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-

Several sympathoexcitatory reflexes, such as the cardiac sympathetic afferent reflex (CSAR) and arterial chemoreflex, are significantly augmented and contribute to elevated sympathetic outflow in chronic heart failure (CHF). This study was undertaken to investigate the interaction between the CSAR and the chemoreflex in CHF and to further identify the involvement of angiotensin II type 1 receptors (AT 1Rs) in the nucleus of the tractus solitarius (NTS) in this interaction. CHF was induced in rats by coronary ligation. Acute experiments were performed in anesthetized rats. The chemoreflex-induced increase in cardiovascular responses was significantly greater in CHF than in sham-operated rats after either chemical or electrical activation of the CSAR. The inhibition of the CSAR by epicardial lidocaine reduced the chemoreflex-induced effects in CHF rats but not in sham-operated rats. Bilateral NTS injection of the AT 1R antagonist losartan (10 and 100 pmol) dosedependently decreased basal sympathetic nerve activity in CHF but not in sham-operated rats. This procedure also abolished the CSARinduced enhancement of the chemoreflex. The discharge and chemosensitivity of NTS chemosensitive neurons were significantly increased following the stimulation of the CSAR in sham-operated and CHF rats, whereas CSAR inhibition by epicardial lidocaine significantly attenuated chemosensitivity of NTS neurons in CHF but not in sham-operated rats. Finally, the protein expression of AT 1R in the NTS was significantly higher in CHF than in sham-operated rats. These results demonstrate that the enhanced cardiac sympathetic afferent input contributes to an excitatory effect of chemoreflex function in CHF, which is mediated by an NTS-AT 1R-dependent mechanism. sympathoexcitatory reflexes; sympathetic activity; angiotensin II type 1 receptor; microinjection; extracellular recording; nucleus of tractus solitarius THE ELEVATED SYMPATHETIC OUTFLOW in the chronic heart failure (CHF) has long been known to be closely associated with the accelerated progression and the poor prognosis of this syndrome (6, 9). The sympathetic hyperactivity is closely related to abnormalities in cardiovascular reflexes in CHF. Sympathoinhibitory cardiovascular reflexes such as the arterial baroreceptor reflex are significantly suppressed in CHF (21, 38). On the other hand, the sympathoexcitatory reflexes including the arterial chemoreceptor reflex and the cardiac sympathetic afferent reflex (CSAR) are exaggerated in CHF (34, 39). Although functional alterations of the above-mentioned reflexes have been independently used to illustrate the sympathoexcitation observed in CHF, the interaction among these reflexes in both the normal and CHF conditions, especially on their contributions to the sympathoexcitatory state in CHF, has been not extensively studied.In CHF, a variety of substances, such as bradykinin, that are released by the myocardium during ischemia can effectively stimulate the cardiac sympathetic afferents and subsequently increase sympathetic outflow (24...

In chronic heart failure (CHF), an overactive skeletal muscle exercise pressor reflex (EPR) results in excessive sympatho‐excitation and exercise intolerance. However, the mechanism(s) of the exaggerated EPR in CHF state remains to be determined. We recently found that muscle NADPH oxidase‐derived reactive oxygen species (ROS) plays an excitatory role in modulation of EPR function in normal rats. In this study, we used the technique of in vivo siRNA to selectively knockdown the protein expression of p22phox (a NADPH oxidase subunit) in hindlimb muscles of CHF rats in order to investigate the contribution of NADPH oxidase‐derived ROS to the exaggerated EPR in CHF. Data from western blotting and immunofluorescence experiments showed that compared with sham rats, the protein expression of p22phox in triceps surae muscle was significantly increased in CHF rats (ratio of p22phox to GAPDH: 0.32±0.03 vs. 0.15±0.02, CHF vs. Sham, n=5, P<0.05). Silencing p22phox in skeletal muscle with in vivo siRNA (1 week) dramatically decreased the protein expression by almost 70% in CHF rats. Animal experiments showed that gene silencing of p22phox in hindlimb muscles significantly attenuated the EPR‐evoked pressor response to a 30‐s static contraction induced by electrical stimulation of L4/L5 ventral roots in CHF rats (+33.1±2.8 vs. +20.8±1.5 mmHg, CHF vs. CHF+siRNA, n=5, P<0.05) whereas treatment with control siRNA has no effect on the EPR function. These data suggest that elevated muscle NADPH oxidase‐derived ROS contributes to the exaggerated EPR in the CHF state.

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.

Muscle metabolic by-products during exercise, such as K+, lactic acid, ATP, H+, and phosphate, are well established to be involved in the reflex cardiovascular response to static muscle contraction. However, the role of muscle reactive oxygen species (ROS), a metabolic by-product during muscle contraction, in the exercise pressor reflex (EPR) has not been investigated in detail. In the present study, we evaluated the role of muscle ROS in the EPR in a decerebrate rat model. We hypothesized that muscle NADPH oxidase-derived ROS contributes to sensitization of the EPR. Thus the rise in blood pressure and heart rate in response to a 30-s static contraction induced by electrical stimulation of L4/L5 ventral roots was compared before and after hindlimb arterial infusion of the redox agents: diethyldithiocarbamate, a superoxide dismutase inhibitor; the superoxide dismutase mimetic 4-hydroxy-2,2,6,6-tetramethyl piperidine 1-oxyl (tempol); the free radical scavenger dimethylthiourea; a NADPH oxidase inhibitor, apocynin; and a xanthine oxidase inhibitor, allopurinol. The EPR-induced pressor response was augmented after treatment with diethyldithiocarbamate and was attenuated after treatment with tempol, dimethylthiourea, and apocynin. Treatment with allopurinol did not affect the EPR function. None of the drug's affected the EPR heart rate response. In addition, neither the pressor response to electrical stimulation of the central end of dorsal roots, nor femoral blood flow was affected by any treatment. These data suggest that NADPH oxidase-derived muscle ROS plays an excitatory role in the EPR control of blood pressure.

Chronic heart failure (CHF) is characterized by decreased cardiac parasympathetic and increased cardiac sympathetic nerve activity. This autonomic imbalance increases the risk of arrhythmias and sudden death in patients with CHF. We hypothesized that the molecular and cellular alterations of cardiac postganglionic parasympathetic (CPP) neurons located in the intracardiac ganglia and sympathetic (CPS) neurons located in the stellate ganglia (SG) possibly link to the cardiac autonomic imbalance in CHF. Rat CHF was induced by left coronary artery ligation. Single-cell real-time PCR and immunofluorescent data showed that L (Ca(v)1.2 and Ca(v)1.3), P/Q (Ca(v)2.1), N (Ca(v)2.2), and R (Ca(v)2.3) types of Ca2+ channels were expressed in CPP and CPS neurons, but CHF decreased the mRNA and protein expression of only the N-type Ca2+ channels in CPP neurons, and it did not affect mRNA and protein expression of all Ca2+ channel subtypes in the CPS neurons. Patch-clamp recording confirmed that CHF reduced N-type Ca2+ currents and cell excitability in the CPP neurons and enhanced N-type Ca2+ currents and cell excitability in the CPS neurons. N-type Ca2+ channel blocker (1 μM ω-conotoxin GVIA) lowered Ca2+ currents and cell excitability in the CPP and CPS neurons from sham-operated and CHF rats. These results suggest that CHF reduces the N-type Ca2+ channel currents and cell excitability in the CPP neurons and enhances the N-type Ca2+ currents and cell excitability in the CPS neurons, which may contribute to the cardiac autonomic imbalance in CHF.

The cardiac "sympathetic afferent" reflex (CSAR) has been reported to increase sympathetic outflow and depress baroreflex function via a central angiotensin II (ANG II) mechanism. In the present study, we examined the role of ANG II type 1 (AT(1)) receptors in the nucleus tractus solitarii (NTS) in mediating the interaction between the CSAR and the baroreflex in anesthetized rats. We examined the effects of bilateral microinjection of AT(1) receptor antagonist losartan (100 pmol) into the NTS on baroreflex control of renal sympathetic nerve activity (RSNA) before and after CSAR activation by epicardial application of capsaicin (0.4 microg). Using single-unit extracellular recording, we further examined the effects of CSAR activation on the barosensitivity of barosensitive NTS neurons and the effects of intravenous losartan (2 mg/kg) on CSAR-induced changes in activity of NTS barosensitive neurons. Bilateral NTS microinjection of losartan significantly attenuated the increases in arterial pressure, heart rate, and RSNA evoked by capsaicin but also markedly (P < 0.01) reversed the CSAR-induced blunted baroreflex control of RSNA (Gain(max) from 1.65 +/- 0.10 to 2.22 +/- 0.11%/mmHg). In 17 of 24 (70.8%) NTS barosensitive neurons, CSAR activation significantly (P < 0.01) inhibited the baseline neuronal activity and attenuated the neuronal barosensitivity. In 11 NTS barosensitive neurons, intravenous losartan effectively (P < 0.01) normalized the decreased neuronal barosensitivity induced by CSAR activation. In conclusion, blockade of NTS AT(1) receptors improved the blunted baroreflex during CSAR activation, suggesting that the NTS plays an important role in processing the interaction between the baroreflex and the CSAR via an AT(1) receptor-dependent mechanism.

Abstract-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. Key Words: exercise Ⅲ baroreflex Ⅲ sympathetic nerve activity Ⅲ reactive oxygen species Ⅲ AT 1 receptor I mpairment of arterial baroreflex function is an important feature in cardiovascular diseases, such as chronic heart failure (CHF) and hypertension. 1,2 This abnormality increases cardiovascular risk. 3 Recent studies indicate that the blunted arterial baroreflex is related to an enhanced central angiotensin II (Ang II) mechanism, because blockade of Ang II type 1 receptors (AT 1 Rs) in the brain restored baroreflex sensitivity. 4 Intracerebroventricular (icv) infusion of Ang II depresses arterial baroreflex function in normal animals. 5 However, specific brain nuclei and the intracellular mechanism for Ang II-induced baroreflex dysfunction have not been fully identified. The paraventricular nucleus (PVN) of the hypothalamus plays an important role in the control of baroreflex function and sympathetic drive. 6 Furthermore, the PVN contains a high density of AT 1 R 7 and is a candidate region to respond to Ang II in the cerebrospinal fluid. 8 Several lines of evidence show that reduced nicotinamideadenine dinucleotide phosphate (NAD[P]H) oxidase-derived reactive oxygen species (ROS) are novel mediators of Ang II signaling in the central nervous system. 9 Pretreatment with adenoviral-mediated superoxide dismutase prevents Ang IIinduced hypertension and the increased superoxide production. 10 On the other hand, the AT 1 R antagonist losartan abolishes Ang II-stimulated superoxide generation. 9 Increases in superoxide production may contribute to impaired arterial baroreflex function. 5,11 Conversely, antioxidant treatment improves baroreflex sensitivity. 11 Exercise training (EX) has been demonstrated to alter neural control of the circulation, including influencing arterial baroreflex function. 12 Although the effects of EX on baroreflex function in normal subjects and animals are increased, 12 decreased, 13,14 or unchanged, 15 EX consistently increases baroreflex sensitivity in CHF and hypertensive subjects. 16 -18 The mechanisms responsible for the training-induced improvement of baroreflex function involve changes in central and peripheral components of the baroreflex arc....