Enhanced angiotensin II-mediated central sympathoexcitation in streptozotocin-induced diabetes: role of superoxide anion

Studies have shown that the superoxide mechanism is involved in angiotensin II (ANG II) signaling in the central nervous system. We hypothesized that ANG II activates sympathetic outflow by stimulation of superoxide anion in the paraventricular nucleus (PVN) of streptozotocin (STZ)-induced diabetic rats. In α-chloralose- and urethane-anesthetized rats, microinjection of ANG II into the PVN (50, 100, and 200 pmol) produced dose-dependent increases in renal sympathetic nerve activity (RSNA), arterial pressure (AP), and heart rate (HR) in control and STZ-induced diabetic rats. There was a potentiation of the increase in RSNA (35.0 ± 5.0 vs. 23.0 ± 4.3%, P < 0.05), AP, and HR due to ANG II type I (AT(1)) receptor activation in diabetic rats compared with control rats. Blocking endogenous AT(1) receptors within the PVN with AT(1) receptor antagonist losartan produced significantly greater decreases in RSNA, AP, and HR in diabetic rats compared with control rats. Concomitantly, there were significant increases in mRNA and protein expression of AT(1) receptor with increased superoxide levels and expression of NAD(P)H oxidase subunits p22(phox), p47(phox), and p67(phox) in the PVN of rats with diabetes. Pretreatment with losartan (10 mg·kg(-1)·day(-1) in drinking water for 3 wk) significantly reduced protein expression of NAD(P)H oxidase subunits (p22(phox) and p47(phox)) in the PVN of diabetic rats. Pretreatment with adenoviral vector-mediated overexpression of human cytoplasmic superoxide dismutase (AdCuZnSOD) within the PVN attenuated the increased central responses to ANG II in diabetes (RSNA: 20.4 ± 0.7 vs. 27.7 ± 2.1%, n = 6, P < 0.05). These data support the concept that superoxide anion contributes to an enhanced ANG II-mediated signaling in the PVN involved with the exaggerated sympathoexcitation in diabetes.     

Microinjection of ANG II into paraventricular nucleus enhances cardiac sympathetic afferent reflex in rats

The aims of present study were to determine whether angiotensin II (ANG II) in the paraventricular nucleus (PVN) is involved in the central integration of the cardiac sympathetic afferent reflex and whether this effect is mediated by the ANG type 1 (AT(1)) receptor. While the animals were under alpha-chloralose and urethane anesthesia, mean arterial pressure, heart rate, and renal sympathetic nerve activity (RSNA) were recorded in sinoaortic-denervated and cervical-vagotomized rats. A cannula was inserted into the left PVN for microinjection of ANG II. The cardiac sympathetic afferent reflex was tested by electrical stimulation (5, 10, 20, and 30 Hz in 10 V and 1 ms) of the afferent cardiac sympathetic nerves or epicardial application of bradykinin (BK) (0.04 and 0.4 microg in 2 microl). Microinjection of ANG II (0.03, 0.3, and 3 nmol) into the PVN resulted in dose-related increases in the RSNA responses to electrical stimulation. The percent change of RSNA response to 20- and 30-Hz stimulation increased significantly at the highest dose of ANG II (3 nmol). The effects of ANG II were prevented by pretreatment with losartan (50 nmol) into the PVN. Microinjection of ANG II (0.3 nmol) into the PVN significantly enhanced the RSNA responses to epicardial application of BK, which was abolished by pretreatment with losartan (50 nmol) into the PVN. These results suggest that exogenous ANG II in the PVN augments the cardiac sympathetic afferent reflex evoked by both electrical stimulation of cardiac sympathetic afferent nerves and epicardial application of BK. These central effects of ANG II are mediated by AT(1) receptors.     

ANG II in the paraventricular nucleus potentiates the cardiac sympathetic afferent reflex in rats with heart failure.

Chronic heart failure (CHF) is characterized by sympathoexcitation, and the cardiac sympathetic afferent reflex (CSAR) is a sympathoexcitatory reflex. Our previous studies have shown that the CSAR was enhanced in CHF. In addition, central angiotensin II (ANG II) is an important modulator of this reflex. This study was performed to determine whether the CSAR evoked by stimulation of cardiac sympathetic afferent nerves (CSAN) in rats with coronary ligation-induced CHF is enhanced by ANG II in the paraventricular nucleus (PVN). Under alpha-chloralose and urethane anesthesia, renal sympathetic nerve activity (RSNA) was recorded. The RSNA responses to electrical stimulation (5, 10, 20, and 30 Hz) of the CSAN were evaluated. Bilateral microinjection of the AT1-receptor antagonist losartan (50 nmol) into the PVN had no significant effects in the sham group, but it abolished the enhanced RSNA response to stimulation in the CHF group. Unilateral microinjection of three doses of ANG II (0.03, 0.3, and 3 nmol) into the PVN resulted in dose-related increases in the RSNA responses to stimulation. Although ANG II also potentiated the RSNA response to electrical stimulation in sham rats, the RSNA responses to stimulation after ANG II into the PVN in rats with CHF were much greater than in sham rats. The effects of ANG II were prevented by pretreatment with losartan into the PVN in CHF rats. These results suggest that the central gain of the CSAR is enhanced in rats with coronary ligation-induced CHF and that ANG II in the PVN augments the CSAR evoked by CSAN, which is mediated by the central angiotensin AT1 receptors in rats with CHF.     

Exercise training normalizes enhanced sympathetic activation from the paraventricular nucleus in chronic heart failure: role of angiotensin II

Exercise training (ExT) normalizes the increased sympathetic outflow in heart failure (HF), but the underlying mechanisms are not known. We hypothesized ExT would normalize the augmented activation of the paraventricular nucleus (PVN) via an angiotensinergic mechanism during HF. Four groups of rats used were the following: 1) sham-sedentary (Sed); 2) sham-ExT; 3) HF-Sed, and 4) HF-ExT. HF was induced by left coronary artery ligation. Four weeks after surgery, 3 wk of treadmill running was performed in ExT groups. The number of FosB-positive cells in the PVN was significantly increased in HF-Sed group compared with the sham-Sed group. ExT normalized (negated) this increase in the rats with HF. In anesthetized condition, the increases in renal sympathetic nerve activity (RSNA), mean arterial pressure (MAP), and heart rate (HR) in response to microinjection of angiotensin (ANG) II (50～200 pmol) in the PVN of HF-Sed group were significantly greater than of the sham-Sed group. In the HF-ExT group the responses to microinjection of ANG II were not different from sham-Sed or sham-ExT groups. Blockade of ANG II type 1 (AT(1)) receptors with losartan in the PVN produced a significantly greater decrease in RSNA, MAP, and HR in HF-Sed group compared with sham-Sed group. ExT prevented the difference between HF and sham groups. AT(1) receptor protein expression was increased 50% in HF-Sed group compared with sham-Sed group. In the HF-ExT group, AT(1) receptor protein expression was not significantly different from sham-Sed or sham-ExT groups. In conclusion, one mechanism by which ExT alleviates elevated sympathetic outflow in HF may be through normalization of angiotensinergic mechanisms within the PVN.     

AT1 receptor block does not affect arterial baroreflex during pregnancy in rabbits

The role of ANG II in the arterial baroreflex control of renal sympathetic nerve activity (RSNA) in eight term-pregnant (P) and eight nonpregnant (NP) conscious rabbits was assessed using sequential intracerebroventricular and intravenous infusions of losartan, an AT1 receptor antagonist. The blood pressure (BP)-RSNA relationship was generated by sequential inflations of aortic and vena caval perivascular occluders. Pregnant rabbits exhibited a lower maximal RSNA reflex gain (-44%) that was primarily due to a reduction in the maximal sympathetic response to hypotension (P, 248 +/- 20% vs. NP, 357 +/- 41% of rest RSNA, P < 0.05). Intracerebroventricular losartan decreased resting BP in P (by 9 +/- 3 mmHg, P < 0.05) but not NP rabbits, and had no effect on the RSNA baroreflex in either group. Subsequent intravenous losartan decreased resting BP in NP and further decreased BP in P rabbits, but had no significant effect on the maximal RSNA reflex gain. ANG II may have an enhanced role in the tonic support of BP in pregnancy, but does not mediate the gestational depression in the arterial baroreflex control of RSNA in rabbits.     

Superoxide anions in the paraventricular nucleus mediate the enhanced cardiac sympathetic afferent reflex and sympathetic activity in renovascular hypertensive rats

An enhanced cardiac sympathetic afferent reflex (CSAR) is involved in the sympathetic activation in renovascular hypertension. The present study was designed to determine the role of superoxide anions in the paraventricular nucleus (PVN) in mediating the enhanced CSAR and sympathetic activity in renovascular hypertension in the two-kidney, one-clip (2K1C) model. Sinoaortic denervation and vagotomy were carried out, and renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) were recorded under anesthesia. The CSAR was evaluated by the response of RSNA to the epicardial application of capsaicin. Superoxide anion levels and NAD(P)H oxidase activity in the PVN increased in 2K1C rats and were much higher in 2K1C rats than in sham-operated (sham) rats after the epicardial application of capsaicin or PVN microinjection of ANG II. In both 2K1C and sham rats, PVN microinjection of the superoxide anion scavenger tempol or the NAD(P)H oxidase inhibitor apocynin abolished the CSAR, whereas the SOD inhibitor diethyldithiocarbamic acid (DETC) potentiated the CSAR. Tempol and apocynin decreased but DETC increased baseline RSNA and MAP. ANG II in the PVN caused larger responses of the CSAR, baseline RSNA, and baseline MAP in 2K1C rats than in sham rats. The effects of ANG II were abolished by pretreatment with tempol or apocynin in both 2K1C and sham rats and augmented by DETC in the PVN in 2K1C rats. These results indicate that superoxide anions in the PVN mediate the CSAR and the effects of ANG II in the PVN. Increased superoxide anions in the PVN contribute to the enhanced CSAR and sympathetic activity in renovascular hypertension.     

Sympathoexcitation by central ANG II: roles for AT1 receptor upregulation and NAD(P)H oxidase in RVLM

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.     

Regulation of angiotensin II-stimulated osteopontin expression in cardiac microvascular endothelial cells: role of p42/44 mitogen-activated protein kinase and reactive oxygen species

Using spontaneously hypertensive and aortic banded rats, we have shown that expression of myocardial osteopontin, an extracellular matrix protein, coincides with the development of heart failure and is inhibited by captopril, suggesting a role for angiotensin II (ANG II). This study tested whether ANG II induces osteopontin expression in adult rat ventricular myocytes and cardiac microvascular endothelial cells (CMEC), and if so, whether induction is mediated via activation of mitogen-activated protein kinases (p42/44 MAPK) and involves reactive oxygen species (ROS). ANG II (1 microM, 16 h) increased osteopontin expression (fold increase 3.3+/-0.34, n = 12, P < 0.01) in CMEC as measured by northern analysis, but not in ARVM. ANG II stimulated osteopontin expression in CMEC in a time- (within 4 h) and concentration-dependent manner, which was prevented by the AT1 receptor antagonist, losartan. ANG II elicited robust phosphorylation of p42/44 MAPK as measured using phospho-specific antibodies, and increased superoxide production as measured by cytochrome c reduction and lucigenin chemiluminescence assays. These effects were blocked by diphenylene iodonium (DPI), an inhibitor of the flavoprotein component of NAD(P)H oxidase. PD98059, an inhibitor of p42/44 MAPK pathway, and DPI each inhibited ANG II-stimulated osteopontin expression. Northern blot analysis showed basal expression of p22phox, a critical component of NADH/NADPH oxidase system, which was increased 40-60% by exposure to ANG II. These results suggest that p42/44 MAPK is a critical component of the ROS-sensitive signaling pathways activated by ANG II in CMEC and plays a key role in the regulation of osteopontin gene expression. Published 2001 Wiley-Liss, Inc.     

Angiotensin-mediated increase in renal sympathetic nerve discharge within the PVN: role of nitric oxide

The paraventricular nucleus (PVN) of the hypothalamus is known to be an important site of integration in the central nervous system for sympathetic outflow. ANG II and nitric oxide (NO) play an important role in regulation of sympathetic nerve activity. The purpose of the present study was to examine how the interaction between NO and ANG II within the PVN affects sympathetic outflow in rats. Renal sympathetic nerve discharge (RSND), arterial blood pressure (AP), and heart rate (HR) were measured in response to administration of ANG II and N(G)-monomethyl-l-arginine (L-NMMA) into the PVN. Microinjection of ANG II (0.05, 0.5, and 1.0 nmol) into the PVN increased RSND, AP, and HR in a dose-dependent manner, resulting in increases of 53 +/- 9%, 19 +/- 3 mmHg, and 32 +/- 12 beats/min from baseline, respectively, at the highest dose. These responses were significantly enhanced by prior microinjection of L-NMMA and were blocked by losartan, an ANG II type 1 receptor antagonist. Similarly, administration of antisense to neuronal NO synthase within the PVN also potentiated the ANG II responses. Conversely, overexpression of neuronal NOS within the PVN with adenoviral gene transfer significantly attenuated ANG II responses. Push-pull administration of ANG II (1 nmol) into the PVN induced an increase in NO release. Our data indicate that ANG II type 1 receptors within the PVN mediate an excitatory effect on RSND, AP, and HR. NO in the PVN, which can be induced by ANG II stimulation, in turn inhibits the ANG II-mediated increase in sympathetic nerve activity. This negative-feedback mechanism within the PVN may play an important role in maintaining the overall balance and tone of sympathetic outflow.     

Inhibition of sympathetic responses at birth in sheep by lesion of the paraventricular nucleus

Birth is characterized by a surge in sympathetic outflow, heart rate (HR), mean arterial blood pressure (MABP) and circulating catecholamines. The paraventricular nucleus (PVN) of the hypothalamus is an important central regulatory site of sympathetic activity, but its role in the regulation of sympathoexcitation at birth is unknown. To test the hypothesis that the PVN regulates sympathetic activity at birth, experiments were performed in chronically instrumented near-term (137- to 142-day gestation, term 145 days) sheep before and after delivery by cesarean section. Stereotaxic guided electrolytic lesioning of the PVN (n = 6) or sham lesioning (n = 6) was performed 48 h before study. At 30 min after birth, renal sympathetic nerve activity (RSNA) increased 128 +/- 26% above fetal values in the sham-lesioned animals (P < 0.05). In contrast, at a similar time point, RSNA decreased to 52 +/- 12% of the fetal value in the PVN-lesioned animals. Lesioning of the PVN did not affect the usual postnatal increases in MABP and epinephrine levels although HR failed to rise above fetal values. ANG II but not arginine vasopressin or norepinephrine levels increased in PVN-lesioned animals after birth, whereas all three hormones increased (P < 0.05) in sham-lesioned animals. Fetal and newborn HR baroreflex responses were similar in both groups. However, the usual postnatal attenuation of baroreflex-mediated inhibition of RSNA was blunted in the PVN-lesioned group. The results of this study demonstrate that ablation of the PVN abolishes sympathoexcitation with birth at near-term gestation. The PVN may play a critical role in physiological adaptation at birth.     

Median preoptic nucleus and subfornical organ drive renal sympathetic nerve activity via a glutamatergic mechanism within the paraventricular nucleus

The paraventricular nucleus (PVN) of the hypothalamus is involved in the neural control of sympathetic drive, but the precise mechanism(s) that influences the PVN is not known. The activation of the PVN may be influenced by input from higher forebrain areas, such as the median preoptic nucleus (MnPO) and the subfornical organ (SFO). We hypothesized that activation of the MnPO or SFO would drive the PVN through a glutamatergic pathway. Neuroanatomical connections were confirmed by the recovery of a retrograde tracer in the MnPO and SFO that was injected bilaterally into the PVN in rats. Microinjection of 200 pmol of N-methyl-d-aspartate (NMDA) or bicuculline-induced activation of the MnPO and increased renal sympathetic activity (RSNA), mean arterial pressure, and heart rate in anesthetized rats. These responses were attenuated by prior microinjection of a glutamate receptor blocker AP5 (4 nmol) into the PVN (NMDA - ΔRSNA 72 ± 8% vs. 5 ± 1%; P < 0.05). Using single-unit extracellular recording, we examined the effect of NMDA microinjection (200 pmol) into the MnPO on the firing activity of PVN neurons. Of the 11 active neurons in the PVN, 6 neurons were excited by 95 ± 17% (P < 0.05), 1 was inhibited by 57%, and 4 did not respond. The increased RSNA after activation of the SFO by ANG II (1 nmol) or bicuculline (200 pmol) was also reduced by AP5 in the PVN (for ANG II - ΔRSNA 46 ± 7% vs. 17 ± 4%; P < 0.05). Prior microinjection of ANG II type 1 receptor blocker losartan (4 nmol) into the PVN did not change the response to ANG II or bicuculline microinjection into the SFO. The results from this study demonstrate that the sympathoexcitation mediated by a glutamatergic mechanism in the PVN is partially driven by the activation of the MnPO or SFO.     

ANG II-stimulated DNA synthesis is mediated by ANG II receptor-dependent Ca(2+)/PKC as well as EGF receptor-dependent PI3K/Akt/mTOR/p70S6K1 signal pathways in mouse embryonic stem cells

Effect of angiotensin II (ANG II) on mouse embryonic stem (ES) cell proliferation was examined. ANG II increased [(3)H] thymidine incorporation in a time- (>4 h) and dose- (>10(-9) M) dependent manner. The ANG II-induced increase in [(3)H] thymidine incorporation was blocked by inhibition of ANG II type 1 (AT(1)) receptor but not by ANG II type 2 (AT(2)) receptor, and AT(1) receptor was expressed. ANG II increased inositol phosphates formation and [Ca(2+)](i), and translocated PKC alpha, delta, and zeta to the membrane fraction. Consequently, the inhibition of PLC/PKC suppressed ANG II-induced increase in [(3)H] thymidine incorporation. The inhibition of EGF receptor kinase or tyrosine kinase prevented ANG II-induced increase in [(3)H] thymidine incorporation. ANG II phosphorylated EGF receptor and increased Akt, mTOR, and p70S6K1 phosphorylation blocked by AG 1478 (EGF receptor kinase blocker). ANG II-induced increase in [(3)H] thymidine incorporation was blocked by the inhibition of p44/42 MAPKs but not by p38 MAPK inhibition. Indeed, ANG II phosphorylated p44/42 MAPKs, which was prevented by the inhibition of the PKC and AT(1) receptor. ANG II increased c-fos, c-jun, and c-myc levels. ANG II also increased the protein levels of cyclin D1, cyclin E, cyclin-dependent kinase (CDK) 2, and CDK4 but decreased the p21(cip1/waf1) and p27(kip1), CDK inhibitory proteins. These proteins were blocked by the inhibition of AT(1) receptor, PLC/PKC, p44/42 MAPKs, EGF receptor, or tyrosine kinase. In conclusion, ANG II-stimulated DNA synthesis is mediated by ANG II receptor-dependent Ca(2+)/PKC and EGF receptor-dependent PI3K/Akt/mTOR/p70S6K1 signal pathways in mouse ES cells.     

Animal model for angiotensin II effects in the internal anal sphincter smooth muscle: mechanism of action

Effect of ANG II was investigated in in vitro smooth muscle strips and in isolated smooth muscle cells (SMC). Among different species, rat internal and sphincter (IAS) smooth muscle showed significant and reproducible contraction that remained unmodified by different neurohumoral inhibitors. The AT(1) antagonist losartan but not AT(2) antagonist PD-123319 antagonized ANG II-induced contraction of the IAS smooth muscle and SMC. ANG II-induced contraction of rat IAS smooth muscle and SMC was attenuated by tyrosine kinase inhibitors genistein and tyrphostin, protein kinase C (PKC) inhibitor H-7, Ca(2+) channel blocker nicardipine, Rho kinase inhibitor Y-27632 or p(44/42) mitogen-activating protein kinase (MAPK(44/42)) inhibitor PD-98059. Combinations of nicardipine and H-7, Y-27632, and PD-98059 caused further attenuation of the ANG II effects. Western blot analyses revealed the presence of both AT(1) and AT(2) receptors. We conclude that ANG II causes contraction of rat IAS smooth muscle by the activation of AT(1) receptors at the SMC and involves multiple intracellular pathways, influx of Ca(2+), and activation of PKC, Rho kinase, and MAPK(44/42).     

Brain sodium channels and ouabainlike compounds mediate central aldosterone-induced hypertension

Central nervous system (CNS) effects of mineralocorticoids participate in the development of salt-sensitive hypertension. In the brain, mineralocorticoids activate amiloride-sensitive sodium channels, and we hypothesized that this would lead to increased release of ouabainlike compounds (OLC) and thereby sympathetic hyperactivity and hypertension. In conscious Wistar rats, intracerebroventricular infusion of aldosterone at 300 or 900 ng/h in artificial cerebrospinal fluid (aCSF) with 0.145 M Na+ for 2 h did not change baseline mean arterial pressure (MAP), renal sympathetic nerve activity (RSNA), or heart rate (HR). Intracerebroventricular infusion of aCSF containing 0.16 M Na+ (versus 0.145 M Na+ in regular aCSF) did not change MAP or RSNA, but significant increases in MAP, RSNA, and HR were observed after intracerebroventricular infusion of aldosterone at 300 ng/h for 2 h. Intracerebroventricular infusion of aCSF containing 0.3 M Na+ increased MAP, RSNA, and HR significantly more after intracerebroventricular infusion of aldosterone versus vehicle. After intracerebroventricular infusion of aldosterone, the MAP, RSNA, and HR responses to intracerebroventricular infusion of aCSF containing 0.16 M Na+ were blocked by blockade of brain OLC with intracerebroventricular infusion of Fab fragments or of brain sodium channels with intracerebroventricular benzamil. Chronic intracerebroventricular infusion of aldosterone at 25 ng/h in aCSF with 0.15 M Na+ for 2 wk increased MAP by 15-20 mmHg and increased hypothalamic OLC by 30% and pituitary OLC by 60%. Benzamil blocked all these responses to aldosterone. These findings indicate that in the brain, mineralocorticoids activate brain sodium channels, with small increases in CSF Na+ leading to increases in brain OLC, sympathetic outflow, and blood pressure.     

AT1 receptor mRNA antisense normalizes enhanced cardiac sympathetic afferent reflex in rats with chronic heart failure

Previous studies showed that the cardiac sympathetic afferent reflex (CSAR) is enhanced in dogs and rats with chronic heart failure (CHF) and that central ANG II type 1 receptors (AT(1)R) are involved in this augmented reflex. The aim of this study was to determine whether intracerebroventricular administration and microinjection of antisense oligodeoxynucleotides targeted to AT(1)R mRNA would attenuate the enhanced CSAR and decrease resting renal sympathetic nerve activity (RSNA) in rats with coronary ligation-induced CHF. The CSAR was elicited by application of bradykinin to the epicardial surface of the left ventricle. Reflex responses to epicardial administration of bradykinin were enhanced in rats with CHF. The response to bradykinin was determined every 50 min after intracerebroventricular administration (lateral ventricle) or microinjection (into paraventricular nucleus) of antisense or scrambled oligonucleotides to AT(1)R mRNA. AT(1)R mRNA and protein levels in the paraventricular nucleus were significantly reduced 5 h after administration of antisense. Antisense significantly decreased resting RSNA and normalized the enhanced CSAR responses to bradykinin in rats with CHF. Scrambled oligonucleotides did not alter resting RSNA or the enhanced responses to bradykinin in rats with CHF. No significant effects were found in sham-operated rats after administration of either antisense or scrambled oligonucleotides. These results strongly suggest that central AT(1)R mRNA antisense reduces expression of AT(1)R protein and normalizes the augmentation of this excitatory sympathetic reflex and that genetic manipulation of protein expression can be used to normalize the sympathetic enhancement in CHF.     

Involvement of enhanced cardiac sympathetic afferent reflex in sympathetic activation in early stage of diabetes

Cardiac sympathetic afferent reflex (CSAR) is involved in sympathetic activation. The present study was designed to investigate the contribution of enhanced CSAR to sympathetic activation in the early stage of diabetes and the involvement of AT(1) receptors in the paraventricular nucleus (PVN). Diabetes was induced by a single intravenous injection of streptozotocin in rats. Acute experiments were carried out under anesthesia after 3 wk. The CSAR was evaluated by the responses of renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) to epicardial application of capsaicin or bradykinin. Sympathetic activity and CSAR were enhanced in diabetic rats. Plasma norepinephrine and angiotensin II were increased, but the transient receptor potential vanilloid 1 (TRPV1) in the left ventricle wall was not significantly increased in diabetic rats. Pericardial injection of resiniferatoxin to desensitize cardiac afferents or PVN microinjection of lidocaine attenuated the CSAR and decreased the RSNA and MAP in diabetic rats. The AT(1) receptor expression in the PVN increased in diabetic rats. Angiotensin II in the PVN caused greater increases in the RSNA and MAP and enhancement in the CSAR in diabetic rats, which were abolished by the losartan pretreatment. Losartan decreased the RSNA and MAP and attenuated the CSAR in diabetic rats but not in control rats. These results indicate that the CSAR is enhanced in the early stage of diabetic rats, which contributes to the sympathetic activation. AT(1) receptors in the PVN are involved in the enhanced CSAR in diabetic rats.     

Blockade of AT1 receptors in the rostral ventrolateral medulla increases sympathetic activity under hypoxic conditions

The role of ANG type 1 (AT1) receptors in the rostral ventrolateral medulla (RVLM) in the maintenance of sympathetic vasomotor tone in normotensive animals is unclear. In this study, we tested the hypothesis that AT1 receptors make a significant contribution to the tonic activity of presympathetic neurons in the RVLM of normotensive rats under conditions where the excitatory input to these neurons is enhanced, such as during systemic hypoxia. In urethane-anesthetized rats, microinjections of the AT1 receptor antagonist candesartan in the RVLM during moderate hypoxia unexpectedly resulted in substantial increases in arterial pressure and renal sympathetic nerve activity (RSNA), whereas under normoxic conditions the same dose resulted in no significant change in arterial pressure and RSNA. Under hypoxic conditions, and after microinjection of the GABA(A) receptor antagonist bicuculline in the RVLM, subsequent microinjection of candesartan in the RVLM resulted in a significant decrease in RSNA. In control experiments, bilateral microinjections in the RVLM of the compound [Sar1,Thr8]ANG II (sarthran), which decreases sympathetic vasomotor activity via a mechanism that is independent of AT1 receptors, significantly reduced arterial pressure and RSNA under both normoxic and hypoxic conditions. The results indicate that, at least under some conditions, endogenous ANG II has a tonic sympathoinhibitory effect in the RVLM, which is dependent on GABA receptors. We suggest that the net effect of endogenous ANG II in this region depends on the balance of both tonic excitatory and inhibitory actions on presympathetic neurons and that this balance is altered in different physiological or pathophysiological conditions.     

NO and endogenous angiotensin II interact in the generation of renal sympathetic nerve activity in conscious rats

Nitric oxide (NO) appears to inhibit sympathetic tone in anesthetized rats. However, whether NO tonically inhibits sympathetic outflow, or whether endogenous angiotensin II (ANG II) promotes NO-mediated sympathoinhibition in conscious rats is unknown. To address these questions, we determined the effects of NO synthase (NOS) inhibition on renal sympathetic nerve activity (RSNA) and heart rate (HR) in conscious, unrestrained rats on normal (NS), high-(HS), and low-sodium (LS) diets, in the presence and absence of an ANG II receptor antagonist (AIIRA). When arterial pressure was kept at baseline with intravenous hydralazine, NOS inhibition with l-NAME (10 mg/kg i.v.) resulted in a profound decline in RSNA, to 42 +/- 11% of control (P < 0.01), in NS animals. This effect was not sustained, and RSNA returned to control levels by 45 min postinfusion. l-NAME also caused bradycardia, from 432 +/- 23 to 372 +/- 11 beats/min postinfusion (P < 0.01), an effect, which, in contrast, was sustained 60 min postdrug. The effects of NOS inhibition on RSNA and HR did not differ between NS, HS, and LS rats. However, when LS and HS rats were pretreated with AIIRA, the initial decrease in RSNA after l-NAME infusion was absent in the LS rats, while the response in the HS group was unchanged by AIIRA. These findings indicate that, in contrast to our hypotheses, NOS activity provides a stimulatory input to RSNA in conscious rats, and that in LS animals, but not HS animals, this sympathoexcitatory effect of NO is dependent on the action of endogenous ANG II.     

Hydrogen Sulfide in Paraventricular Nucleus Enhances Sympathetic Activity and Cardiac Sympathetic Afferent Reflex in Chronic Heart Failure Rats

Background

Intracerebroventricular infusion of NaHS, a hydrogen sulfide (H₂S) donor, increased mean arterial pressure (MAP). This study was designed to determine the roles of H₂S in the paraventricular nucleus (PVN) in modulating sympathetic activity and cardiac sympathetic afferent reflex (CSAR) in chronic heart failure (CHF).

Methodology/Principal Findings

CHF was induced by left descending coronary artery ligation in rats. Renal sympathetic nerve activity (RSNA) and MAP were recorded under anesthesia. CSAR was evaluated by the RSNA and MAP responses to epicardial application of capsaicin. PVN microinjection of low doses of a H₂S donor, GYY4137 (0.01 and 0.1 nmol), had no significant effects on RSNA, MAP and CSAR. High doses of GYY4137 (1, 2 and 4 nmol) increased baseline RSNA, MAP and heart rate (HR), and enhanced CSAR. The effects were greater in CHF rats than sham-operated rats. A cystathionine-β-synthase (CBS) inhibitor, hydroxylamine (HA) in PVN had no significant effect on the RSNA, MAP and CSAR. CBS activity and H₂S level in the PVN were decreased in CHF rats. No significant difference in CBS level in PVN was found between sham-operated rats and CHF rats. Stimulation of cardiac sympathetic afferents with capsaicin decreased CBS activity and H₂S level in the PVN in both sham-operated rats and CHF rats.

Conclusions

Exogenous H₂S in PVN increases RSNA, MAP and HR, and enhances CSAR. The effects are greater in CHF rats than those in sham-operated rats. Endogenous H₂S in PVN is not responsible for the sympathetic activation and enhanced CSAR in CHF rats.     

Aldosterone acts centrally to increase brain renin-angiotensin system activity and oxidative stress in normal rats

Aldosterone acts upon mineralocorticoid receptors in the brain to increase blood pressure and sympathetic nerve activity, but the mechanisms are still poorly understood. We hypothesized that aldosterone increases sympathetic nerve activity by upregulating the renin-angiotensin system (RAS) and oxidative stress in the brain, as it does in peripheral tissues. In Sprague-Dawley rats, aldosterone (Aldo) or vehicle (Veh) was infused for 1 wk via an intracerebroventricular (ICV) cannula, while RU-28318 (selective mineralocorticoid receptor antagonist), Tempol (superoxide dismutase mimetic), losartan [angiotensin II type 1 receptor (AT(1)R) antagonist], or Veh was infused simultaneously via a second ICV cannula. After 1 wk of ICV Aldo, plasma norepinephrine was increased and mean arterial pressure was slightly elevated, but heart rate was unchanged. These effects were ameliorated by ICV infusion of RU-28318, Tempol or losartan. Aldo increased expression of AT(1)R and angiotensin-converting enzyme (ACE) mRNA in hypothalamic tissue. RU-28318 minimized and Tempol prevented the increase in AT(1)R mRNA; RU-28318 prevented the increase in ACE mRNA. Losartan had no effect on AT(1)R or ACE mRNA. Immunohistochemistry revealed Aldo-induced increases in dihydroethidium staining (indicating oxidative stress) and Fra-like activity (indicating neuronal excitation) in neurons of the hypothalamic paraventricular nucleus (PVN). RU-28318 prevented the increases in superoxide and Fra-like activity in PVN; Tempol and losartan minimized these effects. Acute ICV infusions of sarthran (AT(1)R antagonist) or Tempol produced greater sympathoinhibition in Aldo-treated than in Veh-treated rats. Thus aldosterone upregulates key elements of brain RAS and induces oxidative stress in the hypothalamus. Aldosterone may increase sympathetic nerve activity by these mechanisms.