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1.  Disruption of cardiovascular circadian rhythms in mice post myocardial infarction: relationship with central angiotensin II receptor expression 
Physiological Reports  2014;2(11):e12210.
Abstract
Angiotensin II (Ang II) is well known to participate in the abnormal autonomic cardiovascular control that occurs during the development of chronic heart failure (CHF). Disrupted cardiovascular circadian rhythm in CHF is also well accepted; however, the mechanisms underlying and the role of central Ang II type 1 receptors (AT1R) and oxidative stress in mediating such changes are not clear. In a post myocardial infarction (MI) CHF mouse model we investigated the circadian rhythm for mean arterial pressure (MAP), heart rate (HR), and baroreflex sensitivity (BRS) following MI. The cardiovascular parameters represent the middle 6‐h averages during daytime (6:00–18:00) and nighttime (18:00–6:00). HR increased with the severity of CHF reaching its maximum by 12 weeks post‐MI; loss of circadian HR and BRS rhythms were observed as early as 4 weeks post‐MI in conjunction with a significant blunting of the BRS and an upregulation in the AT1R and gp91phox proteins in the brainstem. Loss of MAP circadian rhythm was observed 8 weeks post‐MI. Circadian AT1R expression was demonstrated in sham animals but was lost 8 weeks following MI. Losartan reduced AT1R expression in daytime (1.18 ± 0.1 vs. 0.85 ± 0.1; P < 0.05) with a trend toward a reduction in the AT1R mRNA expression in the nighttime (1.2 ± 0.1 vs. 1.0 ± 0.1; P > 0.05) but failed to restore circadian variability. The disruption of circadian rhythm for HR, MAP and BRS along with the upregulation of AT1 and gp91phox suggests a possible role for central oxidative stress as a mediator of circadian cardiovascular parameters in the post‐MI state.
Increases in central angiotenisn II signaling provide a driving force for sympatho‐excitation in heart failure. In this study, we show a loss of circadian variability in angiotensin type 1 receptor expression in the brainstem of mice post myocardial infarction. These changes correlate with a loss of cardiovascular circadian variability. These data suggest that sympatho‐ excitation may be increased in the post‐MI state at times when sympathetic outflow is normally reduced.
doi:10.14814/phy2.12210
PMCID: PMC4255816  PMID: 25413327
Cardiac dysfunction; cardiovascular reflex; diurnal variability; oxidative stress
2.  The Central RAS and Sympathetic Nerve Activity in Chronic Heart Failure 
Chronic heart failure (CHF) is a multi-factorial disease process that is characterized by over activation of the renin-angiotensin-aldosterone system (RAAS) and the sympathetic nervous system. Both of these systems are chronically activated in CHF. The RAAS consists of an excitatory arm involving Angiotensin II (Ang II), Angiotensin Converting Enzyme (ACE), and the Ang II type 1 Receptor (AT1R). The RAAS also consists of a protective arm consisting of Angiotensin-1-7 (Ang -1-7), the Ang II type 2 receptor (AT2R), ACE2 and the mas receptor. Sympathoexcitation in CHF is driven, in large part, by an imbalance of these two arms, with an increase in the Ang II-AT1R-ACE arm and a decrease in the AT2R-ACE2 arm. This imbalance is manifested in cardiovascular-control regions of the brain such as the rostral ventrolateral medulla and paraventricular nucleus in the hypothalamus. This review focuses on current literature that describes the components of these two arms of the RAAS, and their imbalance in the CHF state. Moreover, this review provides additional evidence for the relevance of ACE2 and Ang-1-7 as key players in the regulation of central sympathetic outflow in CHF. Finally we also examine the effects of exercise training as a therapeutic strategy and the molecular mechanisms at play in CHF, in part, because of the ability of exercise training to restore the balance of the RAAS axis and sympathetic outflow.
doi:10.1042/CS20130294
PMCID: PMC4053944  PMID: 24490814
3.  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
4.  Parallel Changes in Neuronal AT1R and GRK5 Expression Following Exercise Training in Heart Failure 
Hypertension  2012;60(2):354-361.
Although exercise training (ExT) is an important therapeutic strategy for improving quality of life in patients with chronic heart failure (CHF), the central mechanisms by which ExT is beneficial are not well understood. The angiotensin II type 1 receptor (AT1R) plays a pivotal role in the development of CHF, and is upregulated in a number of tissues due in part to transcription factor nuclear factor kappa B (NF-κB). In addition, AT1R is marked for internalization and recycling via G protein coupled receptor kinase (GRK) phosphorylation. Because previous studies have shown that the beneficial effects of ExT in CHF rely on a reduction in Ang II, we hypothesized ExT would decrease AT1R, GRK5 and NF-κB protein expression in the paraventricular nucleus (PVN) and rostral ventrolateral medulla (RVLM) of CHF rats. Following infarction by coronary artery ligation, animals were exercised four weeks post-surgery on a treadmill at a final speed of 25 m/min for 60 minutes, 5 days/week for 6 weeks. Western blot analysis of PVN and RVLM micropunches revealed an upregulation of AT1R, GRK5 and NF-κB in the infarcted group that was reversed by ExT. Furthermore, the relative expression of phosphorylated AT1R and AT1R/GRK5 physical association was increased in the CHF sedentary group, and reversed by ExT. Overexpression of GRK5 in cultured CATH.a neurons blunted angiotensin II-mediated upregulation of AT1R and NF-κB; conversely, silencing of GRK5 exacerbated angiotensin II-mediated AT1R and NF-κB upregulation. Taken together, increased GRK5 may regulate AT1R expression in CHF, and ExT mitigates AT1R and its pathway components.
doi:10.1161/HYPERTENSIONAHA.112.195693
PMCID: PMC3409876  PMID: 22753221
angiotensin II; GRK5; NF-κB; receptor turnover
5.  Neurohumoral Stimulation 
Heart failure clinics  2012;8(1):87-99.
doi:10.1016/j.hfc.2011.08.007
PMCID: PMC3224981  PMID: 22108729
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.  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
8.  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.  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
11.  NF-κB and CREB Are Required for Angiotensin II Type 1 Receptor Upregulation in Neurons 
PLoS ONE  2013;8(11):e78695.
Nuclear factor kappa B (NF-κB) and the Ets like gene-1 (Elk-1) are two transcription factors that have been previously established to contribute to the Angiotensin II mediated upregulation of Angiotensin II type 1 receptor (AT1R) in neurons. The cAMP response element binding protein (CREB) is another transcription factor that has also been implicated in AT1R gene transcription. The goal of the current study was to determine if NF-κB and CREB association was required for AT1R upregulation. We hypothesized that the transcription of the AT1R gene occurs via an orchestration of transcription factor interactions including NF-κB, CREB, and Elk-1. The synergistic role of CREB and NFκB in promoting AT1R gene expression was determined using siRNA-mediated silencing of CREB. Electrophorectic Mobility Shift Assay studies employing CREB and NF-κB demonstrated increased protein – DNA binding as a result of Ang II stimulation which was blunted by siRNA silencing of CREB. Upstream inhibition of p38 mitogen activated protein kinase (p38 MAPK) with SB203580 or inhibition of the calmodulin kinase (CAMK) pathway using KN-62 blunted changes in CREB and NF-κB expression. These findings suggest that Ang II may activate multiple signaling pathways involving p38 MAPK leading to the activation of NF-κB and CREB, which feed back to upregulate the AT1R gene. This study provides insight into the molecular mechanisms involving multiple transcription factor activation in a coordinated fashion which may be partially responsible for sympathoexcitation in clinical conditions associated with increased activation of the renin angiotensin system.
doi:10.1371/journal.pone.0078695
PMCID: PMC3823855  PMID: 24244341
13.  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
14.  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
15.  Muscle reflex in heart failure: the role of exercise training 
Exercise evokes sympathetic activation and increases blood pressure and heart rate (HR). Two neural mechanisms that cause the exercise-induced increase in sympathetic discharge are central command and the exercise pressor reflex (EPR). The former suggests that a volitional signal emanating from central motor areas leads to increased sympathetic activation during exercise. The latter is a reflex originating in skeletal muscle which contributes significantly to the regulation of the cardiovascular and respiratory systems during exercise. The afferent arm of this reflex is composed of metabolically sensitive (predominantly group IV, C-fibers) and mechanically sensitive (predominately group III, A-delta fibers) afferent fibers. Activation of these receptors and their associated afferent fibers reflexively adjusts sympathetic and parasympathetic nerve activity during exercise. In heart failure, the sympathetic activation during exercise is exaggerated, which potentially increases cardiovascular risk and contributes to exercise intolerance during physical activity in chronic heart failure (CHF) patients. A therapeutic strategy for preventing or slowing the progression of the exaggerated EPR may be of benefit in CHF patients. Long-term exercise training (ExT), as a non-pharmacological treatment for CHF increases exercise capacity, reduces sympatho-excitation and improves cardiovascular function in CHF animals and patients. In this review, we will discuss the effects of ExT and the mechanisms that contribute to the exaggerated EPR in the CHF state.
doi:10.3389/fphys.2012.00398
PMCID: PMC3464681  PMID: 23060821
physical training; myocardial infarction; muscle afferents; exercise; sympathetic nerve activity
16.  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
17.  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
18.  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
19.  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
20.  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
21.  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
22.  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
23.  The Mechanism of Adaptation of Left Atrial Stretch Receptors in Dogs with Chronic Congestive Heart Failure 
Journal of Clinical Investigation  1977;60(2):323-331.
Chronic congestive heart failure (CHF) was induced in dogs by the construction of an aorto-caval fistula below the level of the renal arteries. Aorto-caval fistula dogs showed signs of CHF which included ascites, hind limb edema, and pulmonary congestion. Ventricular catheterization indicated a significantly higher left ventricular end diastolic pressure and lower maximum velocity of left ventricular pressure development/left ventricular end diastolic pressure in CHF dogs when compared to sham-operated controls. Heart weight/body weight ratios were significantly higher in CHF dogs. Electrophysiological recordings from medullated left atrial type B receptors from the cervical vagus indicated a depressed sensitivity of these receptors in CHF dogs when compared to sham-operated control dogs. For any given change in left atrial pressure, the discharge of left atrial receptors was significantly reduced in CHF dogs compared with sham-operated controls. The mechanism for this depressed sensitivity was investigated. Sonomicrometry of the left atrial appendage indicated a decreased compliance of the left atrial appendage in the dogs with chronic CHF. In addition, microscope examination of the complex unencapsulated receptor endings taken from the left atrial endocardium indicated a marked alteration in receptor morphology. A loss of the end arborization was the most typical finding. It is concluded that chronic CHF brought about by an aorto-caval fistula results in a depressed left atrial stretch receptor response and that both decreased left atrial compliance and structural alterations in the receptor endings may account for this depressed sensitivity.
Images
PMCID: PMC372372  PMID: 874094

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