angiotensin II-enhanced carotid body chemoreceptor sensitivity in heart failure rabbits 
Cardiovascular research  2007;75(3):546-554.
Objective:
A previous study from this laboratory showed that elevation of endogenous angiotensin II (Ang II) and upregulation of the angiotensin II type 1 (AT1) receptor in the carotid body (CB) are involved in the enhanced peripheral chemoreceptor sensitivity in rabbits with chronic heart failure (CHF). NADPH oxidase-derived superoxide anion mediates the effects of Ang II in many organs. We investigated whether this signaling pathway may mediate the enhanced peripheral chemoreceptor sensitivity induced by Ang II in CHF rabbits.
Methods and results:
By recording single-unit activity from the carotid sinus nerve in isolated preparations, we found that phenylarsine oxide 2 μM (PAO, NADPH oxidase inhibitor) and TEMPOL 1 mM (superoxide dismutase mimetic) significantly decreased not only the Ang II-enhanced CB chemoreceptor responses to different levels of hypoxia in sham rabbits (Δ-12.5 ± 0.8 and Δ-12.8 ± 0.9 imp/s at 40.7 ± 2.3 mm Hg of PO2, and Δ-5.6 ± 0.5 and Δ-5.3 ± 0.4 imp/s at 60.2 ± 3.1 mm Hg of PO2, p<0.05, respectively) but also the CHF-induced elevation of CB chemoreceptor responses to different levels of hypoxia (Δ-13.6 ± 1.1 and Δ-13.7 ± 0.9 imp/s at 40.9 ± 3.1 mm Hg of PO2, and Δ-6.7 ± 1.2 and Δ-6.6 ± 0.8 imp/s at 59.8 ± 3.5 mm Hg of PO2, p<0.05). In addition, mRNA and protein expressions of NADPH oxidase components (gp91phox, p40phox and p47phox) were higher in the CB from CHF rabbits compared to sham rabbits. Furthermore, 100 pM Ang II induced an increase in superoxide production in CB homogenates from sham rabbits, which was similar to that in CB homogenate from CHF rabbits. PAO and Tempol inhibited the Ang II- and CHF-enhanced superoxide anion production.
Conclusions:
These results suggest that the enhanced peripheral chemoreceptor sensitivity mediated by Ang II in CHF rabbits occurs via a NADPH oxidase-superoxide signaling pathway.
doi:10.1016/j.cardiores.2007.04.006
PMCID: PMC2062532  PMID: 17499230
Angiotensin; Reactive oxygen species; autonomic nervous system; chemoreceptor; heart failure
17.  Differential effects of cardiac sympathetic afferent stimulation on neurons in the nucleus tractus solitarius 
Neuroscience letters  2006;409(2):146-150.
Activation of the cardiac “sympathetic afferent” reflex (CSAR) has been reported to depress the arterial baroreflex and enhance the arterial chemoreflex via a central mechanism. In the present study, we used single-unit extracellular recording techniques to examine the effects of stimulation of cardiac sympathetic afferents on baro- or chemosensitive neurons in the nucleus tractus solitarius (NTS) in anesthetized rats. Of 54 barosensitive NTS neurons tested for their response to epicardial application of capsaicin (0.4 μg), 38 were significantly (P<0.01) inhibited by 38 % while 16 did not respond. Of 42 NTS chemosensitive neurons tested for their response to capsaicin, 33 were significantly (P<0.01) excited by 47 % while 9 did not respond. In addition, of 12 both barosensitive and chemosensitive NTS neurons tested for capsaicin, 2 were excited, 7 were inhibited, and 3 did not respond. In conclusion, this study indicates that CSAR activation inhibited NTS barosensitive neurons and excited NTS chemosensitive neurons, suggesting that the NTS plays an important role in processing the interactions between these cardiovascular reflexes.
doi:10.1016/j.neulet.2006.09.032
PMCID: PMC1904510  PMID: 17014954
cardiovascular reflexes; sympathetic activity; capsaicin; extracellular recording; baro-/chemosensitive neuron
18.  Exercise Training Prevents Arterial Baroreflex Dysfunction in Rats Treated With Central Angiotensin II 
Hypertension  2007;49(3):519-527.
Angiotensin II (Ang II)–induced arterial baroreflex dysfunction is associated with superoxide generation in the brain. Exercise training (EX) improves baroreflex function and decreases oxidative stress in cardiovascular diseases linked to elevated central Ang II. The aim of this study was to determine whether previous EX prevents baroreflex impairment caused by central administration of exogenous Ang II via an Ang II–superoxide mechanism. Four groups of rats were used: non-EX artificial cerebrospinal fluid infused, non-EX Ang II infused, EX artificial cerebrospinal fluid infused, and EX Ang II infused. Rats were treadmill trained for 3 to 4 weeks and subjected to intracerebroventricular infusion of Ang II over the last 3 days of EX. Twenty-four hours after the end of EX, the arterial baroreflex was assessed in anesthetized rats. Compared with non-EX artificial cerebrospinal fluid–infused rats, Ang II significantly decreased baroreflex sensitivity (maximum gain: 3.0 ± 0.2% of maximum per millimeter of mercury versus 1.6 ± 0.1% of maximum per millimeter of mercury; P < 0.01), which was abolished by acute intracerebroventricular infusion of the Ang II type 1 receptor antagonist losartan and the reduced nicotinamide-adenine dinucleotide phosphate oxidase inhibitor apocynin. EX prevented the decrease in baroreflex sensitivity and downregulated Ang II type 1 receptor and NADPH oxidase subunit protein expression in the paraventricular nucleus of Ang II–infused rats. Finally, EX decreased superoxide production in the paraventricular nucleus of Ang II–infused rats. These results indicate that EX improves arterial baroreflex function in conditions of high brain Ang II, which is mediated by the central Ang II type 1 receptor and associated with a reduction in central oxidative stress.
doi:10.1161/01.HYP.0000256955.74461.93
PMCID: PMC1904508  PMID: 17224469
exercise; baroreflex; sympathetic nerve activity; reactive oxygen species; AT1 receptor

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