Abstract-Chronic heart failure (CHF) is often associated with excitation of the sympathetic nervous system. This event is thought to be a negative predictor of survival in CHF. Sympathoexcitation and central angiotensin II (Ang II) have been causally linked. Recent studies have shown that NAD(P)H oxidase-derived reactive oxidant species (ROS) are important mediators of Ang II signaling. In the present study, we tested the hypothesis that central Ang II activates sympathetic outflow by stimulation of NAD(P)H oxidase and ROS in the CHF state. CHF was induced in male New Zealand White rabbits by chronic ventricular tachycardia. Using radio telemetry of arterial pressure and intracerebroventricular infusions, experiments were performed in the conscious state. Renal sympathetic nerve activity (RSNA) was recorded as a direct measure of sympathetic outflow. Intracerebroventricular Ang II significantly increased RSNA in sham (131.5Ϯ13.3% of control) and CHF (193.6Ϯ11.9% of control) rabbits. The increase in CHF rabbits was significantly greater than in sham rabbits (PϽ0.01). These responses were abolished by intracerebroventricular losartan, tempol, or apocynin. Resting RSNA was significantly reduced by intracerebroventricular losartan, tempol, or apocynin in CHF rabbits but not in sham rabbits. Intracerebroventricular administration of the superoxide dismutase inhibitor diethyldithio-carbamic acid increased RSNA significantly more in sham compared with CHF rabbits. NADPHdependent superoxide anion production in the rostral ventrolateral medulla (RVLM) was increased by 2.9-fold in CHF rabbits compared with sham rabbits. Finally, increases in the RVLM mRNA and protein expression of Ang II type 1 (AT 1 ) receptor and subunits of NAD(P)H oxidase (p40 phox , p47 phox , and gp91 phox ) were demonstrated in CHF rabbits. These data demonstrate intense radical stress in autonomic areas of the brain in experimental CHF and provide evidence for a tight relationship between Ang II and ROS as contributors to sympathoexcitation in CHF. Key Words: free radicals Ⅲ angiotensin receptors Ⅲ RVLM Ⅲ ventricular pacing I t is now well accepted that neural and humoral excitation are two of the primary and most reproducible sequelae of the chronic heart failure (CHF) syndrome. 1 Excessive sympathetic activation not only exacerbates the heart failure state but also is prognostic of death and complications. 2 A variety of humoral substances have been shown to be elevated in the CHF state, 1,3 of which angiotensin II (Ang II) has been considered a prime candidate for a substance that modulates sympathetic outflow because it has been known for some time that Ang II can alter sympathetic function at several sites from the central nervous system to the periphery. 4 Indeed, many of the current therapeutic targets in the treatment of CHF relate to reducing Ang II generation or blocking the effects of Ang II at its receptor sites. Despite its importance in central regulation of sympathetic outflow and cardiovascular homeostasis in the CHF state, the pre...

Chronic heart failure is often associated with sympathoexcitation and blunted arterial baroreflex function. These phenomena have been causally linked to elevated central ANG II mechanisms. Recent studies have shown that NAD(P)H oxidase-derived reactive oxygen species (ROS) are important mediators of ANG II signaling and therefore might play an essential role in these interactions. The aims of this study were to determine whether central subchronic infusion of ANG II in normal animals has effects on O2- production and expression of NAD(P)H oxidase subunits as well as ANG II type 1 (AT1) receptors in the rostral ventrolateral medulla (RVLM). Twenty-four male New Zealand White rabbits were divided into four groups and separately received a subchronic intracerebroventricular infusion of saline alone, ANG II alone, ANG II with losartan, and losartan alone for 1 wk. On day 7 of intracerebroventricular infusion, mean arterial pressure (MAP), heart rate (HR), and renal sympathetic nerve activity (RSNA) values were recorded, and arterial baroreflex sensitivity was evaluated while animals were in the conscious state. We found that ANG II significantly increased baseline RSNA (161.9%; P< 0.05), mRNA and protein expression of AT1 receptors (mRNA, 66.7%; P < 0.05; protein, 85.1%; P < 0.05), NAD(P)H oxidase subunits (mRNA, 120.0-200.0%; P < 0.05; protein, 90.9-197.0%; P < 0.05), and O2- production (83.2%; P < 0.05) in the RVLM. In addition, impaired baroreflex control of HR (Gain(max) reduced by 48.2%; P < 0.05) and RSNA (Gain(max) reduced by 53.6%; P < 0.05) by ANG II was completely abolished by losartan. Losartan significantly decreased baseline RSNA (-49.5%; P < 0.05) and increased baroreflex control of HR (Gain(max) increased by 64.8%; P < 0.05) and RSNA (Gain(max) increased by 67.9%; P < 0.05), but had no significant effects on mRNA and protein expression of AT1 receptor and NAD(P)H oxidase subunits and O2- production in the RVLM. These data suggest that in normal rabbits, NAD(P)H oxidase-derived ROS play an important role in the modulation of sympathetic activity and arterial baroreflex function by subchronic central treatment of exogenous ANG II via AT1 receptors.

These results indicate that elevation of Ang II and concomitant upregulation of AT1 receptor in the CB contribute to the increased CB chemoreceptor activity and enhanced peripheral chemoreflex function in CHF.

Background-In a recent study, we demonstrated that an increase in oxidative stress in the rostral ventrolateral medulla plays a critical role in the sympathoexcitation observed in chronic heart failure (CHF). Growing evidence indicates that exercise training evokes an antioxidative effect in CHF. In the present study, we therefore hypothesized that long-term exercise exerts its beneficial effect on autonomic activity in CHF via central antioxidative mechanisms. Methods and Results-Experiments were performed on New Zealand White rabbits. All rabbits were instrumented to measure mean arterial pressure, heart rate, and renal sympathetic nerve activity and to test baroreflex sensitivity.

Objective: A previous study from this laboratory showed that elevation of endogenous angiotensin II (Ang II) and upregulation of the angiotensin II type 1 (AT 1 ) 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 PO 2 , and Δ-5.6 ± 0.5 and Δ-5.3 ± 0.4 imp/s at 60.2 ± 3.1 mm Hg of PO 2 , p b 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 PO 2 , and Δ-6.7 ± 1.2 and Δ-6.6 ± 0.8 imp/s at 59.8 ± 3.5 mm Hg of PO 2 , p b 0.05). In addition, mRNA and protein expressions of NADPH oxidase components (gp91 phox , p40 phox and p47 phox ) 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.

Background-In a previous study, we showed that simvastatin (SIM) therapy normalized sympathetic outflow and cardiovascular reflex regulation in chronic heart failure (CHF). However, the precise neural and cellular pathways for these effects are unknown. We hypothesized that SIM exerts its beneficial effect on autonomic function in CHF by downregulating central angiotensin II (Ang II) and superoxide mechanisms. Methods and Results-Experiments were carried out on 36 male New Zealand White rabbits, 13 normal and 23 CHF. All rabbits were identically instrumented to record mean arterial pressure, heart rate, and renal sympathetic nerve activity (RSNA

Abstract-Upregulation of angiotensin II type 1 receptors (AT 1 R) in the rostral ventrolateral medulla (RVLM) contributes to the sympathoexcitation in the chronic heart failure (CHF). However, the role of angiotensin II type 2 receptor (AT 2 R) is not clear. In this study, we measured AT 1 R and AT 2 R protein expression in the RVLM and determined their effects on renal sympathetic nerve activity, blood pressure, and heart rate in anesthetized sham and CHF rats. We found that (1) although AT 1 R expression in the RVLM was upregulated, the AT 2 R was significantly downregulated t is well accepted that chronic heart failure (CHF) is characterized by heightened sympathetic tone. 1 This excessive sympathetic outflow to the heart and peripheral vessels attempts to increase myocardial performance and increases peripheral resistance, thereby contributing to an increase in myocardial oxygen consumption leading to a further deterioration in cardiac function. 2 It has been well established that activation of angiotensin II type 1 receptors (AT 1 R) in the rostral ventrolateral medulla (RVLM) evokes sympathetic excitation and pressor effects in normal animals. [3][4][5] Data from a previous study 6 from our laboratory further suggested that the upregulated AT 1 R expression in the RVLM and its enhanced intracellular signaling transduction plays a critical role in the sympathoexcitation in the CHF state. In addition, Ito et al 7 demonstrated that activation of AT 1 R in the RVLM appears to be important for the maintenance of hypertension in spontaneously hypertensive rats, another animal model of sympathoexcitation.In contrast with the AT 1 R, the functions of central angiotensin II type 2 receptors (AT 2 R) regarding the regulation of autonomic system are not well understood. Although the AT 2 R predominates in the tissues during fetal development, 8 this receptor has been identified to exist in many adult mammalian tissues, including the brain. 9 Further experiments have demonstrated that central regions related to sympathetic function such as the hypothalamus and brainstem exhibit positive AT 2 R mRNA hybridization signals, 10 implying the involvement of AT 2 R in the regulation of sympathetic outflow. Kang et al 11 found that, in the cultured neurons from newborn rat hypothalamus and brainstem, stimulation of AT 2 R significantly increased neuronal voltage-gated potassium channel current (I kv ) and that the third intracellular loop of the AT 2 R is a key component for this effect. 12 This group further determined that the phospholipase A2/arachidonic acid/12-lipoxygenases pathway mediates the modulation of potassium currents by activation of the AT 2 R. 13 These data strongly suggest that the AT 2 R exhibits an inhibitory effect on neuronal function by increasing potassium current and therefore decreasing excitability of neurons. Indeed, a recent study by Matsuura et al 14 demonstrated an AT 2 R-mediated hyper-

An exaggerated exercise pressor reflex (EPR) contributes to exercise intolerance and excessive sympatho-excitation in the chronic heart failure (CHF) state. However, the components of this reflex that are responsible for the exaggerated EPR in CHF remain unknown. To determine whether muscle afferent function is altered in CHF, we recorded the discharge of group III and IV afferents in response to static contraction, passive stretch and hindlimb intra-arterial injection of capsaicin in sham and CHF rats. We also investigated the roles of purinergic 2X receptor (P2X) and the transient receptor potential vanilloid 1 (VR1) in mediating the altered sensitivity of muscle afferents. Compared with sham rats, CHF rats exhibited greater responses of group III afferents to contraction and stretch whereas the responses of group IV afferents to contraction and capsaicin were blunted. Hindlimb intra-arterial infusion of pyridoxal phosphate-6-azophenyl-2 ,4 -disulfonic acid (PPADS), a P2X antagonist, attenuated the responses of group III afferents to contraction and stretch in CHF rats to a greater extent than in sham rats. Western blot data showed that P2X3 receptors were significantly upregulated in doral root ganglion (DRG) of CHF rats whereas VR1 receptors were significantly downregulated. Immunohistochemical evidence showed that immunostaining of the P2X3 receptors was more intense in both IB4-positive (C-fibre) and NF200-positive (A-fibre) neurons in DRG of CHF rats whereas the immunostaining of the VR1 receptors was decreased in IB4-positive neurons. These data suggest that group III afferents are sensitized whereas group IV afferents are desensitized in CHF, which is related to the dysfunction of P2X and VR1 receptors.