Cytokine-induced enhancement of calcium-dependent glutamate release from astrocytes mediated by nitric oxide

Cytokines are produced in the central nervous system (CNS) and exhibit various effects on neurons, microglia, and astrocytes. Astrocytes can release chemical transmitters, including glutamate, in a calcium-dependent manner, which may mediate communication between neurons and astrocytes. To date, no studies have been conducted on the effects of cytokines on calcium-dependent glutamate release from astrocytes. Here, we studied the effects of cytokines on calcium-dependent glutamate release. Cytokines enhanced glutamate release and induced the expression of inducible nitric oxide synthase (iNOS) and the production of nitric oxide (NO). The inhibition of iNOS eliminated the cytokine-induced enhancement of glutamate release, and treatment with a NO donor, even in the absence of cytokines, increased glutamate release. Thus, cytokines enhance glutamate release, and this enhancement is mediated by NO.     

Specific calcium-dependent release of endogenous glutamate from rat striatum is reduced by destruction of the cortico-striatal tract

Summary Rat neostriatal slices exhibited a Ca²⁺-dependent release of endogenous glutamate when depolarized by elevated K⁺. This evoked release was reduced by 30% in tissue from animals subjected to fronto-parietal lesions 3 weeks previously. These results support the proposal that glutamate is the transmitter of the cortico-striatal pathway.Supported by a SRC project grant to P.J.R.     

Nitric oxide up-regulates the release of inflammatory mediators by mouse macrophages

Nitric oxide (NO) plays a key role in mediating macrophage cytotoxicity towards different targets, including tumoral cells and intracellular pathogens. However, its role in macrophage immunoregulation is less well defined. In this study, we have investigated the effect of altering NO levels on the production by mouse macrophages of cytokines, and reactive oxygen intermediates as measured by luminol-dependent chemiluminescence. Our results demonstrate that NO can enhance the release of both tumor necrosis factor-α and interleukin-1α, and chemiluminescence. Thus, in addition to acting as a powerful effector molecule in mediating cytotoxic activities of mouse macrophages, NO can play a role in enhancing the production of a variety of other inflammatory mediators, and thus can contribute both directly and indirectly to the immunopathology of macrophage-dependent inflammation.     

一氧化氮在实验性肝损伤中的作用

目的：观察３种细胞因子对体外培养大鼠肝细胞产生一氧化氮（ＮＯ）的影响及ＮＯ在实验性肝损伤中的作用。方法与结果：给大鼠注射内毒素后分离肝细胞,培养液中加入细胞因子（γ－ＩＦＮ,ＩＬ－２,ＴＮＦ－α）,上清液中ＮＯ含量增加,两种以上细胞因子联合应用,ＮＯ含量增加更明显;大鼠注射内毒素及Ｌ－精氨酸,血清中ＮＯ含量增加,肝损伤减轻;大鼠给硫代乙酰胺（ＴＡＡ）灌胃,并注射ＮＯ生成抑制剂Ｌ－ＮＮＡ,动物出现嗜     

Risperidone attenuates the increase of extracellular nitric oxide and glutamate levels in serotonin syndrome animal models

Serotonin (5-hydroxytryptamine; 5-HT) syndrome is a potentially life-threatening neurotoxic condition provoked by pharmacologically induced excess serotonergic activity. Several studies report that nitric oxide (NO) and glutamate play a role in psychostimulant-induced hyperthermia related to neurotoxicity. In the present study, the involvement of NO and glutamate, as well as the effect of risperidone, a potent 5-HT_2A and D₂ (and a less potent D₁) receptor antagonist, were investigated in animal models of 5-HT syndrome. Two 5-HT syndrome animal models were utilized. The first model was induced by administration of tranylcypromine, a nonselective monoamine oxidase (MAO) inhibitor, and fluoxetine, a selective 5-HT reuptake inhibitor. The second model was induced by the administration of clorgyline, an MAO-A inhibitor, and 5-hydroxy-l-tryptophan, a precursor of 5-HT. Changes in the level of NO metabolites and glutamate in the anterior hypothalamus were measured using microdialysis. In both models, NO metabolite levels significantly increased, and this increase was significantly attenuated by risperidone pretreatment. Extracellular levels of glutamate were increased only in the tranylcypromine and fluoxetine model, and this increase was significantly attenuated by risperidone pretreatment. These results indicate that NO and glutamate may be involved in the development of 5-HT syndrome and that risperidone may be effective against neurotransmitter abnormalities in 5-HT syndrome.     

Borna disease virus P protein inhibits nitric oxide synthase gene expression in astrocytes

Borna disease virus (BDV) is one of the potential infectious agents involved in the development of central nervous system (CNS) diseases. Neurons and astrocytes are the main targets of BDV infection, but little is known about the roles of BDV infection in the biological effects of astrocytes. Here we reported that BDV inhibits the activation of inducible nitric oxide synthase (iNOS) in murine astrocytes induced by bacterial LPS and PMA. To determine which protein of BDV is responsible for the regulation of iNOS expression, we co-transfected murine astrocytes with reporter plasmid iNOS-luciferase and plasmid expressing individual BDV proteins. Results from analyses of reporter activities revealed that only the phosphoprotein (P) of BDV had an inhibitory effect on the activation of iNOS. In addition, P protein inhibits nitric oxide production through regulating iNOS expression. We also reported that the nuclear factor kappa B (NF-kappaB) binding element, AP-1 recognition site, and interferon-stimulated response element (ISRE) on the iNOS promoter were involved in the repression of iNOS gene expression regulated by the P protein. Functional analysis indicated that sequences from amino acids 134 to 174 of the P protein are necessary for the regulation of iNOS. These data suggested that BDV may suppress signal transduction pathways, which resulted in the inhibition of iNOS activation in astrocytes.     

Enhanced GLT-1 mediated glutamate uptake and migration of primary astrocytes directed by fibronectin-coated electrospun poly-l-lactic acid fibers

Bioengineered fiber substrates are increasingly studied as a means to promote regeneration and remodeling in the injured central nervous system (CNS). Previous reports largely focused on the ability of oriented scaffolds to bridge injured regions and direct outgrowth of axonal projections. In the present work, we explored the effects of electrospun microfibers on the migration and physiological properties of brain astroglial cells. Primary rat astrocytes were cultured on either fibronectin-coated poly-l-lactic acid (PLLA) films, fibronectin-coated randomly oriented PLLA electrospun fibers, or fibronectin-coated aligned PLLA electrospun fibers. Aligned PLLA fibers strongly altered astrocytic morphology, orienting cell processes, actin microfilaments, and microtubules along the length of the fibers. On aligned fibers, astrocytes also significantly increased their migration rates in the direction of fiber orientation. We further investigated if fiber topography modifies astrocytic neuroprotective properties, namely glutamate and glutamine transport and metabolism. This was done by quantifying changes in mRNA expression (qRT-PCR) and protein levels (Western blotting) for a battery of relevant biomolecules. Interestingly, we found that cells grown on random and/or aligned fibers increased the expression levels of two glutamate transporters, GLAST and GLT-1, and an important metabolic enzyme, glutamine synthetase, as compared to the fibronectin-coated films. Functional assays revealed increases in glutamate transport rates due to GLT-1 mediated uptake, which was largely determined by the dihydrokainate-sensitive GLT-1. Overall, this study suggests that aligned PLLA fibers can promote directed astrocytic migration, and, of most importance, our in vitro results indicate for the first time that electrospun PLLA fibers can positively modify neuroprotective properties of glial cells by increasing rates of glutamate uptake.     

一氧化氮/诱导型一氧化氮合酶在动脉粥样硬化过程中的作用

 目的：探讨一氧化氮（NO）/诱导型一氧化氮合酶（iNOS）在动脉粥样硬化（atherosclerosis,AS）过程中的动态变化,分析其对动脉粥样硬化形成过程的影响。方法：将60只SD大鼠随机分成2组：对照组及AS组,每组30只。AS组采用维生素D3腹腔注射联合高脂饲料饲养的方法构建动脉粥样硬化模型。用相关生化方法检测血清各项生化指标：总胆固醇、甘油三酯、高密度脂蛋白胆固醇、低密度脂蛋白胆固醇、空腹血糖和钙离子,比色法检测血清NO浓度,并对主动脉行HE染色,免疫组化技术检测iNOS蛋白表达,将所得数据进行统计分析,用简单线性相关分析NO与钙离子及动脉粥样硬化指数的相关性。结果：90 d后成功构建了主动脉中膜钙化型动脉粥样硬化模型。血清NO浓度在动脉粥样硬化过程中逐步下降,各组间差异均有统计学意义（均P<0.05）。动脉粥样硬化过程中动脉粥样硬化指数与钙离子呈正相关,与NO呈负相关。在90 d的AS组粥样斑块区免疫组化技术检测到iNOS蛋白表达。结论:在动脉粥样硬化形成过程中,主动脉粥样斑块区iNOS蛋白高表达,但血清NO浓度逐渐降低,NO抗动脉粥样硬化作用减弱。     

脑挫伤后一氧化氮合酶阳性细胞及一氧化氮含量的变化

目的　探讨脑挫伤后一氧化氮合酶 (NOS)阳性细胞和一氧化氮 (NO)的变化和意义。方法　采用自由落体法致Wistar大鼠顶叶皮质挫裂伤动物模型。伤后 2 4h、72h和 7d取脑 ,制作冰冻切片 ,采用NADPH组织化学染色 ,显示脑挫伤区NOS阳性细胞。用硝酸还原酶法测定血液和脑组织中NO含量。结果　脑挫伤后 72h ,NOS阳性细胞数密度 (Nv)和面密度(Sv)明显增高 (P <0 0 5 ) ,而且 7d时仍无明显下降。血液和脑中NO含量也增高 ,并与NOS细胞呈平行关系。结论　脑挫伤后不同时间NOS细胞数目和NO含量有明显改变 ,提示NOS和NO参与了脑挫伤的病理过程     

Possible release of nitric oxide from cholinergic axons in the thalamus by stimulation of the rat laterodorsal tegmental nucleus as measured with voltammetry

By means of the differential direct current voltammetry technique with carbon fiber electrodes in urethane-anesthetized rats, we monitored nitric oxide (NO) concentrations in the thalamus in the basal condition and following electrical stimulation of the laterodorsal tegmental nucleus (LDT), whose neurons have the strongest activity of NADPH-diaphorase, or NO synthase, together with acetylcholine. NO levels, measured as the height of the peak at +970–1000 mV in the voltammetry (NO was soon oxidized in vivo to be detected at the voltage of this peak, so that NO levels in this report are, in the strict sense, levels of the oxidized metabolites reflecting very possibly those of NO in physiological conditions; see Section 2, Methods), increased just after repetitive stimulation of the LDT. Stimulation of the surrounding areas or the cerebellum produced virtually no change in NO levels. An intravenous injection of -nitroarginine methyl ester reduced the basal level of NO, but stimulation of the LDT still increased NO levels, which may be due to very strong activity of NO synthase in the LDT neurons. These results are consistent with the notion that NO can be released from axons of the LDT neurons by their excitation.     

The role of nitric oxide in multiple sclerosis

 During the past decade nitric oxide has emerged as an important mediator of physiological and pathophysiological processes. Elevated nitric oxide biosynthesis has been associated with nonspecific immune-mediated cellular cytotoxicity and the pathogenesis of chronic, inflammatory autoimmune diseases including rheumatoid arthritis, insulin-dependent diabetes, inflammatory bowel disease, and mutiple sclerosis. Recent evidence suggests, however, that nitric oxide is also immunoregulatory and suppresses the function of activated proinflammatory macrophages and T lymphocytes involved in these diseases. This article reviews the role of nitric oxide in the biology of central nervous system glial cells (astrocytes and microglia) as it pertains to the pathogenesis of multiple sclerosis in humans and experimental allergic encephalitis, the animal model of this disease. Although nitric oxide has been clearly implicated as a potential mediator of microglia-dependent primary demyelination, a hallmark of multiple sclerosis, studies with nitric oxide synthase inhibitors in the encephalitis model have been equivocal. These data are critically reviewed in the context of what is know from clinical research on the nitric oxide pathway in multiple sclerosis. Specific recommendations for future preclinical animal model research and clinical research on the nitric oxide pathway in patients are suggested. These studies are necessary to further define the role of nitric oxide in the pathology of multiple sclerosis and to fully explore the potential for nitric oxide synthase inhibitors as novel therapeutics for this disease. Received: 6 June 1996 / Accepted: 24 September 1996     

Hypoxia-induced nitric oxide protects chondrocytes from damage by hydrogen peroxide

Objective:Because articular cartilage has no vascular supply, chondrocytes are hypoxic under normal physiological conditions. Nitric oxide (NO) plays an important role in chondrocyte damage, such as apoptosis. Although oxygen stress with hydrogen peroxide was found to cause chondrocyte damage, these data were obtained under normoxic (21% O₂) conditions. We investigated the effects of hypoxia on hydrogen peroxide-induced chondrocyte damage Methods:Bovine articular chondrocytes were used in this study. Proteoglycan (PG) synthesis and the induction of apoptosis were analyzed with [³⁵S]-sulfate incorporation and annexin V staining, respectively. The induction of NO was examined using a fluorescent probe and RT-PCR. Results:Cells maintained at 5% O₂ had the maximum PG synthesis. Under normoxic conditions, hydrogen peroxide inhibited PG synthesis and induced annexin V positive cells in a dose-dependent fashion. However, in those cells cultured under hypoxic (5%) conditions, the hydrogen peroxide-induced annexin V expression was attenuated. Chondrocytes exposed to hypoxia showed induction of NO. When the hypoxia-induced NO was inhibited, the hypoxia-enhanced PG synthesis was abolished and hydrogen peroxide clearly induced cell damage. Conclusions:Endogenous NO induced by hypoxia protects chondrocytes from apoptosis induced by an oxidative stress.Received 18 August 2003; returned for revision 25 September 2003; accepted by W. van den Berg 26 February 2004     

Polysaccharide-based biomaterials with on-demand nitric oxide releasing property regulated by enzyme catalysis

The regulatory role of nitric oxide (NO) in cell signaling has been well recognized. Clinically, NO deficiency is known to be associated with severe vascular disorders, especially in patients with long-term diabetes. Exogenous compensation of NO is a promising therapeutic strategy, although the lack of stable NO compounds often lead to unsatisfactory clinical outcomes. In the present study, we report a stable comb-shaped polymer (CS–NO) using glycosylated NO compound as pendent chains and chitosan (CS) as backbone for controlled NO release. The on-demand release of NO is achieved by controlling the decomposition process of the CS–NO polymer, which is blocked by galactose and only occurs in the presence of glycosidase, making the NO releasing kinetic closely correlate with the glycosidase concentration. In addition, due to its high stability, the CS–NO polymers can also be processed into supportive membrane or injectable hydrogel, further demonstrating its clinical potential. Indeed, we report that the NO-releasing membrane inhibited platelet adhesion, prolonged activated partial thromboplastin time (APTT) as shown in the platelet-rich-plasma (PRP) assay. We also observe enhanced human umbilical vein endothelial cell growth yet suppressed vascular smooth muscle cell proliferation on the NO-contained membrane in vitro. Furthermore, in vivo administration of CS–NO solution significantly enhanced angiogenesis in diabetic mice with hind-limb ischemia. Protective effect of CS–NO was also observed against limb necrosis. Given the physiological importance of NO, the CS–NO polymer may be considered a promising option in therapeutic development against vascular disorders and diabetic feet.     

Hemodialysis and nitric oxide

Conclusion The discovery of the strong vasodilating agent NO stimulated research on its role in blood pressure regulation in a variety of hypertensive and hypotensive disorders, including erythropoietin-induced hypertension or acute hypotension in HD patients. Besides its role in blood pressure regulation, NO possesses other functions as an antimicrobial defense or an oxidant stress. Therefore, further studies may be undertaken to evaluate possible roles of NO in HD-related complications.     

多巴胺通过mTOR-EAAT2通路影响星形胶质细胞谷氨酸摄取能力

目的:研究多巴胺(dopamine,DA)对星形胶质细胞谷氨酸(glutamate,Glu)摄取能力的影响,以及DA通过哺乳动物雷帕霉素靶蛋白(m TOR)-兴奋性氨基酸转运体2(EAAT2)信号通路对星形胶质细胞Glu摄取能力的影响。方法:采用Amplex Red谷氨酸测定试剂盒检测经过干预的原代皮层星形胶质细胞对Glu摄取含量的变化,RT-q PCR、Western blot和免疫荧光染色等检测EAAT2和m TOR mRNA和蛋白质相对表达量,m TOR拮抗剂雷帕霉素或m TOR兴奋剂MHY1485干预在DA中共培养的星形胶质细胞,检测m TOR和EAAT2的表达情况,以及培养上清液Glu的含量。结果:DA干预的原代星形胶质细胞中m TOR表达下调,EAAT2表达下调,培养上清液Glu水平上升;雷帕霉素干预后,EAAT2表达下调,培养上清液中Glu的含量增加;MHY1485干预后,EAAT2表达上调,培养上清液中Glu的含量下降。结论:DA通过与星形胶质细胞m TOR-EAAT2通路相互作用,减弱星形胶质细胞摄取Glu的能力,引起细胞外Glu蓄积,最终损伤星形胶质细胞的功能。     

一氧化氮对星形胶质细胞轴突生长因子-1表达及迁移的影响

目的 探讨一氧化氮(NO)对星形胶质细胞中轴突生长因子-1(netri-1)的表达变化以及对细胞迁移的影响。方法 采用硝普钠（SNP）作为NO供体处理星形胶质细胞,通过振荡培养和差速贴壁法分离纯化新生SD大鼠星形胶质细胞,接种至培养板,分为实验组和对照组,每组6个样本,实验组用50μmol/L SNP处理星形胶质细胞,通过划痕法观察细胞的迁移,并采用Western blotting 和免疫细胞化学方法分别检测处理前后的星形胶质细胞中netrin-1蛋白的表达变化。 结果 SNP处理后,实验组的星形胶质细胞与对照组相比,划痕区细胞明显增多,星形胶质细胞netrin-1表达随着时间的推移出现先升高后降低趋势,于48 h达到峰值 (P<0.01)。 结论 SNP使星形胶质细胞netrin-1表达水平发生变化和影响星形胶质细胞迁移,提示netrin-1可能通过NO的调控而影响星形胶质细胞迁移。     

Role of nitric oxide in mast cells

Mast cells (MC) are important effector cells in allergic disorders. Recenty, the role of MC in innate and adaptive immunity is gaining prominence. Nitric oxide is an important signaling molecule and its production in mast cell has been reported widely. However, controversy exists about whether MC produce NO. This review addresses the role of NO in MC biology and the reasons behind the controversy and discusses effects of NO in regulation of MC phenotype and function.     

Central release of nitric oxide mediates antinociception induced by aerobic exercise

Nitric oxide (NO) is a soluble gas that participates in important functions of thecentral nervous system, such as cognitive function, maintenance of synapticplasticity for the control of sleep, appetite, body temperature, neurosecretion, andantinociception. Furthermore, during exercise large amounts of NO are released thatcontribute to maintaining body homeostasis. Besides NO production, physical exercisehas been shown to induce antinociception. Thus, the present study aimed toinvestigate the central involvement of NO in exercise-induced antinociception. Inboth mechanical and thermal nociceptive tests, central [intrathecal(it) and intracerebroventricular (icv)]pretreatment with inhibitors of the NO/cGMP/K_ATP pathway (L-NOArg, ODQ,and glybenclamide) prevented the antinociceptive effect induced by aerobic exercise(AE). Furthermore, pretreatment (it, icv) withspecific NO synthase inhibitors (L-NIO, aminoguanidine, and L-NPA) also preventedthis effect. Supporting the hypothesis of the central involvement of NO inexercise-induced antinociception, nitrite levels in the cerebrospinal fluid increasedimmediately after AE. Therefore, the present study suggests that, during exercise,the NO released centrally induced antinociception.     

内皮型一氧化氮合酶基因转染对人吞噬细胞释放细胞因子和生成环核苷酸的影响

目的和方法:将内皮型一氧化氮合酶基因转染吞噬细胞,观察该基因表达对吞噬细胞释放细胞因子和cAMP的影响。 结果:内皮型一氧化氮合酶可在吞噬细胞获得稳定表达,但该产物在活细胞中不产生一氧化氮。一氧化氮合酶基因表达可上调TNF-α的释放,下调IL-10和cAMP的生成,使用一氧化氮合酶抑制剂不改变这种变化趋势。结论:内皮型一氧化氮合酶基因产物的功能具有细胞特异性。导致吞噬细胞功能变化的效应分子可能不是一氧化氮。