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Ubiquitin-specific protease 2 (USP2) participates in glucose metabolism in peripheral tissues such as the liver and skeletal muscles. However, the glucoregulatory role of USP2 in the CNS is not well known. In this study, we focus on USP2 in the ventromedial hypothalamus (VMH), which has dominant control over systemic glucose homeostasis. ISH, using a Usp2 -specific probe, showed that Usp2 mRNA is present in VMH neurons, as well as other glucoregulatory nuclei, in the hypothalamus of male mice. Administration of a USP2-selective inhibitor ML364 (20 ng/head), into the VMH elicited a rapid increase in the circulating glucose level in male mice, suggesting USP2 has a suppressive role on glucose mobilization. ML364 treatment also increased serum norepinephrine concentration, whereas it negligibly affected serum levels of insulin and corticosterone. ML364 perturbated mitochondrial oxidative phosphorylation in neural SH-SY5Y cells and subsequently promoted the phosphorylation of AMP-activated protein kinase (AMPK). Consistent with these findings, hypothalamic ML364 treatment stimulated AMPKα phosphorylation in the VMH. Inhibition of hypothalamic AMPK prevented ML364 from increasing serum norepinephrine and blood glucose. Removal of ROS restored the ML364-evoked mitochondrial dysfunction in SH-SY5Y cells and impeded the ML364-induced hypothalamic AMPKα phosphorylation as well as prevented the elevation of serum norepinephrine and blood glucose levels in male mice. These results indicate hypothalamic USP2 attenuates perturbations in blood glucose levels by modifying the ROS-AMPK-sympathetic nerve axis. SIGNIFICANCE STATEMENT Under normal conditions (excluding hyperglycemia or hypoglycemia), blood glucose levels are maintained at a constant level. In this study, we used a mouse model to identify a hypothalamic protease controlling blood glucose levels. Pharmacological inhibition of USP2 in the VMH caused a deviation in blood glucose levels under a nonstressed condition, indicating that USP2 determines the set point of the blood glucose level. Modification of sympathetic nervous activity accounts for the USP2-mediated glucoregulation. Mechanistically, USP2 mitigates the accumulation of ROS in the VMH, resulting in attenuation of the phosphorylation of AMPK. Based on these findings, we uncovered a novel glucoregulatory axis consisting of hypothalamic USP2, ROS, AMPK, and the sympathetic nervous system., (Copyright © 2022 the authors.)

Heart failure (HF) represents the end-stage condition of several structural and functional cardiovascular diseases, characterized by reduced myocardial pump function and increased pressure load. The dysregulation of neurohormonal systems, especially the hyperactivity of the cardiac adrenergic nervous system (ANS), constitutes a hallmark of HF and exerts a pivotal role in its progression. Indeed, it negatively affects patients' prognosis, being associated with high morbidity and mortality rates, with a tremendous burden on global healthcare systems. To date, all the techniques proposed to assess the cardiac sympathetic nervous system are burdened by intrinsic limits that hinder their implementation in clinical practice. Several biomarkers related to ANS activity, which may potentially support the clinical management of such a complex syndrome, are slow to be implemented in the routine practice for several limitations due to their assessment and clinical impact. Lymphocyte G-protein-coupled Receptor Kinase 2 (GRK2) levels reflect myocardial β-adrenergic receptor function in HF and have been shown to add independent prognostic information related to ANS overdrive. In the present manuscript, we provide an overview of the techniques currently available to evaluate cardiac ANS in HF and future perspectives in this field of relevant scientific and clinical interest.

The molecular mechanisms regulating sympathetic innervation of the heart during embryogenesis and its importance for cardiac development and function remain to be fully elucidated. We generated mice in which conditional knockout (CKO) of the Hif1a gene encoding the transcription factor hypoxia-inducible factor 1α (HIF-1α) is mediated by an Islet1-Cre transgene expressed in the cardiac outflow tract, right ventricle and atrium, pharyngeal mesoderm, peripheral neurons, and hindlimbs. These Hif1aCKO mice demonstrate significantly decreased perinatal survival and impaired left ventricular function. The absence of HIF-1α impaired the survival and proliferation of preganglionic and postganglionic neurons of the sympathetic system, respectively. These defects resulted in hypoplasia of the sympathetic ganglion chain and decreased sympathetic innervation of the Hif1aCKO heart, which was associated with decreased cardiac contractility. The number of chromaffin cells in the adrenal medulla was also decreased, indicating a broad dependence on HIF-1α for development of the sympathetic nervous system., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Background: The hypothalamic paraventricular nucleus (PVN) is the center of the regulation of autonomic nervous system functions and cardiovascular activity. Phosphoinositide-3 kinase (PI3K)-AKT pathway in PVN contributes to mediate sympathetic nerve activity and is activated in spontaneously hypertensive rats. Overactivation of the sympathetic output was considered as an important mechanism of the arrhythmias. In the present study, we aimed to explore whether targeted regulation of sympathetic activity in PVN could reduce the peripheral sympathoexcitatory and attenuate the ventricular arrhythmias (VAs) in myocardial infarction (MI) rats via PI3K-AKT pathway., Methods: A stainless steel gauge guide cannula was stereotaxically implanted into the PVN, and 7 days later, rats were randomly divided into the following 4 groups: group A, control+dimethyl sulfoxide (DMSO); group B, control+LY294002; group C, MI surgery+DMSO; and group D, MI surgery+LY294002. Studies were conducted seven days post-MI. Myocardial function, infarct size, inducible VAs by programmed electrical stimulation, renal sympathetic nerve activity (RSNA), and protein level of PI3K and AKT were measured., Results: MI increased the protein ratios of p-PI3K-to-total-PI3K and p-AKT-to-total-AKT in PVN but can be reduced by LY294002 treatment. Inhibition of sympathetic nerve activity in PVN led to a reversion in plasma norepinephrine, RSNA and inducible VAs in MI rats., Conclusions: PI3K-AKT pathway in the PVN was a main mechanism in regulating sympathetic activity and arrhythmias in MI rats. Targeted inhibition of sympathetic activity in PVN may be a potential treatment for the VAs via PI3K-AKT pathway., (Copyright © 2018 Japanese College of Cardiology. Published by Elsevier Ltd. All rights reserved.)

Salivary alpha-amylase (sAA) is a digestive enzyme mainly responsible for the hydrolysis of starch and glycogen in the oral cavity. Since the secretion of sAA is largely under the control of the sympathetic nervous system, sAA activity is also considered to be a non-invasive marker of sympathetic activation. However, the direct association between sAA activity and other sympathetic parameters remains questionable. Therefore, we employed the corticotropin-releasing hormone (CRH) stimulation test to pharmacologically activate the sympathetic nervous system and to analyze plasma noradrenaline response together with sAA activity. Thirty-one healthy male volunteers (mean age of 25.2±3.1years) were randomized into two groups and received injections with either CRH (100μg, N=17) or placebo (0.9% NaCl, N=14). Blood samples were taken at baseline and 15, 30, 60, 120min after injection. Results showed that CRH administration increased plasma noradrenaline and cortisol concentrations, sAA activity, heart rate, as well as self-reported side effects (i.e. flushing in the facial area, heart rate changes, giddiness, malaise and restlessness) and stress perception, while plasma adrenaline levels remained unaffected. In the CRH group, the total increase of sAA activity significantly correlated with noradrenaline release, indicating that sAA activity reflects pharmacologically induced sympathetic activation., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The effect of renal denervation on the efficacy of antihypertensive drugs has not yet been elucidated. Twenty-week-old spontaneously hypertensive rats were treated with metoprolol, losartan, indapamide, or saline (controls) and assigned to renal denervation or a sham procedure. Acute hemodynamic measurements were performed ten days later. Series showing a significant interaction between renal denervation and the drugs were repeated with chronic telemetry measurements. In the saline series, denervated rats showed a significantly lower mean arterial blood pressure (blood pressure) than the sham-operated rats. In contrast, in the metoprolol series denervated rats showed a significantly higher blood pressure than sham rats. There were no differences in blood pressure between denervated and sham rats in the losartan and indapamide series. In chronic studies, a 4-week treatment with metoprolol caused a decrease in blood pressure. Renal denervation and sham denervation performed 10 days after the onset of metoprolol treatment did not affect blood pressure. Denervated rats showed markedly reduced renal nerve tyrosine hydroxylase levels. In conclusion, renal denervation decreases blood pressure in hypertensive rats. The hypotensive action of metoprolol, indapamide, and losartan is not augmented by renal denervation, suggesting the absence of synergy between renal denervation and the drugs investigated in this study.

Standards in quantitative fluorescent imaging are vaguely recognized and receive insufficient discussion. A common best practice is to acquire images at Nyquist rate, where highest signal frequency is assumed to be the highest obtainable resolution of the imaging system. However, this particular standard is set to insure that all obtainable information is being collected. The objective of the current study was to demonstrate that for quantification purposes, these correctly set acquisition rates can be redundant; instead, linear size of the objects of interest can be used to calculate sufficient information density in the image. We describe optimized image acquisition parameters and unbiased methods for processing and quantification of medium-size cellular structures. Sections of rabbit aortas were immunohistochemically stained to identify and quantify sympathetic varicosities, >2 μm in diameter. Images were processed to reduce background noise and segment objects using free, open-access software. Calculations of the optimal sampling rate for the experiment were based on the size of the objects of interest. The effect of differing sampling rates and processing techniques on object quantification was demonstrated. Oversampling led to a substantial increase in file size, whereas undersampling hindered reliable quantification. Quantification of raw and incorrectly processed images generated false structures, misrepresenting the underlying data. The current study emphasizes the importance of defining image-acquisition parameters based on the structure(s) of interest. The proposed postacquisition processing steps effectively removed background and noise, allowed for reliable quantification, and eliminated user bias. This customizable, reliable method for background subtraction and structure quantification provides a reproducible tool for researchers across biologic disciplines.

The activin receptor-like kinase 1 (ALK-1) is a type I cell-surface receptor for the transforming growth factor-β (TGF-β) family of proteins. Hypertension is related to TGF-β1, because increased TGF-β1 expression is correlated with an elevation in arterial pressure (AP) and TGF-β expression is upregulated by the renin-angiotensin-aldosterone system. The purpose of this study was to assess the role of ALK-1 in regulation of AP using Alk1 haploinsufficient mice (Alk1(+/-)). We observed that systolic and diastolic AP were significantly higher in Alk1(+/-) than in Alk1(+/+) mice, and all functional and structural cardiac parameters (echocardiography and electrocardiography) were similar in both groups. Alk1(+/-) mice showed alterations in the circadian rhythm of AP, with higher AP than Alk1(+/+) mice during most of the light period. Higher AP in Alk1(+/-) mice is not a result of a reduction in the NO-dependent vasodilator response or of overactivation of the peripheral renin-angiotensin system. However, intracerebroventricular administration of losartan had a hypotensive effect in Alk1(+/-) and not in Alk1(+/+) mice. Alk1(+/-) mice showed a greater hypotensive response to the β-adrenergic antagonist atenolol and higher concentrations of epinephrine and norepinephrine in plasma than Alk1(+/+) mice. The number of brain cholinergic neurons in the anterior basal forebrain was reduced in Alk1(+/-) mice. Thus, we concluded that the ALK-1 receptor is involved in the control of AP, and the high AP of Alk1(+/-) mice is explained mainly by the sympathetic overactivation shown by these animals, which is probably related to the decreased number of cholinergic neurons., (© 2015. Published by The Company of Biologists Ltd.)

Introduction: We tested the hypothesis that subcutaneous nerve activity (SCNA) of the thorax correlates with the stellate ganglion nerve activity (SGNA) and can be used to estimate the sympathetic tone., Methods and Results: We implanted radio transmitters in 11 ambulatory dogs to record left SGNA, left thoracic vagal nerve activity (VNA), and left thoracic SCNA, including 3 with simultaneous video monitoring and nerve recording. Two additional dogs were studied under general anesthesia with apamin injected into the right stellate ganglion while the right SGNA and the right SCNA were recorded. There was a significant positive correlation between integrated SGNA (iSGNA) and integrated SCNA (iSCNA) in the first 7 ambulatory dogs, with correlation coefficient of 0.70 (95% confidence interval [CI] 0.61-0.84, P < 0.05 for each dog). Tachycardia episodes (heart rate exceeding 150 bpm for ≥3 seconds) were invariably preceded by SGNA and SCNA. There was circadian variation of both SCNA and SGNA. Crosstalk was ruled out because SGNA, VNA, and SCNA bursts had different timing and activation patterns. In an eighth dog, closely spaced bipolar subcutaneous electrodes also recorded SCNA, but with reduced signal to noise ratio. Video monitoring in additional 3 dogs showed that movement was not a cause of high frequency SCNA. The right SGNA correlated strongly with right SCNA and heart rate in 2 anesthetized dogs after apamin injection into the right stellate ganglion., Conclusions: SCNA recorded by bipolar subcutaneous electrodes correlates with the SGNA and can be used to estimate the sympathetic tone., (© 2014 Wiley Periodicals, Inc.)

Rationale: Dopamine β-hydroxylase (DBH) catalyzes the conversion of dopamine to norepinephrine in the central nervous system and peripherally. DBH variants are associated with large changes in circulating DBH and implicated in multiple disorders; yet causal relationships and tissue-specific effects remain unresolved., Objective: To characterize regulatory variants in DBH, effect on mRNA expression, and role in modulating sympathetic tone and disease risk., Methods and Results: Analysis of DBH mRNA in human tissues confirmed high expression in the locus coeruleus and adrenal gland, but also in sympathetically innervated organs (liver>lung>heart). Allele-specific mRNA assays revealed pronounced allelic expression differences in the liver (2- to 11-fold) attributable to promoter rs1611115 and exon 2 rs1108580, but only small differences in locus coeruleus and adrenals. These alleles were also associated with significantly reduced mRNA expression in liver and lung. Although DBH protein is expressed in other sympathetically innervated organs, mRNA levels were too low for analysis. In mice, hepatic Dbh mRNA levels correlated with cardiovascular risk phenotypes. The minor alleles of rs1611115 and rs1108580 were associated with sympathetic phenotypes, including angina pectoris. Testing combined effects of these variants suggested protection against myocardial infarction in 3 separate clinical cohorts., Conclusions: We demonstrate profound effects of DBH variants on expression in 2 sympathetically innervated organs, liver and lung, but not in adrenals and brain. Preliminary results demonstrate an association of these variants with clinical phenotypes responsive to peripheral sympathetic tone. We hypothesize that in addition to endocrine effects via circulating DBH and norepinephrine, the variants act in sympathetically innervated target organs., (© 2014 American Heart Association, Inc.)

Recently, involvement of the sympathetic nervous system in bone metabolism has attracted attention. β2-Adrenergic receptor (β2-AR) is presented on osteoblastic and osteoclastic cells. We previously demonstrated that β-AR blockers at low dose improve osteoporosis with hyperactivity of the sympathetic nervous system via β2-AR blocking, while they may have a somewhat inhibitory effect on osteoblastic activity at high doses. In this study, the effects of butoxamine (BUT), a specific β2-AR antagonist, on tooth movement were examined in spontaneously hypertensive rats (SHR) showing osteoporosis with hyperactivity of the sympathetic nervous system. We administered BUT (1 mg/kg) orally, and closed-coil springs were inserted into the upper-left first molar. After sacrifice, we calculated the amount of tooth movement and analyzed the trabecular microarchitecture and histomorphometry. The distance in the SHR control was greater than that in the Wistar-Kyoto rat group, but no significant difference was found in the SHR treated with BUT compared with the Wistar-Kyoto rat control. Analysis of bone volume per tissue volume, trabecular number, and osteoclast surface per bone surface in the alveolar bone showed clear bone loss by an increase of bone resorption in SHR. In addition, BUT treatment resulted in a recovery of alveolar bone loss. Furthermore, TH-immunoreactive nerves in the periodontal ligament were increased by tooth movement, and BUT administration decreased TH-immunoreactive nerves. These results suggest that BUT prevents alveolar bone loss and orthodontic tooth movement via β2-AR blocking., (© International & American Associations for Dental Research.)

Background: The effect of phosphodiesterase-3 (PDE-3) inhibitors on arrhythmia remains controversial, so the purpose of this study was to determine their differential effects on sympathetic hyperinnervation and the involved mechanisms in a rat model of myocardial infarction., Methods and Results: After ligating the coronary artery, male Wistar rats were randomized to cilostazol or milrinone, chemically unrelated inhibitors of PDE-3, or vehicle for 4 weeks. The postinfarction period was associated with increased myocardial norepinephrine levels and oxidant release, as measured by myocardial superoxide level and dihydroethidine fluorescence staining. Infarcted rats in the milrinone- and cilostazol-treated groups had favorable ventricular remodeling with similar potency. Compared with milrinone, cilostazol significantly increased interstitial adenosine levels and reduced the production of myocardial cAMP and superoxide. Cilostazol significantly blunted sympathetic hyperinnervation, as assessed by immunofluorescent analysis of sympathetic innervation, and western blotting and real-time quantitative RT-PCR of nerve growth factor. Furthermore, the inhibitory effect of cilostazol on nerve growth factor was reversed by 8-cyclopentyl-1,3-dipropylxanthine, a selective A1 receptor antagonist, and enhanced by tempol administration. In spite of similar arrhythmic vulnerability during programmed stimulation in both the vehicle-and cilostazol-treated groups, cilostazol did not have proarrhythmic effects compared with milrinone., Conclusions: Unlike milrinone, cilostazol has therapeutic neutrality in arrhythmias because of adenosine uptake inhibition, which antagonizes the PDE-3-induced increase of sympathetic reinnervation via mediation of an adenosine A1 receptor-mediated antioxidation.

Cyclooxygenase (COX)-derived prostanoids contribute to angiotensin II (ANG II) hypertension (HTN). However, the specific mechanisms by which prostanoids act are unclear. ANG II-induced HTN is caused partly by increased sympathetic nervous system activity, especially in a setting of high dietary salt intake. This study tested the hypothesis that COX-derived prostanoids cause ANG II-salt sympathoexcitation and HTN. Experiments were conducted in conscious rats. Infusion of ANG II (150 ng·kg(-1)·min(-1) sc) caused a marked HTN in rats on 2% salt diet, but a much smaller increase in blood pressure in rats on 0.4% salt diet. The nonselective COX inhibitor ketoprofen (2 mg/kg sc) given throughout the ANG-II infusion period attenuated HTN development in rats on 2% NaCl diet, but not in rats on 0.4% NaCl diet. The acute depressor response to ganglion blockade was used to assess neurogenic pressor activity in rats on 2% NaCl diet. Ketoprofen-treated rats showed a smaller fall in arterial pressure in response to ganglion blockade during ANG-II infusion than did nontreated controls. In additional experiments, ketoprofen-treated rats exhibited smaller increases in plasma norepinephrine levels and whole body norepinephrine spillover than we previously reported in ANG II-salt HTN. Finally, the effects of the selective COX-1 inhibitor SC560 (10 mg·kg(-1)·day(-1) ip) and the selective COX-2 inhibitor nimesulide (10 mg·kg(-1)·day(-1) ip) were investigated. Treatment with SC560 but not nimesulide significantly reduced blood pressure and the depressor response to ganglion blockade in ANG II-salt HTN rats. The results suggest that COX-1 products are critical for sympathoexcitation and the full development of ANG II-salt HTN in rats.

Nitric oxide, derived from nitric oxide synthase (NOS), plays an important role in regulating sympathetic nerve activity. Neuronal NOS (nNOS) is expressed throughout the central and peripheral nervous system. nNOS has a sympathoinhibitory effect under physiological conditions by acting on different sites of the nervous system, including the paraventricular nucleus, the nucleus of the solitary tract, the rostral ventrolateral medulla, the carotid body and nerves in the kidney. nNOS is sympathoinhibitory in a range of diseases including chronic heart failure, chronic renal failure, hypertension and diabetes. nNOS is believed to mediate sympathoinhibitory effects induced by a range of signaling pathways including those promoted by angiotensin-converting enzyme 2 over-expression; statin therapy; angiotensin II type 1 receptor blockers; exercise training; tumor necrosis factor-α blockade; superoxide dismutase mimetics; and estrogen replacement therapy. Increase in nNOS can increase sympathoinhibitory γ-aminobutyric acid activity and decrease sympathoexcitatory angiotensin II signaling and glutamate activity. nNOS may have sympathoexcitatory effects in some circumstances such as chronic heart failure induced by prolonged high salt treatment. The effectiveness of nNOS upregulation in treating sympathetic overactive conditions including chronic heart failure needs to be further investigated.

Serum ghrelin concentration is generally reduced in obesity. We aimed to assess whether this alteration is present in rats predisposed to obesity because of moderate undernutrition during gestation, and to explore whether this could be related with alterations in stomach sympathetic innervation, which is involved in gastric ghrelin secretion. Offspring of control and 20% gestational calorie-restricted dams (CR) exposed to normal-fat-diet from weaning onward were studied. Circulating ghrelin levels were measured at 25 days and 4 months of age. Morphometry, number of ghrelin-positive (ghrelin(+)) cells, ghrelin mRNA and protein levels, and tyrosine hydroxylase (TH) protein levels in stomach were determined at 25 days. Adult CR male animals, but not females, exhibited greater body-weight (BW) than their controls, but both males and females showed lower circulating ghrelin levels. This alteration in ghrelin levels was already present at 25 days, prior to any difference in BW. At this juvenile age, no differences in gastric morphometry, number of ghrelin(+) cells or ghrelin mRNA/protein levels were found between control and CR animals, however, CR animals showed lower TH stomach content. These results suggest that circulating ghrelin concentration is early altered in rats prenatally programmed to develop obesity. This does not seem to be associated with lower ghrelin production capacity but with specific alterations in sympathetic drive to the stomach., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: AMPK activation promotes glucose and lipid metabolism., Results: Hepatic AMPK activities were decreased in fatty liver from lipodystrophic mice, and leptin activated the hepatic AMPK via the α-adrenergic effect., Conclusion: Leptin improved the fatty liver possibly by activating hepatic AMPK through the central and sympathetic nervous systems., Significance: Hepatic AMPK plays significant roles in the pathophysiology of lipodystrophy and metabolic action of leptin. Leptin is an adipocyte-derived hormone that regulates energy homeostasis. Leptin treatment strikingly ameliorates metabolic disorders of lipodystrophy, which exhibits ectopic fat accumulation and severe insulin-resistant diabetes due to a paucity of adipose tissue. Although leptin is shown to activate 5'-AMP-activated protein kinase (AMPK) in the skeletal muscle, the effect of leptin in the liver is still unclear. We investigated the effect of leptin on hepatic AMPK and its pathophysiological relevance in A-ZIP/F-1 mice, a model of generalized lipodystrophy. Here, we demonstrated that leptin activates hepatic AMPK through the central nervous system and α-adrenergic sympathetic nerves. AMPK activities were decreased in the fatty liver of A-ZIP/F-1 mice, and leptin administration increased AMPK activities in the liver as well as in skeletal muscle with significant reduction in triglyceride content. Activation of hepatic AMPK with A769662 also led to a decrease in hepatic triglyceride content and blood glucose levels in A-ZIP/F-1 mice. These results indicate that the down-regulation of hepatic AMPK activities plays a pathophysiological role in the metabolic disturbances of lipodystrophy, and the hepatic AMPK activation is involved in the therapeutic effects of leptin.

Angiotensin (ANG)-converting enzyme (ACE)2 in brain regions such as the paraventricular nucleus (PVN) controlling cardiovascular function may be involved in the regulation of sympathetic outflow in chronic heart failure (CHF). The purpose of this study was to determine if ACE2 plays a role in the central regulation of sympathetic outflow by regulating neuronal nitric oxide (NO) synthase (nNOS) in the PVN. We investigated ACE2 and nNOS expression within the PVN of rats with CHF. We then determined the effects of ACE2 gene transfer in the PVN on the contribution of NO-mediated sympathoinhibition in rats with CHF. The results showed that there were decreased expressions for ACE2, the ANG-(1-7) receptor, and nNOS within the PVN of rats with CHF. After the application of adenovirus vectors encoding ACE2 (AdACE2) into the PVN, the increased expression of ACE2 in the PVN was confirmed by Western blot analysis. AdACE2 transfection significantly increased nNOS protein levels (change of 50 ± 5%) in the PVN of CHF rats. In anesthetized rats, AdACE2 treatment attenuated the responses of renal sympathetic nerve activity (RSNA), mean arterial pressure, and heart rate to the NOS inhibitor N-monomethyl-L-arginine in rats with CHF (RSNA: 28 ± 3% vs. 16 ± 3%, P < 0.05) compared with CHF + AdEGFP group. Furthermore, neuronal NG-108 cells incubated with increasing doses of AdACE2 showed a dose-dependent increase in nNOS protein expression (60% at the highest dose). Taken together, our data highlight the importance of increased expression and subsequent interaction of ACE2 and nNOS within the PVN, leading to a reduction in sympathetic outflow in the CHF condition.

In this study, we examined the effect of intracerebroventricular (ICV) injection of 5-aminoimidazole-4-carboxamide 1-beta-d-ribofuranoside (AICAR), an AMP-activated protein kinase (AMPK) activator, or compound C (CC), an AMPK inhibitor, on the activity of sympathetic nerves innervating the adrenal gland and kidney in urethane-anesthetized rats to elucidate the role of AMPK in sympathetic nervous system function. We found that an ICV injection of AICAR or CC significantly stimulated renal sympathetic nerve activity (RSNA) and adrenal sympathetic nerve activity (ASNA) in a dose-dependent manner. Following this, we examined the role of AMPK on the sympatho-excitation caused by leptin injection. Pretreatment with AICAR or CC eliminated the leptin-induced increase in RSNA, however, neither pretreatment with AICAR or CC affected the leptin-induced increase in ASNA. Our data suggest that AMPK may regulate the sympathetic nerve system, and that the stimulating effect of leptin on sympathetic nerve activity in kidney may depend on central AMPK., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Proinflammatory cytokines, including tumor necrosis factor (TNF)-α, augment the progression of heart failure (HF) that is characterized by sympathoexcitation. In this study, we explored the role of TNF-α in hypothalamic paraventricular nucleus (PVN) in the exaggerated sympathetic activity observed in HF. Heart failure rats were made by ligating the left anterior descending coronary artery. The expression levels of angiotensin type 1 receptor (AT1-R) and neurotransmitters were analyzed in the PVN of HF rats that received direct PVN infusion of a TNF-α blocker (pentoxifylline or etanercept) or vehicle. Sham-operated control (SHAM) or HF rats were treated for 4 weeks through PVN infusion with each TNF-α blocker or vehicle. Rats with HF had higher levels of glutamate, norepinephrine, AT1-R and tyrosine hydroxylase (TH), and lower levels of gamma-aminobutyric acid (GABA), neuronal nitric oxide synthase (nNOS) and the 67-kDa isoform of glutamate decarboxylase (GAD67) in the PVN when compared to SHAM rats. Plasma levels of cytokines, norepinephrine and angiotensin II and renal sympathetic nerve activity (RSNA) were increased in HF rats. PVN infusion of pentoxifylline or etanercept attenuated the decreases in PVN GABA, nNOS and GAD67, and the increases in RSNA and PVN glutamate, norepinephrine, TH and AT1-R observed in HF rats. We have developed a novel method for chronic and continuous infusion of drugs directly into the PVN and provided evidence that TNF-α in the PVN modulates neurotransmitters and the expression of AT1 receptor, which could account for exaggerated sympathetic activity in HF.

Generally, sympathetic distribution in the pterygopalatine fossa (PPF) is considered to be via the pterygopalatine ganglion (PPG) sympathetic root and branches. We hypothesized that there may be a dual sympathetic path within the PPF, through the vidian nerve and the PPG and through the periarterial plexuses. We dissected 10 human adult cadavers, fixed and unfixed; we applied antibodies for tyrosine hydroxylase (TH) to 5 human adult samples of PPF contents dissected from cadavers at autopsy. We identified TH(+) nerves and fibers distributed through the neuronal clusters of the PPG and also bundles extrinsic to these clusters, distributed along the maxillary artery. Also, TH(+) reactions were identified at the level of the neuronal capsules of the PPG. All the arteries within the PPF presented TH(+) fibers, periadventitial and intramural-the periarterial plexuses were also identified during dissections, a major one being that along the descending palatine artery, distinctive to the greater palatine nerve. Thus, concerning the sympathetic entry to the PPF, this one seems to use both the path of the external carotid artery (via the maxillary artery plexus) and the path of the internal carotid artery, via the vidian nerve supplying the PPG and reinforcing the maxillary artery plexus. The sympathetic exit of the PPF uses the neural scaffolding of the PPG branches and also the arterial scaffolding. The complex trigeminal-autonomic, anatomic content of the PPF may be involved in several distinctive facial algias and thus the pain may be relieved by routine approaches to the PPF, based on updated anatomical knowledge and a correct diagnostic., (Copyright (c) 2009 Elsevier GmbH. All rights reserved.)

Oxidative stress in the rostral ventrolateral medulla (RVLM) increases sympathetic nervous system activity (SNA). Oral treatment with atorvastatin decreases SNA through antioxidant effects in the RVLM of stroke-prone spontaneously hypertensive rats (SHRSP). We aimed to examine whether centrally administered atorvastain reduces SNA in SHRSP and, if so, to determine whether it is associated with the reduction of oxidative stress induced by alteration of activities of nicotinamide adenine dinucleotide phosphate [NAD(P)H] oxidase and superoxide dismutase (SOD) in the RVLM of SHRSP. SHRSP received atorvastatin (S-ATOR) or vehicle (S-VEH) by continuous intracerebroventricular infusion for 14 days. Mean blood pressure, heart rate, and SNA were significantly lower in S-ATOR than in S-VEH. Oxidative stress, Rac1 activity, NAD(P)H oxidase activity, Rac1, gp91(phox) and p22(phox) expression in the membrane fraction, and p47(phox) and p40(phox) expression in the cytosolic fraction in the RVLM were significantly lower in S-ATOR than in S-VEH. Rac1 expression in the cytosolic fraction and Mn-SOD activity, however, were significantly higher in S-ATOR than in S-VEH. Our findings suggest that centrally administered atorvastatin decreases SNA and is associated with decreasing NAD(P)H oxidase activity and upregulation of Mn-SOD activity in the RVLM of SHRSP, leading to suppressing oxidative stress.

Objective: Social isolation is regarded as one of the most relevant causes of diseases in mammalian species. The activation of the sympathoneural system represents one of the key components of the stress response. The sympathetic nervous system is one of the major pathways involved in immune-neuroendocrine interactions. The aim of the present study was to determine plasma epinephrine and norepinephrine in individually housed rats, as well as to find out whether splenic gene expression of catecholamine synthesizing enzymes and their protein levels are affected by chronic psychosocial stress., Methods: Tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT) mRNA levels were quantified by quantitative real-time RT-PCR. The TH, DBH and PNMT immunoproteins were assayed by Western blot., Results: Chronic social isolation of adult male rats produced a significant increase in plasma catecholamine levels and a decrease in splenic TH mRNA, DBH mRNA and PNMT mRNA. Protein levels of TH, DBH and PNMT were also reduced., Conclusion: These results suggest that increased plasma catecholamines and decreased gene expression and protein levels of catecholamine biosynthetic enzymes in the spleen of chronically individually housed animals might reduce catecholamine synthesis, thus leaving the immunocompetent tissues depleted of catecholamines and consequently leading to an impairment of immune response.

Corticotropin-releasing factor (CRF) in the brain has been shown to stimulate sympathetic activity, leading to elevations of blood pressure, heart rate and plasma catecholamine levels and neuronal activation of the sympathetic ganglia and adrenal medulla. We previously reported that brain cyclooxygenase (COX), the rate-limiting enzyme in the synthesis of prostanoids, is involved in centrally administered CRF-induced sympathetic activation in rats. Therefore, the present study was designed to reveal the effect of centrally administered CRF (1.5 nmol/animal) on the expression of COX isozymes, COX-1 and COX-2, in spinally projecting neurons until 6h after the administration, using rats microinjected with a monosynaptic retrograde tracer into the intermediolateral cell column of the thoracic spinal cord. Retrogradely labeled neurons were detected in the paraventricular hypothalamic nucleus (PVN), locus coeruleus (LC), raphe pallidus nucleus and rostral ventrolateral medulla. Centrally administered CRF significantly increased the number of spinally projecting PVN neurons expressing COX-1 throughout the experimental period and those expressing COX-2 during only the late phase. CRF also increased the number of spinally projecting LC neurons expressing COX-2 throughout the experimental period. In other regions, the CRF administration had no effect on COXs expression in spinally projecting neurons. These results suggest that COX-1 and COX-2 in the PVN and COX-2 in the LC play roles in the CRF-induced sympathetic regulation in rats.

Animal studies have indicated that nitric oxide is a key signalling molecule involved in the tonic restraint of central sympathetic outflow from the brainstem. Extension of these findings to humans has been difficult because systemic infusion of nitric oxide synthase (NOS) inhibitors increases blood pressure due to inhibition of endothelial NOS, resulting in activation of the arterial baroreflex and subsequent inhibition of central sympathetic outflow. To overcome this confounding inhibitory influence of the baroreflex, in the current study we directly measured skin sympathetic nerve activity (SNA), which is not under baroreceptor control. Healthy, normotensive humans were studied before, during a 60 min intravenous infusion of the NOS inhibitor N(G)-nitro-l-arginine methyl ester (l-NAME; 4 mg kg(1)), and for 120 min following the infusion (i.e. 180 min total). Skin SNA and arterial blood pressure (BP) were continuously measured. BP was increased from baseline at the end of the l-NAME infusion (14 +/- 2 mmHg; P < 0.05) and remained significantly elevated for the remainder of the experiment (18 +/- 3 mmHg; P < 0.05). Similarly, systemic NOS inhibition produced time-dependent increases in skin SNA, such that skin SNA was elevated at the end of the l-NAME infusion (total activity, 200 +/- 22% baseline; P = 0.08) and was further increased at the end of the study protocol (total activity, 350 +/- 41% baseline; P < 0.05). Importantly, skin SNA remained unchanged during time and hypertensive (phenylephrine) control experiments. These findings indicate that pharmacological inhibition of NOS causes sympathetic activation and support a role of nitric oxide in central sympathetic control in humans.

Our previous studies have shown that nitric oxide (NO) synthase (NOS)-containing neurons in the rostral ventrolateral medulla (rVLM) are activated during cardiac sympathoexcitatory reflexes (Refs. 12 and 13). However, the precise function of NO in the rVLM in regulation of these reflexes has not been defined. Three isoforms of NOS, including neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS), are located in the rVLM. We explored the role of NO, derived from different NOS isoforms in the rVLM, in processing cardiac-sympathetic reflexes using whole animal reflex and electrophysiological approaches. We found that, in anesthetized cats, increased mean arterial blood pressure and renal sympathetic nerve activity elicited by epicardial application of bradykinin (BK; 1-10 microg/ml, 50 microl) were significantly attenuated following unilateral rVLM microinjection of the nonselective NOS inhibitor, N(omega)-nitro-L-arginine methyl ester (50 nmol/50 nl), or a specific nNOS inhibitor, 7-nitroindazole (7-NI; 5-10 pmol/50 nl; both P < 0.05). In contrast, the responses of mean arterial blood pressure and renal sympathetic nerve activity to cardiac BK stimulation were unchanged by unilateral rVLM microinjection of N(omega)-nitro-D-arginine methyl ester (inactive isomer of N(omega)-nitro-L-arginine methyl ester, 50 nmol/50 nl), 3-6% methanol (7-NI vehicle), N(6)-(1-iminoethyl)-L-lysine (250 pmol/50 nl; iNOS inhibitor), or N(5)-(1-iminoethyl)-L-ornithine (250 nmol/50 nl; eNOS inhibitor). Furthermore, in separate cats, we noted that iontophoresis of 7-NI (0.1 mM) reduced the increased discharge of cardiovascular sympathoexcitatory rVLM neurons in response to cardiac stimulation with BK (P < 0.05). These neurons were characterized by their responses to inputs from baroreceptors, and their cardiac rhythmicity was determined through frequency and time domain analyses, correlating their discharge to arterial blood pressure and cardiac sympathetic efferent nerve activity. These data suggest that NO, specifically nNOS, mediates sympathetic cardiac-cardiovascular responses through its action in the rVLM.

Inflammatory cytokines that act through glycoprotein (gp)130 are elevated in the heart after myocardial infarction and in heart failure. These cytokines are potent regulators of neurotransmitter and neuropeptide production in sympathetic neurons but are also important for the survival of cardiac myocytes after damage to the heart. To examine the effect of gp130 cytokines on cardiac nerves, we used gp130(DBH-Cre/lox) mice, which have a selective deletion of the gp130 cytokine receptor in neurons expressing dopamine beta-hydroxylase (DBH). Basal sympathetic parameters, including norepinephrine (NE) content, tyrosine hydroxylase expression, NE transporter expression, and sympathetic innervation density, appeared normal in gp130(DBH-Cre/lox) compared with wild-type mice. Likewise, basal cardiovascular parameters measured under isoflurane anesthesia were similar in both genotypes, including mean arterial pressure, left ventricular peak systolic pressure, dP/dt(max), and dP/dt(min). However, pharmacological interventions revealed an autonomic imbalance in gp130(DBH-Cre/lox) mice that was correlated with an increased incidence of premature ventricular complexes after reperfusion. Stimulation of NE release with tyramine and infusion of the beta-agonist dobutamine revealed blunted adrenergic transmission that correlated with decreased beta-receptor expression in gp130(DBH-Cre/lox) hearts. Due to the developmental expression of the DBH-Cre transgene in parasympathetic ganglia, gp130 was eliminated. Cholinergic transmission was impaired in gp130(DBH-Cre/lox) hearts due to decreased parasympathetic drive, but tyrosine hydroxylase immunohistochemistry in the brain stem revealed that catecholaminergic nuclei appeared grossly normal. Thus, the apparently normal basal parameters in gp130(DBH-Cre/lox) mice mask an autonomic imbalance that includes alterations in sympathetic and parasympathetic transmission.

The proportion of sympathetic preganglionic neurons (SPN) showing nitric oxide synthase (NOS) immunoreactivity appears to vary with innervation target and blood pressure level. For normotensive Sprague-Dawley rats (SD), we evaluated peroxidase immunolabelling for choline acetyltransferase (ChAT) plus NOS in spinal cord segments T1-L2 and assessed NOS immunofluorescence in SPN retrogradely labelled with cholera toxin B subunit from the adrenal medulla (AM) or superior cervical (SCG), coeliac (CG), or major pelvic (MPG) ganglia. We also compared the distributions and numbers of NOS-positive and NOS-negative/ChAT-positive lateral horn neurons in SD with those in normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). In SD, WKY, and SHR, rostrocaudal, dorsoventral, and mediolateral differences occurred in the distributions of NOS-positive and NOS-negative/ChAT-positive neurons in the intermediolateral cell column (IML), whereas the two groups were similarly distributed throughout the central autonomic area (CAA). Among the four retrogradely labelled populations of SPN, the percentages showing NOS immunoreactivity differed (CG-projecting, 54.8% +/- 0.7%; SCG-projecting, 75.3% +/- 1.2%; MPG-projecting, 89% +/- 1.1% and AM-projecting, 98.6% +/- 0.2%). Within each retrogradely labelled group of SPN, the NOS-positive proportion also varied with subnuclear location (e.g., 25.5% +/- 4.0% of CG-projecting SPN in the CAA vs. 82.7% +/- 7.6% of CG-projecting SPN in the dorsolateral funiculus). The numbers of NOS-positive and NOS-negative/ChAT-positive neurons in T9-T11 were the same in SD and SHR but differed in WKY. Our results show that the expression of NOS within SPN varies depending on the target that they innervate and also on their subnuclear location. Our data indicate that there are no anatomical differences between nitric oxide-synthesizing SPN in normotensive SD and hypertensive SHR.

Heightened sympathetic excitation and diminished parasympathetic suppression of heart rate, cardiac contractility and vascular tone are all associated with cardiovascular diseases such as hypertension and ischemic heart disease. This phenotype often exists before these disease states have been established and is a strong correlate of mortality in the population. However, the causal role of the autonomic phenotype in the development and maintenance of hypertension and myocardial ischemia remains a subject of debate, as are the mechanisms responsible for regulating sympathovagal balance. Emerging evidence suggests oxidative stress and reactive oxygen species (such as nitric oxide (NO) and superoxide) play important roles in the modulation of autonomic balance, but so far the most important sites of action of these ubiquitous signaling molecules are unclear. In many cases, these mediators have opposing effects in separate tissues rendering conventional pharmacological approaches non-efficacious. Novel techniques have recently been used to augment these signaling pathways experimentally in a targeted fashion to central autonomic nuclei, cardiac neurons, and myocytes using gene transfer of NO synthase. This review article discusses these recent advances in the understanding of the roles of NO and its oxidative metabolites on autonomic imbalance in models of cardiovascular disease.

Evidence from patients with inflammatory bowel disease (IBD) and animal models suggests that inflammation alters blood flow to the mucosa, which precipitates mucosal barrier dysfunction. Impaired purinergic sympathetic regulation of submucosal arterioles, the resistance vessels of the splanchnic vasculature, is one of the defects identified during IBD and in mouse models of IBD. We hypothesized that this may be a consequence of upregulated catabolism of ATP during colitis. In vivo and in vitro video microscopy techniques were employed to measure the effects of purinergic agonists and inhibitors of CD39, an enzyme responsible for extracellular ATP catabolism, on the diameter of colonic submucosal arterioles from control mice and mice with dextran sodium sulfate [DSS, 5% (wt/vol)] colitis. Using a luciferase-based ATP assay, we examined the degradation of ATP and utilized real-time PCR, Western blotting, and immunohistochemistry to examine the expression and localization of CD39 during colitis. Arterioles from mice with DSS colitis did not constrict in response to ATP (10 microM) but did constrict in the presence of its nonhydrolyzable analog alpha,beta-methylene ATP (1 microM). alpha,beta-Methylene ADP (100 microM), an inhibitor of CD39, restored ATP-induced vasoconstriction in arterioles from mice with DSS-induced colitis. CD39 protein and mRNA expression was markedly increased during colitis. Immunohistochemical analysis demonstrated that, in addition to vascular CD39, F4/80-immunoreactive macrophages accounted for a large proportion of submucosal CD39 staining during colitis. These data implicate upregulation of CD39 in impaired sympathetic regulation of gastrointestinal blood flow during colitis.

Imbalanced protease expression and activities may contribute to the development of cancers, including neuroblastoma (NB). NB is a fatal childhood cancer of the sympathetic nervous system that frequently overexpresses mitogenic peptides, chemokines and their receptors. Dipeptidyl peptidase IV (DPPIV), a cell surface serine protease, inactivates or degrades some of these bioactive peptides and chemokines, thereby regulating cell proliferation and survival. Our studies show that DPPIV is expressed in normal neural crest-derived structures, including superior cervical and dorsal root ganglion cells, sciatic nerve, and in adrenal glands, but its expression is greatly decreased or lost in cells derived from NB, their malignant counterpart. Restoration of DPPIV expression in NB cells led to their differentiation in association with increased expression of the neural marker MAP2 and decreased expression of chemokines, including stromal-derived factor 1 (SDF1) and its receptor CXCR4. Furthermore, DPPIV promoted apoptosis, and inhibited SDF1-mediated in vitro cell migration and angiogenic potential. These changes were accompanied by caspase activation and decreased levels of phospho-Akt and MMP9 activity, which are downstream effectors of SDF1-CXCR4 signaling. Importantly, DPPIV suppressed the tumorigenic potential of NB cells in a xenotransplantation mouse model. These data support a potential role for DPPIV in inhibiting NB growth and progression.

Rho-kinase is involved in the pathogenesis of hypertension and left ventricular remodelling after myocardial infarction (MI). In an earlier study, we had demonstrated that Rho-kinase in the brainstem contributes to hypertensive mechanisms via the sympathetic nervous system; however, it is not known whether Rho-kinase in the brainstem also contributes to sympathetic nerve activation after MI. Male Institute of Cancer Research mice (8-10 weeks old) were used for the study. Two days before coronary artery occlusion (MI group), the left ventricular function was estimated by echocardiography. Following this, Y-27632 (0.5 mM, 0.25 microL/h), a specific Rho-kinase inhibitor, or a vehicle was intracisternally infused in the mice using an osmotic mini-pump. Nine days after coronary artery occlusion, we evaluated the 24-hour urinary norepinephrine excretion (U-NE) as a marker of sympathetic nerve activity. Ten days after coronary artery occlusion, we measured organ weight and evaluated Rho-kinase activity in the brainstem by measuring the amount of phosphorylated ezrin/radixin/moesin proteins, one of the substrates of Rho-kinase. The control group underwent a sham operation. Rho-kinase activity, U-NE, and lungs and liver weight were significantly greater in the MI group compared with the control group. Left ventricular size increased and percent fractional shortening decreased in the MI group compared with the control group. Y-27632 significantly decreased Rho-kinase activity and attenuated the increase in U-NE after MI. These results demonstrate that Rho-kinase is activated in the brainstem after MI and that the activation of this pathway is involved in the resulting enhanced sympathetic drive.

Integral cytophotometry was used to measure lactate dehydrogenase (LDH) and its H-and M-isoforms in neurons and satellite gliocytes in tissue sections from the cranial cervical sympathetic ganglion of the rabbit in normal conditions and after experimental partial and complete pharmacological blockade of nicotinic cholinoreceptors (n-CR). In normal conditions, both cell types showed both the H-and M-type isoforms, though the isoenzyme profiles differed--neurons showed a dominance of H-isoform activity while the M-isoform was more active in satellite gliocytes. In partial and complete blockade, the activity of LDH and its H-and M-isoforms decreased significantly in proportion to the number of blocked n-CR. In satellite gliocytes, increases in the extent of blockade were associated with decreases in the activity only of the M-isoform, while the activity of the H-isoform did not change. In partial blockade, the LDH isoenzyme profile of satellite gliocytes shifted towards the neuronal isoform, while in complete blockade there was no difference from the LDH isoenzyme profile of intact neurons. These data led to the suggestion that the formation of lactate in satellite gliocytes is induced by nicotinic cholinergic synapses directly involved in neuron-glial interactions and in controlling the activity of the LDH enzyme system in sympathetic neurons.

It was hypothesized that renal sympathetic nerve activity (RSNA) and neuronal nitric oxide synthase (nNOS) are involved in the acute inhibition of renin secretion and the natriuresis following slow NaCl loading (NaLoad) and that RSNA participates in the regulation of arterial blood pressure (MABP). This was tested by NaLoad after chronic renal denervation with and without inhibition of nNOS by S-methyl-thiocitrulline (SMTC). In addition, the acute effects of renal denervation on MABP and sodium balance were assessed. Rats were investigated in the conscious, catheterized state, in metabolic cages, and acutely during anesthesia. NaLoad was performed over 2 h by intravenous infusion of hypertonic solution (50 micromol.min(-1).kg body mass(-1)) at constant body volume conditions. SMTC was coinfused in amounts (20 microg.min(-1).kg(-1)) reported to selectively inhibit nNOS. Directly measured MABPs of acutely and chronically denervated rats were less than control (15% and 9%, respectively, P < 0.005). Plasma renin concentration (PRC) was reduced by renal denervation (14.5 +/- 0.2 vs. 19.3 +/- 1.3 mIU/l, P < 0.005) and by nNOS inhibition (12.4 +/- 2.3 vs. 19.6 +/- 1.6 mlU/l, P < 0.005). NaLoad reduced PRC (P < 0.05) and elevated MABP modestly (P < 0.05) and increased sodium excretion six-fold, irrespective of renal denervation and SMTC. The metabolic data demonstrated that renal denervation lowered sodium balance during the first days after denervation (P < 0.001). These data show that renal denervation decreases MABP and renin secretion. However, neither renal denervation nor nNOS inhibition affects either the renin down-regulation or the natriuretic response to acute sodium loading. Acute sodium-driven renin regulation seems independent of RSNA and nNOS under the present conditions.

This study investigated whether selective endothelin (ET) type A (ET(A)) or nonselective ET(A)/ET(B) receptor blockade exerted antiarrhythmic effects through attenuated sympathetic reinnervation after infarction. Twenty-four hours after ligation of the left anterior descending artery, male Wistar rats received either vehicle, ABT-627 (selective ET(A) receptor antagonist), bosentan (nonselective ET(A)/ET(B) receptor antagonist), or hydralazine for 4 wk. The measurement of myocardial ET-1 levels at the remote zone revealed a significant increase in vehicle-treated infarcted rats compared with sham-operated rats, consistent with increased activities of ET-1 after infarction. Sympathetic nerve function changes assessed by the norepinephrine content of myocardium and the dialysate and plasma dihydroxyphenylglycol levels were parallel to ET-1 levels. Immunohistochemical analysis for tyrosine hydroxylase, growth-associated protein 43, and neurofilament also confirmed the change of nerve function. This was accompanied with a significant upregulation of nerve growth factor protein expression and mRNA in the vehicle-treated infarcted rats, which reduced after the administration of either ET(A) or ET(A)/ET(B) blockade to a similar extent. The beneficial effects of ET receptor antagonists on sympathetic nerve function and structures were dissociated from their blood pressure-lowering effect because ET receptor antagonists and hydralazine reduced arterial pressure similarly. Arrhythmic severity during programmed stimulation in ET receptor antagonists-treated rats was significantly lower than that in vehicle-treated infarcted rats. Our data indicate that the ET system, especially via ET(A) receptors, plays an important role in attenuating sympathetic reinnervation after infarction. Independent of their hemodynamic effects, a chronic use of either ET(A) or ET(A)/ET(B) antagonists may modify the arrhythmogenic response to programmed electrical stimulation.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is an excitatory neuropeptide present in the rat brain stem. The extent of its localization within catecholaminergic groups and bulbospinal sympathoexcitatory neurons is not established. Using immunohistochemistry and in situ hybridization, we determined the extent of any colocalization with catecholaminergic and/or bulbospinal projections from the brain stem was determined. PACAP mRNA was found in tyrosine hydroxylase-immunoreactive (TH-ir) neurons in the C1-C3 cell groups. In the rostral ventrolateral medulla (RVLM), PACAP mRNA was found in 84% of the TH-ir neurons and 82% of bulbospinal TH-ir neurons. The functional significance of these PACAP mRNA positive bulbospinal neurons was tested by intrathecal administration of PACAP-38 in anaesthetized rats. Splanchnic sympathetic nerve activity doubled (110%) and heart rate rose significantly (19%), although blood pressure was unaffected. In addition, as previously reported, PACAP was found in the A1 cell group but not in the A5 cell group or in the locus coeruleus. The RVLM is the primary site responsible for the tonic and reflex control of blood pressure through the activity of bulbospinal presympathetic neurons, the majority of which contain TH. The results indicate 1) that pontomedullary neurons containing both TH and PACAP that project to the intermediolateral cell column originate from C1-C3 and not A5, and 2) intrathecal PACAP-38 causes a prolonged, sympathoexcitatory effect.

Although the presence of neurotrophin p75 receptor on sympathetic nerves is a well-recognised feature, there is still a scarcity of details of the distribution of the receptor on cerebrovascular nerves. This study examined the distribution of p75 receptor on perivascular sympathetic nerves of the middle cerebral artery and the basilar artery of healthy young rats using immunohistochemical methods at the laser confocal microscope and transmission electron microscope levels. Immunofluorescence methods of detection of tyrosine hydroxylase (TH) in sympathetic nerves, p75 receptor associated with the nerves, and also S-100 protein in Schwann cells were applied in conjunction with confocal microscopy, while the pre-embedding single and double immunolabelling methods (ExtrAvidin and immuno-gold-silver) were applied for the electron microscopic examination. Immunofluorescence studies revealed "punctuate" distribution of the p75 receptor on sympathetic nerves including accompanying Schwann cells. Image analysis of the nerves showed that the level of co-localization of p75 receptor and TH was low. Immunolabelling applied at the electron microscope level also showed scarce co-localization of TH (which was intra-axonal) and p75. Immunoreactivity for p75 receptor was present on the cell membrane of perivascular axons and to a greater extent on the processes of accompanying Schwann cells. Some Schwann cell processes were adjacent to each other displaying strong immunoreactivity for p75 receptor; immunoreactivity was located on the extracellular sites of the adjacent cell membranes suggesting that the receptor was involved in cross talk between these. It is likely that variability of locations of p75 receptor detected in the study reflects diverse interactions of p75 receptor with axons and Schwann cells. It might also imply a diverse role for the receptor and/or the plasticity of sympathetic cerebrovascular nerves to neurotrophin signalling.

Retrograde neuronal tracing and immunohistochemical methods were used to define the neurochemical content of sympathetic neurons projecting to the sow retractor clitoridis muscle (RCM). Differently from the other smooth muscles of genital organs, the RCM is an isolated muscle that is tonically contracted in the rest phase and relaxed in the active phase. This peculiarity makes it an interesting experimental model. The fluorescent tracer fast blue was injected into the RCM of three 50 kg subjects. After a one-week survival period, the ipsilateral paravertebral ganglion S1, that in a preliminary study showed the greatest number of cells projecting to the muscle, was collected from each animal. The co-existence of tyrosine hydroxylase with choline acetyltransferase, neuronal nitric oxide synthase, calcitonin gene-related peptide, leu-enkephalin, neuropeptide Y, substance P and vasoactive intestinal polypeptide was studied under a fluorescent microscope on cryostat sections. Tyrosine hydroxylase was present in about 58% of the neurons projecting to the muscle and was found to be co-localized with each of the other tested substances. Within fast blue-labelled cells negative to the adrenergic marker, small populations of neurons singularly containing each of the other enzymatic markers or peptides were also observed. The present study documents the complexity of the neurochemical interactions that regulate the activity of the smooth myocytes of the RCM and their vascular components.

The balance between norepinephrine (NE) synthesis, release, and reuptake is disrupted after acute myocardial infarction, resulting in elevated extracellular NE. Stimulation of sympathetic neurons in vitro increases NE synthesis and the synthetic enzyme tyrosine hydroxylase (TH) to a greater extent than it increases NE reuptake and the NE transporter (NET), which removes NE from the extracellular space. We used TGR(ASrAOGEN) transgenic rats, which lack postinfarct sympathetic hyperactivity, to test the hypothesis that increased cardiac sympathetic nerve activity accounts for the imbalance in TH and NET expression in these neurons after myocardial infarction. TH and NET mRNA levels were identical in the stellate ganglia of unoperated TGR(ASrAOGEN) rats compared with Sprague Dawley (SD) controls, but the threefold increase in TH and twofold increase in NET mRNA seen in the stellate ganglia of SD rats 1 wk after ischemia-reperfusion was absent in TGR(ASrAOGEN) rats. Similarly, the increase in TH and NET protein observed in the base of the SD ventricle was absent in the base of the TGR (ASrAOGEN) ventricle. Neuronal TH content was depleted in the left ventricle of both genotypes, whereas NET was unchanged. Basal heart rate and cardiac function were similar in both genotypes, but TGR(ASrAOGEN) hearts were more sensitive to the beta-agonist dobutamine. Tyramine-induced release of endogenous NE generated similar changes in ventricular pressure and contractility in both genotypes, but postinfarct relaxation was enhanced in TGR(ASrAOGEN) hearts. These data support the hypothesis that postinfarct sympathetic hyperactivity is the major stimulus increasing TH and NET expression in cardiac neurons.

Changes in the patterns of production and in the effects of signal substances may be involved in the development of tendinosis, a chronic condition of pain in human tendons. There is no previous information concerning the patterns of sympathetic innervation in the human patellar tendon. In this study, biopsies of normal and tendinosis patellar tendons were investigated with immunohistochemical methods, including the use of antibodies against tyrosine hydroxylase (TH) and neuropeptide Y, and against alpha1-, alpha2A-, and beta1-adrenoreceptors. It was noticed that most of the sympathetic innervation was detected in the walls of the blood vessels entering the tendon through the paratendinous tissue, and that the tendon tissue proper of the normal and tendinosis tendons was very scarcely innervated. Immunoreactions for adrenergic receptors were noticed in nerve fascicles containing both sensory and sympathetic nerve fibers. High levels of these receptors were also detected in the blood vessel walls; alpha1-adrenoreceptor immunoreactions being clearly more pronounced in the tendinosis tendons than in the tendons of controls. Interestingly, immunoreactions for adrenergic receptors and TH were noted for the tendon cells (tenocytes), especially in tendinosis tendons. The findings give a morphological correlate for the occurrence of sympathetically mediated effects in the patellar tendon and autocrine/paracrine catecholamine mechanisms for the tenocytes, particularly, in tendinosis. The observation of adrenergic receptors on tenocytes is interesting, as stimulation of these receptors can lead to cell proliferation, degeneration, and apoptosis, events which are all known to occur in tendinosis. Furthermore, the results imply that a possible source of catecholamine production might be the tenocytes themselves

Although sympathetic neurons are a well-studied model for neuronal apoptosis, the role of the apoptosome in activating caspases in these neurons remains debated. We find that the ability of sympathetic neurons to undergo apoptosis in response to nerve growth factor (NGF) deprivation is completely dependent on having an intact apoptosome pathway. Genetic deletion of Apaf-1, caspase-9, or caspase-3 prevents apoptosis after NGF deprivation, and importantly, allows these neurons to recover and survive long-term following readdition of NGF. The inability of caspase-3 deficient sympathetic neurons to undergo apoptosis is particularly striking, as apoptosis in dermal fibroblasts and cortical neurons proceeds even in the absence of caspase-3. Our results show that in contrast to dermal fibroblasts and cortical neurons, sympathetic neurons express no detectable levels of caspase-7. The strict requirement for an intact apoptosome, coupled with a lack of effector caspase redundancy, provides sympathetic neurons with a markedly increased control over their apoptotic pathway.

Gene transfer of neuronal nitric oxide synthase (nNOS) with nonspecific adenoviral vectors can cause promiscuous transduction. We provide direct evidence that nNOS targeted only to cardiac sympathetic neurons inhibits sympathetic neurotransmission. An adenovirus constructed with a noradrenergic neuron-specific promoter (PRSx8), driving nNOS or enhanced green fluorescence protein (eGFP) gene expression caused exclusive expression in tyrosine hydroxylase (TH) positive rat cardiac sympathetic neurons. There was no detectable leakage of transgene expression in other cell types in the preparation nor did the transgene express in choline acetyltransferase (CHAT)-positive intracardiac cholinergic ganglia. Functionally, Ad.PRS-nNOS gene transfer increased nNOS activity and significantly reduced norephinephrine release evoked by field stimulation of isolated right atria. These effects were reversed by the NOS inhibitor N(omega)-Nitro-L-arginine. Our results demonstrate that noradrenergic cell-specific gene transfer with nNOS can inhibit cardiac sympathetic neurotransmission. This targeted technique may provide a novel method for reducing presynaptic sympathetic hyperactivity.

The enzyme nitric oxide synthase (NOS) which is necessary for the production of nitric oxide from L-arginine exists in three isoforms: neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). Our previous studies have demonstrated the roles of nNOS and eNOS within the rostral (RVLM) and caudal ventrolateral medulla (CVLM) in modulating cardiovascular responses during static skeletal muscle contraction via altering localized glutamate and GABA levels (Brain Res. 977 (2003) 80-89; Neuroscience Res. 52 (2005) 21-30). In this study, we investigated the role of iNOS within the RVLM and CVLM on cardiovascular responses and glutamatergic/GABAergic neurotransmission during the exercise pressor reflex. Bilateral microdialysis of a selective iNOS antagonist, aminoguanidine (AGN; 1.0 microM), for 60 min into the RVLM attenuated increases in mean arterial pressure (MAP), heart rate (HR), and extracellular glutamate levels during a static muscle contraction. Levels of GABA within the RVLM were increased. After 120 min of discontinuation of the drug, MAP and HR responses and glutamate/GABA concentrations recovered to baseline values during a subsequent muscle contraction. In contrast, bilateral application of AGN (1.0 microM) into CVLM potentiated cardiovascular responses and glutamate concentration while attenuating levels of GABA during a static muscle contraction. All values recovered after 120 min of discontinuation of the drug. These results demonstrate that iNOS within the ventrolateral medulla plays an important role in modulating cardiovascular responses and glutamatergic/GABAergic neurotransmission that regulates the exercise pressor reflex.

The contribution of nitric oxide (NO) to the antinatriuresis and antidiuresis caused by low-level electrical stimulation of the renal sympathetic nerves (RNS) was investigated in rats anaesthetized with chloralose-urethane. Groups of rats, n= 6, were given i.v. infusions of vehicle, l-NAME (10 microg kg(-1) min(-1)), 1400W (20 microg kg(-1) min(-1)), or S-methyl-thiocitrulline (SMTC) (20 microg kg(-1) min(-1)) to inhibit NO synthesis non-selectively or selectively to block the inducible or neuronal NOS isoforms (iNOS and nNOS, respectively). Following baseline measurements of blood pressure (BP), renal blood flow (RBF), glomerular filtration rate (GFR), urine flow (UV) and sodium excretion (U(Na)V), RNS was performed at 15 V, 2 ms duration with a frequency between 0.5 and 1.0 Hz. RNS did not cause measurable changes in BP, RBF or GFR in any of the groups. In untreated rats, RNS decreased UV and U(Na)V by 40-50% (both P < 0.01), but these excretory responses were prevented in l-NAME-treated rats. In the presence of 1400W i.v., RNS caused reversible reductions in both UV and U(Na)V of 40-50% (both P < 0.01), while in SMTC-treated rats, RNS caused an inconsistent fall in UV, but a significant reduction (P < 0.05) in U(Na)V of 21%. These data demonstrated that the renal nerve-mediated antinatriuresis and antidiuresis was dependent on the presence of NO, generated in part by nNOS. The findings suggest that NO importantly modulates the neural control of fluid reabsorption; the control may be facilitatory at a presynaptic level but inhibitory on tubular reabsorptive processes.

Unlike the thoracic and lumbar sympathetic nervous systems with paravertebral ganglions in individual spinal segments, the cervical sympathetic nervous system lacks segmental structures corresponding to the spinal segments and only three ganglions, namely the upper and middle cervical ganglions and the stellate ganglion, are present. Single axons have been observed in the ganglions using an anterograde-labeling method to analyze their expansion in order to investigate the relationship between the cervical sympathetic ganglions and the spinal cord in rats. Although segmental structures were not confirmed in the upper cervical ganglion, segmental structures were demonstrated in the stellate ganglion. Next, it was determined that some sympathetic preganglionic neurons, nitric oxide synthetase-positive preganglionic neurons, form dense nerve endings on the upper cervical ganglion neurons that project onto organs closely related to glandular secretion in the head and neck region. Finally, the relationship between the cell body size of upper cervical ganglion neurons and the size of the target was investigated for the three major salivary glands in rats and it was determined that no direct relationship was present.

Existing evidence led us to hypothesize that increases in p85alpha, a regulatory subunit of PI3-kinase, in presympathetic brain areas contribute to hypertension. PI3-kinase p85alpha, p110alpha, and p110delta mRNA was 1.5- to 2-fold higher in the paraventricular nucleus (PVN) of spontaneously hypertensive rats (SHR) compared with their controls, Wistar Kyoto rats (WKY). The increase in p85alpha/p110delta was attenuated in SHR treated with captopril, an angiotensin (Ang)-converting enzyme inhibitor, from in utero to 6 months of age. In the rostral ventrolateral medulla (RVLM), p110delta mRNA was approximately 2-fold higher in SHR than in WKY. Moreover, the increases in mRNA were associated with higher PI3-kinase activity in both nuclei. The functional relevance was studied in neuronal cultures because SHR neurons reflect the augmented p85alpha mRNA and PI3-kinase activity. Expression of a p85 dominant-negative mutant decreased norepinephrine (NE) transporter mRNA and [3H]NE uptake by approximately 60% selectively in SHR neurons. In summary, increased p85alpha/p110delta expression in the PVN and RVLM is associated with increased PI3-kinase activity in the SHR. Furthermore, normalized PI3-kinase p85alpha/p110delta expression within the PVN might contribute to the overall effect of captopril, perhaps attributable to a consequent decrease in NE availability.

Object: The present study was undertaken to elucidate the extent and precise distribution of the postganglionic sympathetic fibers in the cranial nerves projecting to the orbit and to reconstruct sympathetic routes in the orbit in humans. For this purpose, the authors made an immunohistochemical determination of the sympathetic fibers by using an antibody against norepinephrine-synthetic enzyme, tyrosine hydroxylase (TH)., Methods: Specimens containing the orbit and the cavernous sinus were obtained from formalin-fixed human cadavers. First, it was confirmed that the superior cervical ganglion contained strongly immunostained TH-positive neuronal cell bodies and fibers. After careful dissection of the cranial nerves projecting to the orbit, different segments of each cranial nerve were processed for immunohistochemical analysis for TH. All of the intraorbital cranial nerves contained TH-positive sympathetic fibers, although the amounts were very different in each cranial nerve. At the proximal site of the common tendinous ring, TH-positive fibers were found mainly in the abducent and trochlear nerves. At the distal site of this ring, TH-positive fibers were lost or markedly reduced in number in the abducent and trochlear nerves and were distributed mostly in the ophthalmic and oculomotor nerves. Among the cranial nerves projecting to the orbit, the ophthalmic nerve and its bifurcated nerves--frontal, lacrimal, and nasociliary--contained numerous TH-positive fibers., Conclusions: The authors conclude that the postganglionic sympathetic fibers are distributed to all cranial nerves projecting to the orbit and that the ophthalmic nerve provides a major sympathetic route in the orbital cavity in humans.

To clarify the paradoxic effects of cerulenin, namely its in vitro inhibitory effects on fat catabolism and its in vivo reduction of fat mass, we studied the in vivo and in vitro effects of cerulenin on carnitine palmitoyltransferase-1 (CPT-1) activity, the rate-limiting enzyme of fatty acid oxidation. A single ip injection of cerulenin significantly reduced body weight and increased core temperature without significantly reducing food intake. In situ hybridization study revealed that a single injection of cerulenin did not affect the expression of orexigenic neuropeptide mRNA. Cerulenin's effect on CPT-1 activity was biphasic in the liver and muscle: early suppression during the first 1 h and late stimulation in the 3-5 h after ip treatment. In vitro cerulenin treatment reduced CPT-1 activity, which was overcome by cotreating with catecholamine. Intracerebroventricular injection of cerulenin increased CPT-1 activity significantly in soleus muscle, and this effect was sustained for up to 3 h. Pretreatment with alpha-methyl-p-tyrosine inhibited the cerulenin-induced increase in core temperature and the late-phase stimulating effect of cerulenin on CPT-1 activity. In adrenalectomized mice, cerulenin also increased the activity. In vivo cerulenin treatment enhanced muscle CPT-1 activity in monosodium glutamate-treated arcuate nucleus lesioned mice but not in gold thioglucose-treated ventromedial hypothalamus lesioned mice. These findings suggest that cerulenin-induced late-phase stimulating effects on CPT-1 activity and energy expenditure is mediated by the activation of innervated sympathetic nervous system neurons through the firing of undefined neurons of the ventromedial hypothalamus, rather than the arcuate nucleus.

NGF activates several signaling cascades in sympathetic neurons. We examined how activation of one of these cascades, the ERK/MAP (extracellular signal-regulated kinase/mitogen-activated protein) kinase pathway, affects dendritic growth in these cells. Dendritic growth was induced by exposure to NGF and BMP-7 (bone morphogenetic protein-7). Exposure to NGF increased phosphorylation of ERK1/2. Unexpectedly, two MEK (MAP kinase kinase) inhibitors (PD 98059 and U 0126) enhanced dendritic growth, and a ligand, basic FGF, that activates the ERK pathway inhibited the growth of these processes. The enhancement of dendritic growth by PD 98059 was associated with an increase in the number of axo-dendritic synapses, and it appeared to represent a specific morphogenic effect because neither axonal growth nor cell survival was affected. In addition, increased dendritic growth was not observed after exposure to inhibitors of other signaling pathways, including the phosphatidylinositol-3-kinase inhibitor LY 294002. Dendritic growth was also increased in cells transfected with dominant-negative mutants of MEK1 and ERK2 but not with dominant-negative mutants of MEK5 and ERK5, suggesting that ERK1/2 is the primary mediator of this effect. Exposure to BMP-7 induces nuclear translocation of Smad1 (Sma- and Mad-related protein 1), and PD 98059 treatment potentiated nuclear accumulation of Smad-1 induced by BMP-7 in sympathetic neurons, suggesting a direct enhancement of BMP signaling in cells treated with an MEK inhibitor. These observations indicate that one of the signaling cascades activated by NGF can act in an antagonistic manner in sympathetic neurons and reduce the dendritic growth induced by other NGF-sensitive pathways.