nNOS Activity Research Articles

Design and synthesis of benzo[d]thiazol-2-yl-methyl-4-(substituted)-piperazine-1-carbothioamide as novel neuronal nitric oxide inhibitors and evaluation of their neuroprotecting effect in 6-OHDA-induced unilateral lesioned rat model of Parkinson's disease

Neuronal nitric oxide synthase (nNOS) is an enzyme constitutively expressed in the mammalian brain and skeletal muscles. The excessive activation of nNOS in the neurons results in oxidative and nitrosative stress associated with neuronal loss in various neurological disorders. Several nNOS inhibitors have been reported to limit the excessive activation of nNOS. In the present work, we have designed and carried the synthesis of benzo[d]thiazol-2-yl-methyl-4-(substituted)-piperazine-1-carbothioamide as novel neuronal nitric oxide inhibitors (5–28, twenty-four compounds). Stably transfected HEK 293 cells expressing NOS isoforms treated with the compounds (5–28) showed that the eight compounds exhibited > 95% cell survival in the MTT assay. nNOS inhibition assay of the eight compounds illustrated that the compound 18 was most selective for nNOS (nNOS=66.73 ± 1.51; eNOS=28.70 ± 1.39; iNOS =13.26 ± 1.01) in HEK 293 cells expressing NOS isoforms. 6-OHDA-induced unilaterally lesioned rats treated with the compound 18 showed the improvement in motor and non-motor functions. Furthermore, the compound 18 showed the increased levels of dopamine and decreased levels of glutamate and nitrite ions in the isolated rat brain. In the docking analysis, the compound 18 showed the significant binding affinity with the nNOS binding site (the ∆G value = - 9.0 kcal/mol). Overall results demonstrated that the N-(benzo[d]thiazol-2-ylmethyl)-4-(4-nitrophenyl) piperazine-1-carbothioamide (the compound 18) possessed significant nNOS inhibiting activity and neuroprotecting potential in 6-OHDA-induced unilaterally lesioned rat model of PD and more work will be required to establish the role of the compound 18 in the therapy of PD and other neurodegenerative disorders.

Dual effect of amitriptyline in the control of vascular tone: Direct blockade of calcium channel in smooth muscle cells and reduction of TLR4-dependent NO production in endothelial cells

Background and purposeAmitriptyline (AM) is a classical and typical tricyclic antidepressant drug. Despite its well-known effects on the nervous system, it has been described to work as a TLR4 antagonist and several clinical works suggested some unexpected cardiovascular effects. The role of amitriptyline on vascular tone is not clear, thus we hypothesized that amitriptyline has a double effect on vascular tone by both endothelial TLR4-dependent nitric oxide down-regulation and calcium channel blockade in smooth muscle cells. Experimental approachChanges in isometric tension were recorded on a wire myograph. NO production was evaluated by fluorescence microscopy and flow cytometry in the mouse aorta and EAhy926 cells using DAF fluorescence intensity. Calcium influx was evaluated in A7r5 cells by flow cytometry. Western blot was used to analyze eNOS and nNOS phosphorylation. Key resultsAM reduced PE-induced contraction by calcium influx diminution in smooth muscle cells (F/F0 = 225.6 ± 15.9 and 118.6 ± 17.6 to CT and AM, respectively). AM impaired Ach-dependent vasodilation (Emax = 95.8 ± 1.4; 78.1 ± 1.8; 60.4 ± 2.9 and −7.4 ± 1.0 for CT, 0.01, 0,1 and 1 μmol/L AM, respectively) through reduction of calcium influx and NO availability and TLR4 antagonism in a concentration-dependent manner. AM or TLR4 gene deletion significantly reduced NO production (Fluorescence = 9503 ± 871.7, 2561 ± 282, 4771 ± 728 and 1029 ± 103 to CT, AM, TLR4−/− and AM + TLR4−/−, respectively) by an increase in nNOSser852 and reduction in eNOSser1177 phosphorylation in endothelial cells. Conclusions and implicationsOur data show that amitriptyline impaired vascular function through two different mechanisms: blockade of TLR4 in endothelial cells and consequent decrease in NO production and calcium influx reduction in smooth muscle and endothelial cells. We also suggest, for the first time, nNOS activity reduction by AM in non-neuronal cells.

Neuronal nitric oxide synthases in the pathogenesis of metabolic syndrome

The study of the molecular mechanisms of metabolic syndrome (MS) and its complications are among the most acute problems of modern endocrinology. Functional changes in the expression, activity, and regulatory properties of neuronal NO synthase (nNOS), which catalyzes the formation of the most important secondary mediator, nitric oxide (NO), and its dependent NO/cGMP signaling pathways in the brain, myocardium, and skeletal muscles, play a key role among the molecular causes of MS. In the brain, nNOS is associated with NMDA receptors, the hyperactivation of which in MS leads to excessive stimulation of nNOS and hyperproduction of NO, which leads to NO-induced damage to neurons and disruption of the central regulation of physiological processes and neurodegeneration. In the myocardium with MS, there are changes in the expression and localization of nNOS, as well as its functional interaction with cytoskeletal proteins, which leads to disorders of myocardial contraction and hypertrophy. In skeletal muscles, nNOS controls their contraction, oxidative metabolism, is involved in the regulation of vascular relaxation, and also participates in the regulation of glucose transport. A decrease in the expression and activity of nNOS, as well as dysregulation of its activity in MS, cause disturbances of these processes and make a significant contribution to the development of insulin resistance and deterioration of glucose homeostasis. Thus, nNOS can be considered an important therapeutic target in the treatment of MS and other metabolic disorders, as well as to prevent their complications from the nervous and cardiovascular systems and the musculoskeletal system.

Caveolin 3 suppresses phosphorylation-dependent activation of sarcolemmal nNOS

Mutations of the caveolin 3 gene cause autosomal dominant limb-girdle muscular dystrophy (LGMD)1C. In mice, overexpression of mutant caveolin 3 leads to loss of caveolin 3 and results in myofiber hypotrophy in association with activation of neuronal nitric oxide synthase (nNOS) at the sarcolemma. Here, we show that caveolin 3 directly bound to nNOS and suppressed its phosphorylation-dependent activation at a specific residue, Ser1412 in the nicotinamide adenine dinucleotide phosphate (NADPH)−flavin adenine dinucleotide (FAD) module near the C-terminus of the reduction domain, in vitro. Constitutively active nNOS enhanced myoblast fusion, but not myogenesis, in vitro. Phosphorylation-dependent activation of nNOS occurred in muscles from caveolin 3-mutant mice and LGMD1C patients. Mating with nNOS-mutant mice exacerbated myofiber hypotrophy in the caveolin 3-mutant mice. In nNOS-mutant mice, regenerating myofibers after cardiotoxin injury became hypotrophic with reduced myoblast fusion. Administration of NO donor increased myofiber size and the number of myonuclei in the caveolin 3-mutant mice. Exercise also increased myofiber size accompanied by phosphorylation-dependent activation of nNOS in wild-type and caveolin 3-mutant mice. These data indicate that caveolin 3 inhibits phosphorylation-dependent activation of nNOS, which leads to myofiber hypertrophy via enhancing myoblast fusion. Hypertrophic signaling by nNOS phosphorylation could act in a compensatory manner in caveolin 3-deficient muscles.

The argininosuccinate synthetase can differentially regulate nitric oxide synthase in yellow catfish Pelteobagrus fulvidraco

Fish are at high risk of exposure to ammonia in aquaculture systems. When ammonia stress occurs, fish are more prone to disease outbreaks, but the mechanism is not very clear. The argininosuccinate synthetase (ASS) plays an important role in the regulation of urea synthesis and nitric oxide synthesis. We speculated that there must be some relationship between ASS expression and disease outbreak. In this study, ASS was cloned from the yellow catfish. The full-length cDNAs of ASS was 1558 bp, with open reading frames of 1236 bp. The mRNA expression of ASS gene was the highest in liver, kidney and brain. This study consists of two parts: 1) For ammonia challenge in vivo, yellow catfish (15.00 ± 1.50 g) were divided into control group, low ammonia group (1/10 96 h LC50), and high ammonia group (1/2 96 h LC50). The experiment continued for 192 h. The results showed that ammonia stress elevated serum ammonia content, and inhibited urea synthesis enzymes activities but up-regulated the expression levels of related genes except ARG, and induced arginine accumulation and nitric oxide synthase (nNOS and iNOS) different expression, and decreased resistance to Aeromonas hydrophage; 2) For ammonia challenge in vitro, the primary culture of liver cell was divided into four groups: control group, BPP group (Bj-BPP-10c was added as ASS activator), Amm group (96 h LC50), and Amm + BPP group. The experiment continued for 96 h. The results showed that the Bj-BPP-10c can inhibit nNOS activity and improve cell survival rate, and enhance iNOS activity and immune response (lysozyme, complement, respiratory burst, and phagocytic index) by activate ASS when ammonia stress occurred. Our results indicated that targeted regulation of ASS can improve iNOS activity, and enhance the immune response of yellow catfish under ammonia stress.

Neuronal Nitric Oxide Synthase Suppression Confers the Prolonged Analgesic Effect of Sciatic Nerve Block With Perineural Dexamethasone in Postoperative Pain Model Mice

Dexamethasone supplementation to local anesthetics prolongs its action, yet the underlying mechanism is unclear. Previous studies have reported that increased p-p38 mitogen-activated protein kinase (MAPK) in the dorsal root ganglia (DRG) is associated with pain-associated behavior and that nitric oxide (NO), which is known to be a pronociceptive substance, directly inhibits sciatic nerve conduction. Here, we investigated the temporal changes in the hyperalgesic effect and p-p38 MAPK and NO synthase (NOS) expression levels in the DRG when dexamethasone was added to ropivacaine used for a sciatic nerve block (SNB) in postoperative pain model mice. Dexamethasone supplementation to ropivacaine significantly prolonged the analgesic effect of SNB via glucocorticoid receptor activation. Histological examination revealed that ropivacaine suppressed p-p38 MAPK expression in the DRG regardless of dexamethasone supplementation, suggesting that p-p38 MAPK was not involved in the prolonging effect of dexamethasone on nerve block. Contrastingly, plantar incision markedly increased the expression of neuronal NOS (nNOS) in DRG, and dexamethasone supplementation to ropivacaine significantly suppressed nNOS expression. Supplementation of L-NAME, an inhibitor of NOS, to ropivacaine markedly prolonged the effect of SNB, similar to dexamethasone. These results suggest that dexamethasone supplementation to local anesthetics prolongs the analgesic effect by inhibiting nNOS activity. PerspectiveThe current study revealed that dexamethasone supplementation to local anesthetics prolongs the analgesic effect by inhibiting the activity of neuronal NOS and that p-p38 MAPK may not be involved in this phenomenon. Our findings offer a new target for the discovery of long-acting local anesthetics.

Active nNOS Is Required for Grp94-Induced Antioxidant Cytoprotection: A Lesson from Myogenic to Cancer Cells.

The endoplasmic reticulum (ER) chaperone Grp94/gp96 appears to be involved in cytoprotection without being required for cell survival. This study compared the effects of Grp94 protein levels on Ca2+ homeostasis, antioxidant cytoprotection and protein–protein interactions between two widely studied cell lines, the myogenic C2C12 and the epithelial HeLa, and two breast cancer cell lines, MDA-MB-231 and HS578T. In myogenic cells, but not in HeLa, Grp94 overexpression exerted cytoprotection by reducing ER Ca2+ storage, due to an inhibitory effect on SERCA2. In C2C12 cells, but not in HeLa, Grp94 co-immunoprecipitated with non-client proteins, such as nNOS, SERCA2 and PMCA, which co-fractionated by sucrose gradient centrifugation in a distinct, medium density, ER vesicular compartment. Active nNOS was also required for Grp94-induced cytoprotection, since its inhibition by L-NNA disrupted the co-immunoprecipitation and co-fractionation of Grp94 with nNOS and SERCA2, and increased apoptosis. Comparably, only the breast cancer cell line MDA-MB-231, which showed Grp94 co-immunoprecipitation with nNOS, SERCA2 and PMCA, increased oxidant-induced apoptosis after nNOS inhibition or Grp94 silencing. These results identify the Grp94-driven multiprotein complex, including active nNOS as mechanistically involved in antioxidant cytoprotection by means of nNOS activity and improved Ca2+ homeostasis.

Inhibition of nNOS in the paraventricular nucleus of hypothalamus decreases exercise-induced hyperthermia

The paraventricular nucleus of the hypothalamus (PVN) is an important site for autonomic control, which integrates thermoregulation centers and sympathetic outflow to thermoeffector organs. PVN neurons express the neuronal isoform of nitric oxide synthase (nNOS) whose expression is locally upregulated by physical exercise. Thus, the aim of the present study was to evaluate the role of nNOS in the PVN in the exercise-induced hyperthermia. Seven days after surgery, male Wistar rats received bilateral intra-PVN microinjections of the selective nNOS inhibitor Nw-Propyl-L-Arginine (NPLA) or vehicle (saline) and were submitted to an acute progressive exercise session on a treadmill until fatigue. Abdominal and tail skin temperature (Tabd and Ttail, respectively) were measured, and the threshold (Hthr; °C) and sensitivity (Hsen) for heat dissipation calculated. Performance variables were also collected. During the progressive exercise protocol, all animals displayed an increase in the Tabd. However, compared to vehicle group, the microinjection of NPLA in the PVN attenuated the exercise-induced hyperthermia. There was no difference in Ttail or Hthr between NPLA and control rats. In contrast, Hsen was increased in the NPLA group compared to vehicle. In addition, heat storage was lower in NPLA-treated animals. Despite the temperature differences, inhibition of nNOS in the PVN did not affect running performance on the treadmill. These results suggest that nitrergic signaling within the PVN, under nNOS activation, drives the increase of body temperature, being necessary for the proper thermal regulatory mechanisms during progressive exercise-induced hyperthermia.

Nitrergic modulation of neuronal excitability in the mouse hippocampus is mediated via regulation of Kv2 and voltage-gated sodium channels.

Regulation of neuronal activity is a necessity for communication and information transmission. Many regulatory processes which have been studied provide a complex picture of how neurons can respond to permanently changing functional requirements. One such activity-dependent mechanism involves signaling mediated by nitric oxide (NO). Within the brain, NO is generated in response to neuronal NO synthase (nNOS) activation but NO-dependent pathways regulating neuronal excitability in the hippocampus remain to be fully elucidated. This study was set out to systematically assess the effects of NO on ion channel activities and intrinsic excitabilities of pyramidal neurons within the CA1 region of the mouse hippocampus. We characterized whole-cell potassium and sodium currents, both involved in action potential (AP) shaping and propagation and determined NO-mediated changes in excitabilities and AP waveforms. Our data describe a novel signaling by which NO, in a cGMP-independent manner, suppresses voltage-gated Kv2 potassium and voltage-gated sodium channel activities, thereby widening AP waveforms and reducing depolarization-induced AP firing rates. Our data show that glutathione, which possesses denitrosylating activity, is sufficient to prevent the observed nitrergic effects on potassium and sodium channels, whereas inhibition of cGMP signaling is also sufficient to abolish NO modulation of sodium currents. We propose that NO suppresses both ion channel activities via redox signaling and that an additional cGMP-mediated component is required to exert effects on sodium currents. Both mechanisms result in a dampened excitability and firing ability providing new data on nitrergic activities in the context of activity-dependent regulation of neuronal function following nNOS activation.

Alcohol hangover induces nitric oxide metabolism changes by impairing NMDA receptor-PSD95-nNOS pathway

Alcohol hangover is defined as the combination of mental and physical symptoms experienced the day after a single episode of heavy drinking, starting when blood alcohol concentration approaches zero. We previously evidenced increments in free radical generation and an imbalance in antioxidant defences in non-synaptic mitochondria and synaptosomes during hangover. It is widely known that acute alcohol exposure induces changes in nitric oxide (NO) production and blocks the binding of glutamate to NMDAR in central nervous system. Our aim was to evaluate the residual effect of acute ethanol exposure (hangover) on NO metabolism and the role of NMDA receptor-PSD95-nNOS pathway in non-synaptic mitochondria and synaptosomes from mouse brain cortex. Results obtained for the synaptosomes fraction showed a 37% decrease in NO total content, a 36% decrease in NOS activity and a 19% decrease in nNOS protein expression. The in vitro addition of glutamate to synaptosomes produced a concentration-dependent enhancement of NO production which was significantly lower in samples from hangover mice than in controls for all the glutamate concentrations tested. A similar patter of response was observed for nNOS activity being decreased both in basal conditions and after glutamate addition. In addition, synaptosomes exhibited a 64% and 15% reduction in NMDA receptor subunit GluN2B and PSD-95 protein expression, respectively. Together with this, glutamate-induced calcium entry was significant decreased in synaptosomes from alcohol-treated mice. On the other hand, in non-synaptic mitochondria, no significant differences were observed in NO content, NOS activity or nNOS protein expression. The expression of iNOS remained unaltered in synaptosomes and non-synaptic mitochondria. Here we demonstrated that hangover effects on NO metabolism are strongly evidenced in synaptosomes probably due to a disruption in NMDAR/PSD-95/nNOS pathway.

Serine/Threonine Phosphatases in LTP: Two B or Not to Be the Protein Synthesis Blocker-Induced Impairment of Early Phase.

Dephosphorylation of target proteins at serine/threonine residues is one of the most crucial mechanisms regulating their activity and, consequently, the cellular functions. The role of phosphatases in synaptic plasticity, especially in long-term depression or depotentiation, has been reported. We studied serine/threonine phosphatase activity during the protein synthesis blocker (PSB)-induced impairment of long-term potentiation (LTP). Established protein phosphatase 2B (PP2B, calcineurin) inhibitor cyclosporin A prevented the LTP early phase (E-LTP) decline produced by pretreatment of hippocampal slices with cycloheximide or anisomycin. For the first time, we directly measured serine/threonine phosphatase activity during E-LTP, and its significant increase in PSB-treated slices was demonstrated. Nitric oxide (NO) donor SNAP also heightened phosphatase activity in the same manner as PSB, and simultaneous application of anisomycin + SNAP had no synergistic effect. Direct measurement of the NO production in hippocampal slices by the NO-specific fluorescent probe DAF-FM revealed that PSBs strongly stimulate the NO concentration in all studied brain areas: CA1, CA3, and dentate gyrus (DG). Cyclosporin A fully abolished the PSB-induced NO production in the hippocampus, suggesting a close relationship between nNOS and PP2B activity. Surprisingly, cyclosporin A alone impaired short-term plasticity in CA1 by decreasing paired-pulse facilitation, which suggests bi-directionality of the influences of PP2B in the hippocampus. In conclusion, we proposed a minimal model of signaling events that occur during LTP induction in normal conditions and the PSB-treated slices.

Positive Correlation between nNOS and Stress-Activated Bowel Motility Is Confirmed by In Vivo HiBiT System.

Neuronal nitric oxide synthase (nNOS) has various roles as a neurotransmitter. However, studies to date have produced insufficient data to fully support the correlation between nNOS and bowel motility. This study aimed to investigate the correlation between nNOS expression and gastrointestinal (GI) tract motility using a stress-induced neonatal maternal separation (NMS) mouse model. In this study, we generated a genetically modified mouse with the HiBiT sequence knock-in into the nNOS gene using CRISPR/Cas9 for analyzing accurate nNOS expression. nNOS expression was measured in the stomach, small intestine, large intestine, adrenal gland, and hypothalamus tissues after establishing the NMS model. The NMS model exhibited a significant increase in nNOS expression in large intestine, adrenal gland, and hypothalamus. Moreover, a significant positive correlation was observed between whole gastrointestinal transit time and the expression level of nNOS. We reasoned that NMS induced chronic stress and consequent nNOS activation in the hypothalamic-pituitary-adrenal (HPA) axis, and led to an excessive increase in intestinal motility in the lower GI tract. These results demonstrated that HiBiT is a sensitive and valuable tool for analyzing in vivo gene activation, and nNOS could be a biomarker of the HPA axis-linked lower intestinal tract dysfunction.

MTOR Attenuation with Rapamycin Reverses Neurovascular Uncoupling and Memory Deficits in Mice Modeling Alzheimer's Disease.

Vascular dysfunction is a universal feature of aging and decreased cerebral blood flow has been identified as an early event in the pathogenesis of Alzheimer's disease (AD). Cerebrovascular dysfunction in AD includes deficits in neurovascular coupling (NVC), a mechanism that ensures rapid delivery of energy substrates to active neurons through the blood supply. The mechanisms underlying NVC impairment in AD, however, are not well understood. We have previously shown that mechanistic/mammalian target of rapamycin (mTOR) drives cerebrovascular dysfunction in models of AD by reducing the activity of endothelial nitric oxide synthase (eNOS), and that attenuation of mTOR activity with rapamycin is sufficient to restore eNOS-dependent cerebrovascular function. Here we show mTOR drives NVC impairments in an AD model through the inhibition of neuronal NOS (nNOS)- and non-NOS-dependent components of NVC, and that mTOR attenuation with rapamycin is sufficient to restore NVC and even enhance it above WT responses. Restoration of NVC and concomitant reduction of cortical amyloid-β levels effectively treated memory deficits in 12-month-old hAPP(J20) mice. These data indicate that mTOR is a critical driver of NVC dysfunction and underlies cognitive impairment in an AD model. Together with our previous findings, the present studies suggest that mTOR promotes cerebrovascular dysfunction in AD, which is associated with early disruption of nNOS activation, through its broad negative impact on nNOS as well as on non-NOS components of NVC. Our studies highlight the potential of mTOR attenuation as an efficacious treatment for AD and potentially other neurologic diseases of aging.SIGNIFICANCE STATEMENT Failure of the blood flow response to neuronal activation [neurovascular coupling (NVC)] in a model of AD precedes the onset of AD-like cognitive symptoms and is driven, to a large extent, by mammalian/mechanistic target of rapamycin (mTOR)-dependent inhibition of nitric oxide synthase activity. Our studies show that mTOR also drives AD-like failure of non-nitric oxide (NO)-mediated components of NVC. Thus, mTOR attenuation may serve to treat AD, where we find that neuronal NO synthase is profoundly reduced early in disease progression, and potentially other neurologic diseases of aging with cerebrovascular dysfunction as part of their etiology.

BH4 Increases nNOS Activity and Preserves Left Ventricular Function in Diabetes

In diabetic patients, heart failure with predominant left ventricular (LV) diastolic dysfunction is a common complication for which there is no effective treatment. Oxidation of the NOS (nitric oxide synthase) cofactor tetrahydrobiopterin (BH4) and dysfunctional NOS activity have been implicated in the pathogenesis of the diabetic vascular and cardiomyopathic phenotype. Using mice models and human myocardial samples, we evaluated whether and by which mechanism increasing myocardial BH4 availability prevented or reversed LV dysfunction induced by diabetes. In contrast to the vascular endothelium, BH4 levels, superoxide production, and NOS activity (by liquid chromatography) did not differ in the LV myocardium of diabetic mice or in atrial tissue from diabetic patients. Nevertheless, the impairment in both cardiomyocyte relaxation and [Ca2+]i (intracellular calcium) decay and in vivo LV function (echocardiography and tissue Doppler) that developed in wild-type mice 12 weeks post-diabetes induction (streptozotocin, 42-45 mg/kg) was prevented in mGCH1-Tg (mice with elevated myocardial BH4 content secondary to trangenic overexpression of GTP-cyclohydrolase 1) and reversed in wild-type mice receiving oral BH4 supplementation from the 12th to the 18th week after diabetes induction. The protective effect of BH4 was abolished by CRISPR/Cas9-mediated knockout of nNOS (the neuronal NOS isoform) in mGCH1-Tg. In HEK (human embryonic kidney) cells, S-nitrosoglutathione led to a PKG (protein kinase G)-dependent increase in plasmalemmal density of the insulin-independent glucose transporter GLUT-1 (glucose transporter-1). In cardiomyocytes, mGCH1 overexpression induced a NO/sGC (soluble guanylate cyclase)/PKG-dependent increase in glucose uptake via GLUT-1, which was instrumental in preserving mitochondrial creatine kinase activity, oxygen consumption rate, LV energetics (by 31phosphorous magnetic resonance spectroscopy), and myocardial function. We uncovered a novel mechanism whereby myocardial BH4 prevents and reverses LV diastolic and systolic dysfunction associated with diabetes via an nNOS-mediated increase in insulin-independent myocardial glucose uptake and utilization. These findings highlight the potential of GCH1/BH4-based therapeutics in human diabetic cardiomyopathy. Graphic Abstract: A graphic abstract is available for this article.

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The organ-specific nitric oxide synthase activity in the interaction with sympathetic nerve activity: a hypothesis.

The sympathetic nerve activity (SNA) is augmented in hypertension. SNA is regulated by neuronal nitric oxide synthase (nNOS) or endothelial nitric oxide synthase (eNOS) activity in hypothalamic paraventricular nuclei (PVN) and/or brainstem rostral ventrolateral medulla. High nNOS or eNOS activity within these brain regions lowers the SNA, whereas low cerebral nNOS and/or eNOS activity causes SNA augmentation. We hypothesize that the decreased cerebral nNOS/eNOS activity, which allows the enhancement of SNA, leads to the augmentation of renal eNOS/nNOS activity. Similarly, when the cerebral nNOS/eNOS activity is increased and SNA is suppressed, the renal eNOS/nNOS activity is suppressed as well. The activation of endothelial alpha(2)-adrenoceptors, may be a possible mechanism involved in the proposed regulation. Another possible mechanism might be based on nitric oxide, which acts as a neurotransmitter that tonically activates afferent renal nerves, leading to a decreased nNOS activity in PVN. Furthermore, the importance of the renal nNOS/eNOSactivity during renal denervation is discussed. In conclusion, the presented hypothesis describes the dual organ-specific role of eNOS/nNOS activity in blood pressure regulation and suggests possible connection between cerebral NOS and renal NOS via activation or inhibition of SNA, which is an innovative idea in the concept of pathophysiology of hypertension.

Nox2 signaling and muscle fiber remodeling are attenuated by losartan administration during skeletal muscle unloading.

Reduced mechanical loading results in atrophy of skeletal muscle fibers. Increased reactive oxygen species (ROS) are causal in sarcolemmal dislocation of nNOS and FoxO3a activation. The Nox2 isoform of NADPH oxidase and mitochondria release ROS during disuse in skeletal muscle. Activation of the angiotensin II type 1 receptor (AT1R) can elicit Nox2 complex formation. The AT1R blocker losartan was used to test the hypothesis that AT1R activation drives Nox2 assembly, nNOS dislocation, FoxO3a activation, and thus alterations in morphology in the unloaded rat soleus. Male Fischer 344 rats were divided into four groups: ambulatory control (CON), ambulatory + losartan (40 mg kg−1 day−1) (CONL), 7 days of tail‐traction hindlimb unloading (HU), and HU + losartan (HUL). Losartan attenuated unloading‐induced loss of muscle fiber cross‐sectional area (CSA) and fiber‐type shift. Losartan mitigated unloading‐induced elevation of ROS levels and upregulation of Nox2. Furthermore, AT1R blockade abrogated nNOS dislocation away from the sarcolemma and elevation of nuclear FoxO3a. We conclude that AT1R blockade attenuates disuse remodeling by inhibiting Nox2, thereby lessening nNOS dislocation and activation of FoxO3a.

Роль нейрональной NO-синтазы в реализации респираторных эффектов провоспалительного цитокина ФНО-α

In our previous studies we showed that an increase in the systemic or cerebral level of proinflammatory cytokines has a modulating effect on the reflex control of respiration. The aim of this study was to investigate the possible involvement of neuronal nitric oxide synthase (nNOS) in the hypoxic ventilatory response induced by an increased systemic level of the proinflammatory cytokine Tumor necrosis factor — α (TNF-α). To achieve this goal, we conducted experiments on urethane anesthetized rats with intravenous administration of TNF-α before and after pretreatment with 7-nitroindazole specific inhibitor of nNOS. The hypoxic ventilation response was assessed by rebreathing with a hypoxic gas mixture before and after administration of TNF-α. We found that TNF-α increased the lung ventilation in normoxia but decreased the ventilatory response to hypoxia. Pretreatment with nNOS inhibitor reduced respiratory effects of TNF-α. We believe that TNF-α can participate in the modulation of peripheral hypoxic chemoreflex not only in pathological, but also in normal physiological conditions through the activation of constitutive nNOS in the glomus cells of the carotid bodies. This makes the respiratory system urgently respond to an increase in the level of pro-inflammatory cytokines in the circulatory system. The obtained data made it possible to improve the understanding of how systemic inflammation affects the respiratory function.

Beta3-Adrenergic Receptor Activation Alleviates Cardiac Dysfunction in Cardiac Hypertrophy by Regulating Oxidative Stress.

Background Excessive myocardial oxidative stress could lead to the congestive heart failure. NADPH oxidase is involved in the pathological process of left ventricular (LV) remodeling and dysfunction. β3-Adrenergic receptor (AR) could regulate cardiac dysfunction proved by recent researches. The molecular mechanism of β3-AR regulating oxidative stress, especially NADPH oxidase, remains to be determined. Methods Cardiac hypertrophy was constructed by the transverse aortic constriction (TAC) model. ROS and NADPH oxidase subunits expression were assessed after β3-AR agonist (BRL) or inhibitor (SR) administration in cardiac hypertrophy. Moreover, the cardiac function, fibrosis, heart size, oxidative stress, and cardiomyocytes apoptosis were also detected. Results β3-AR activation significantly alleviated cardiac hypertrophy and remodeling in pressure-overloaded mice. β3-AR stimulation also improved heart function and reduced cardiomyocytes apoptosis, oxidative stress, and fibrosis. Meanwhile, β3-AR stimulation inhibited superoxide anion production and decreased NADPH oxidase activity. Furthermore, BRL treatment increased the neuronal NOS (nNOS) expression in cardiac hypertrophy. Conclusion β3-AR stimulation alleviated cardiac dysfunction and reduced cardiomyocytes apoptosis, oxidative stress, and fibrosis by inhibiting NADPH oxidases. In addition, the protective effect of β3-AR is largely attributed to nNOS activation in cardiac hypertrophy.

Post-treatment with glycyrrhizin can attenuate hepatic mitochondrial damage induced by acetaminophen in mice.

Overdose of acetaminophen (APAP) is responsible for the most cases of acute liver failure worldwide. Hepatic mitochondrial damage mediated by neuronal nitric oxide synthase- (nNOS) induced liver protein tyrosine nitration plays a critical role in the pathophysiology of APAP hepatotoxicity. It has been reported that pre-treatment or co-treatment with glycyrrhizin can protect against hepatotoxicity through prevention of hepatocellular apoptosis. However, the majority of APAP-induced acute liver failure cases are people intentionally taking the drug to commit suicide. Any preventive treatment is of little value in practice. In addition, the hepatocellular damage induced by APAP is considered to be oncotic necrosis rather than apoptosis. In the present study, our aim is to investigate if glycyrrhizin can be used therapeutically and the underlying mechanisms of APAP hepatotoxicity protection. Hepatic damage was induced by 300 mg/kg APAP in balb/c mice, followed with administration of 40, 80, or 160 mg/kg glycyrrhizin 90 min later. Mice were euthanized and harvested at 6 h post-APAP. Compared with model controls, glycyrrhizin post-treatment attenuated hepatic mitochondrial and hepatocellular damages, as indicated by decreased serum glutamate dehydrogenase, alanine aminotransferase, and aspartate aminotransferase activities as well as ameliorated mitochondrial swollen, distortion, and hepatocellular necrosis. Notably, 80 mg/kg glycyrrhizin inhibited hepatic nNOS activity and its mRNA and protein expression levels by 16.9, 14.9, and 28.3%, respectively. These results were consistent with the decreased liver nitric oxide content and liver protein tyrosine nitration indicated by 3-nitrotyrosine staining. Moreover, glycyrrhizin did not affect the APAP metabolic activation, and the survival rate of ALF mice was increased by glycyrrhizin. The present study indicates that post-treatment with glycyrrhizin can dose-dependently attenuate hepatic mitochondrial damage and inhibit the up-regulation of hepatic nNOS induced by APAP. Glycyrrhizin shows promise as drug for the treatment of APAP hepatotoxicity.