Effect of curcumin on nitric oxide synthase expression in Iipopolysaccharide-activated microglia cells and the anti-oxidative effect of curcumin

BACKGROUND: It has been demonstrated that curcumin can increase the activities of various anti-oxidase in blood and tissue, effectively eliminate various free radicals, reduce the production of peroxisome, and alleviate oxidative stress reaction. Whether it has the same effect on microglia? OBJECTIVE: To observe the effects of curcumin on the expressions of inducible nitric oxide synthase (iNOS), nuclear factor-κB (NF-κB), and superoxide dismutase (SOD) in microglial cell line BV stimulated by lipopolysaccharide (LPS). DESIGN: An observational comparative study. SETTING: Research Room of Biochemistry, Medical College of Nantong University. MATERIALS: Mice microglia cell line BV, iNOS and NF-κB reporter gene plasmids were presented by Dr. Bhat.NR. from the Medical University of South Carolina (USA). Curcumin was produced by the Xi'an Branch of China Chengdu Scholar Bio-Tech. Co.,Ltd.; LPS (E.Coli O26:B6), anti-mice iNOS monoclonal antibody, horseradish peroxidase labeled goat-anti-mice IgG were the products of Sigma Company (USA). METHODS: The experiments were carried out in the Research Room of Biochemistry, Medical College of Nantong University from May 2006 to April 2007. ① Detection of iNOS: The cells were seeded onto 24-well plate at the density of 1×105, After the cells had adhered to the cover glasses, the cells were grouped as negative control group (the primary antibody was replaced by phosphate buffered solution PBS); normal control group (the cells were normally cultured); LPS-treated group (the cells were treated with LPS for 24 hours); curcumin+LPS group (the cells were treated with curcumin for 1 hour and LPS for 24 hours). The expressions of iNOS protein were detected with immunocytochemical staining. ② Determination of iNOS and NF-κB gene activities: According to the introduction of the kit for transfection, iNOS or NF-κB report gene plasmids were transiently transfected with LipofectamineTM2000 liposomes into the cells in the 24-well plate for 24 hours. The cells were divided into normal control group (the cells were normally cultured after transfected with report gene plasmids); blank plasmid group (the cells were normally cultured after transfected with blank plasmids); LPS-treated group (the cells were treated with LPS for 4 hours after transfected with report gene plasmids); curcumin+LPS group (the cells were treated with curcumin for 1 hour and LPS for 24 hours after transfected with report gene plasmids). The content of luciferase in the cell lysis buffer was determined after cell lysis. ③ Determination of SOD activity: The cells were seeded into culture bottle at the density of 1×106, and the divided into four groups, including normal control group (the cells were normally cultured); LPS-treated group (the cells were treated with LPS for 24 hours); curcumin+LPS group (the cells were treated with curcumin for 1 hour and LPS for 24 hours); vitamin C+LPS group (the cells were treated with vitamin C for 1 hour and LPS for 24 hours). The SOD activity was determined with xanthine oxidase and quantitative colorimetric assay. MAIN OUTCOME MEASURES: The expressions of iNOS protein, iNOS and NF-κB, and the activity of SOD were observed. RESULTS: ① Expression of iNOS protein in microglia: The expression of iNOS protein in the LPS-treated group was obviously higher than that in the negative control group (P < 0.01); Those in the curcumin+LPS group were significantly decreased as compared with that in the LPS-treated group (P < 0.01). ② Expressions of iNOS and NF-κB genes: The expressions of iNOS and NF-κB genes in the LPS-treated group were significantly higher than those in the normal control group (P < 0.01); Those in the curcumin+LPS group were significantly lower than those in the LPS-treated group (P < 0.01). ③ SOD activity: The activity of SOD in the LPS-treated group was significantly lower than those in the normal control group (P < 0.01). It in the curcumin+LPS group and vitamin C +LPS group was significantly higher than that in the LPS-treated group (P < 0.01). CONCLUSION: Curcumin could inhibit the expression of iNOS in the activated microglia, and it also has the abilities in eliminating free radicals and antagonizing lipid peroxidation.     

Differential effects of the nonsteroidal antiinflammatory drug flurbiprofen and its nitric oxide-releasing derivative, nitroflurbiprofen, on prostaglandin E(2), interleukin-1beta, and nitric oxide synthesis by activated microglia.

Increasing experimental, clinical, and epidemiological studies point to the pivotal role of inflammation in the pathogenesis of acute and chronic neurodegenerative diseases and to the protective effects of nonsteroidal antiinflammatory drug (NSAID) therapies. Nonetheless, NSAID long-term therapies are limited by their significant adverse effects on gastrointestinal tract and kidneys. Nitroflurbiprofen (NO-flurbiprofen) belongs to a novel class of antiinflammatory agents obtained by derivatization of conventional NSAIDs with a nitric oxide (NO)-releasing moiety, which strongly reduces their untoward side effects without altering the antiinflammatory effectiveness. The recent evidence of neuroprotective effects of NO-NSAIDs in animal models of chronic brain inflammation prompted us to investigate the activities of NO-flurbiprofen and its parent molecule flurbiprofen on activated rat microglia, the brain resident macrophages. We found that NO-flurbiprofen was as potent as flurbiprofen in preventing prostaglandin E(2) synthesis in lipopolysaccharide-activated microglial cultures. At variance with previous observations on peripheral macrophages is that NO-flurbiprofen did not show any additional capacity to inhibit interleukin-1beta synthesis compared with flurbiprofen. Moreover, NO enhanced the expression of the inducible NO synthase; this effect was most likely attributable to the NO released from the drug, as suggested by experiments performed in the presence of the NO donor Deta-NONOate, which similarly to NO-flurbiprofen is characterised by a slow and long-lasting release. Our findings indicate that NO-NSAIDs may differently affect peripheral and brain macrophages. Given their potential therapeutic role in brain inflammation, further in vivo and in vitro studies are required to understand fully their mechanism of action in the CNS.     

青白散对慢性软组织损伤模型大鼠骨骼肌一氧化氮合酶系统和一氧化氮的影响*☆

背景：对慢性软组织损伤后一氧化氮合酶系统和一氧化氮的研究目前较少。 目的：观察青白散对大鼠慢性软组织损伤模型骨骼肌中一氧化氮合酶系统和一氧化氮的影响。 方法：雄性 SD 大鼠随机分为对照组、模型组、氨基胍组、青白散组。后3组采用机械损伤法制备慢性骨骼肌损伤动物模型,分别予以生理盐水 10 mL/kg,0.10 g/kg氨基胍,0.54 g/kg青白散,1次/d,连续 14 d。于给药后1,2,3周,分别检测大鼠肌组织一氧化氮含量、总一氧化氮合酶和诱导型一氧化氮合酶的活性。 结果与结论：骨骼肌损伤修复过程中,模型组大鼠骨骼肌中一氧化氮含量、总一氧化氮合酶和诱导型一氧化氮合酶的活性较对照组显著增高;而青白散组和氨基胍组大鼠骨骼肌中一氧化氮含量、总一氧化氮合酶和诱导型一氧化氮合酶的活性均较模型组显著降低。说明青白散可能通过阻抑诱导型一氧化氮合酶诱导过量一氧化氮的产生,为慢性软组织损伤的修复创造了有利条件。     

Cervical sympathetic trunk transection affects inducible nitric oxide synthase expression in rat hippocampus following focal cerebral ischemia/reperfusion injury

BACKGROUND： The stellate ganglion block （SGB） plays a protective role in focal cerebral ischemia/reperfusion injury. The human SGB can be simulated by transection of the cervical sympathetic trunk （TCST） in rats. OBJECTIVE： To observe the effects of TCST on inducible nitric oxide synthase （iNOS） levels and cerebral infarct volume in the hippocampus of rats with cerebral ischemia/reperfusion injury, and to analyze the mechanism of action. DESIGN, TIME AND SETTING： A completely randomized, controlled, neuropathological experiment was performed at the Institute of Neurological Disease, Taihe Hospital, Yunyang Medical College between March and September 2006. MATERIALS： A total of 93 Wistar rats, aged 1718 weeks, of either gender, were used for this study. 2, 3, 5-triphenyl tetrazolium chloride was purchased from Changsha Hongyuan Biological Reagent Company China. Rabbit iNOS antibody and goat anti-rabbit IgG antibody were the products of Wuhan Boster Biological Reagent Co., Ltd., China. METHODS： Ten rats were randomly selected for the sham-operated group. Cerebral ischemia/reperfusion injury was induced by middle cerebral artery occlusion （MCAO） using the suture method in the remaining rats. Forty successful rat models were randomly and equally divided into the following two groups： （1） TCST group： subsequent to TCST, MCAO was performed for 2 hours, followed by 24 hours reperfusion; （2） model group： rats underwent experimental procedures similar to the TCST group, with the exception of TCST. Rats in the sham-operated group were subjected to experimental procedures similar to the model group; however, the thread was only introduced to a depth of 10 mm. MAIN OUTCOME MEASURES： Following 24 hours of reperfusion, functional neurological deficits were scored. Brain tissue sections from ten rats of each group were used to measure cerebral infarct volume by TTC staining. Hippocampal tissue sections of an additional ten rats from each group were used to detect iNOS levels using the s     

依达拉奉预处理对小鼠脑缺血再灌注损伤后皮质一氧化氮合酶表达的影响

目的探讨依达拉奉预处理对小鼠脑缺血再灌注(IR)损伤后皮质一氧化氮合酶(NOS)表达的影响。方法 48只健康ICR小鼠被分为假手术组、对照组和依达拉奉组。依达拉奉组和对照组分别给予依达拉奉3 mg/(kg.d)和同等体积的生理盐水腹腔注射共7 d,然后建立小鼠IR模型;缺血1 h、再灌注24 h时应用2,3,5-氯化三苯基四氮唑(TTC)染色法测量各组脑梗死体积,应用免疫组化法检测各组小鼠皮质神经元型、、诱导型和内皮型NOS(nNOS、iNOS、eNOS)阳性细胞数。结果与假手术组比较,对照组小鼠皮质nNOS、iNOS和eNOS阳性细胞数明显增多(均P<0.05);与对照组比较,依达拉奉组脑梗死体积明显缩小,皮质nNOS和iNOS阳性细胞数明显减少,eNOS阳性细胞数明显增多(均P<0.05)。结论依达拉奉预处理可以影响IR小鼠皮质nNOS、iNOS和eNOS的表达,发挥神经保护作用。     

Absence of linkage with the neuronal nitric oxide synthase (NOS1) gene in 41 multiplex Swedish MS families

Several genetic factors are likely to play a role in the aetiology of multiple sclerosis (MS), although so far only the HLA gene complex has been clearly identified as important. In addition, several studies support the importance of nitric oxide synthase (NOS) as a component in the pathogenesis of MS. We have investigated the role of the neuronal nitric oxide synthase (NOS1) gene in 41 Swedish multiplex MS families by parametric and nonparametric linkage analysis with two polymorphic intragenic markers. Two-point lod scores were clearly negative, effectively excluding a role of the NOS1 gene in most models tested. Non-parametric linkage analysis (NPL), affected pedigree member (APM) analysis and extended transmission disequilibrium test (ETDT) also failed to provide evidence for a susceptibility locus in this gene. Thus, a contribution of the NOS1 gene to the genetic background of MS is unlikely in this population.     

Chlorogenic acid protection of neuronal nitric oxide synthase-positive neurons in the hippocampus of mice with impaired learning and memory

BACKGROUND: Clinical practice and modern pharmacology have confirmed that ehlorogenic acid can ameliorate learning and memory impairments. OBJECTIVE: To observe the effects of chlorogenic acid on neuronal nitric oxide synthase (nNOS)-positive neurons in the mouse hippocampus, and to investigate the mechanisms underlying the beneficial effects of chlorogenic acid on learning and memory. DESIGN, TIME AND SETTING: The present randomized, controlled, neural cell morphological observation was performed at the Institute of Neurobiology, Central South University between January and May 2005.MATERIALS: Forty-eight female, healthy, adult, Kunming mice were included in this study. Learning and memory impairment was induced with an injection of 0.5 μL kainic acid (0.4 mg/mL) into the hippocampus.METHODS: The mice were randomized into three groups (n = 16): model, control, and chlorogenic acid-treated. At 2 days following learning and memory impairment induction, intragastric administration of physiological saline or chlorogenic acid was performed in the model and chlorogenic acid-treated groups, respectively. The control mice were administered 0.5 μ L physiological saline into the hippocampus, and 2 days later, they received an intragastric administration of physiological saline. Each mouse received two intragastric administrations (1 mL solution once) per day, for a total of 35 days. MAIN OUTCOME MEASURES: Detection of changes in hippocampal and cerebral cortical nNOS neurons by immunohistochemistry; determination of spatial learning and memory utilizing the Y-maze device.RESULTS: At day 7 and 35 after intervention, there was no significant difference in the number of nNOS-positive neurons in the cerebral cortex between the model, chlorogenic acid, and control groups (P > 0.05). Compared with the control group, the number of nNOS-positive neurons in the hippocampal CA1-4 region was significantly less in the model group (P < 0.05). However, the control group was not different from the ehlorogenic acid-treated group (P > 0.05). At day 7 following intervention, the number of correct responses in the Y-maze test was greater in the ehlorogenic acid-treated group than in the model group. CONCLUSION: Chlorogenic acid protects kainic acid-induced injury to nNOS-positive neurons in the hippocampal CA1-4 regions, thereby ameliorating learning and memory impairment.     

Chlorogenic acid protection of neuronal nitric oxide synthase-positive neurons in the hippocampus of mice with impaired learning and memory☆

BACKGROUND： Clinical practice and modern pharmacology have confirmed that chlorogenic acid can ameliorate learning and memory impairments. OBJECTIVE： To observe the effects of chlorogenic acid on neuronal nitric oxide synthase （nNOS）-positive neurons in the mouse hippocampus, and to investigate the mechanisms underlying the beneficial effects of chlorogenic acid on learning and memory. DESIGN, TIME AND SETTING： The present randomized, controlled, neural cell morphological observation was performed at the Institute of Neurobiology, Central South University between January and May 2005. MATERIALS： Forty-eight female, healthy, adult, Kunming mice were included in this study. Learning and memory impairment was induced with an injection of 0.5 uL kainic acid （0.4 mg/mL） into the hippocampus. METHODS： The mice were randomized into three groups （n = 16）： model, control, and chlorogenic acid-treated. At 2 days following learning and memory impairment induction, intragastric administration of physiological saline or chlorogenic acid was performed in the model and chlorogenic acid-treated groups, respectively. The control mice were administered 0.5uL physiological saline into the hippocampus, and 2 days later, they received an intragastfic administration of physiological saline. Each mouse received two intragastric administrations （1 mL solution once） per day, for a total of 35 days. MAIN OUTCOME MEASURES： Detection of changes in hippocampal and cerebral cortical nNOS neurons by immunohistochemistry; determination of spatial learning and memory utilizing the Y-maze device. RESULTS： At day 7 and 35 after intervention, there was no significant difference in the number of nNOS-positive neurons in the cerebral cortex between the model, chlorogenic acid, and control groups （P 〉 0.05）. Compared with the control group, the number of nNOS-positive neurons in the hippocampal CA1-4 region was significantly less in the model group （P 〈 0.05）. However, the control group was not different fro     

Selective protection against oxidative damage in brain of mice with a targeted disruption of the neuronal nitric oxide synthase gene

Nitric oxide (NO) is an essential messenger molecule in brain, where it is produced in neurons mostly by the activity of the neuronal isoform of nitric oxide synthase (nNOS). To understand the participation of the different isoforms of NOS in physiological functioning and in pathological processes, mice with null mutations for each of the NOS isoforms have been generated. In the present paper, we report that there is a selective protection from oxidative damage in the brain of mice with a targeted disruption of the nNOS gene. The cerebellum of these mice shows reduced levels of lipid peroxidation (LP) at the different ages tested, compared with wild-type mice, and also a reduction in the formation of reactive oxygen species (ROS). We observed a decrease of LP in cortex, and no effect on either LP or ROS formation was observed in striatum of knockout mice compared with wild type. We also report increased spontaneous motor activity of knockout mice. The expression and activity of nNOS are crucial to maintain redox status in brain, and we consider that the alteration in oxidative damage may help us to explain the phenotypical characteristics of nNOS knockout mice and their differential susceptibility to brain insults.     

Functional activity and expression of inducible nitric oxide synthase (iNOS) in muscle of the isolated distal colon of mdx mice

The inducible isoform of nitric oxide (NO) synthase (iNOS), expressed in endothelium, epithelium, and inflammatory cells, produces a large amount of NO. Previous studies on mouse intestine indicate that a muscular iNOS may have a role in the storage of intraluminal content. In this study we investigated the presence and function of iNOS in the colonic smooth muscle cells of 2- and 12-month-old dystrophic (mdx) mice. By using an in vitro isovolumic technique, and immunohistochemical and Western blot analysis, we demonstrated that iNOS is expressed and active in the smooth muscle cells of normal mouse and defective in young adult (2-month-old) mdx mice. Therefore, an altered activity of the muscle iNOS might explain the motility disorders observed in the colon of mdx mice and, from a clinical point of view, the impairment of intestinal function in dystrophic patients.     

Cytokine-stimulated inducible nitric oxide synthase expression in astroglia: role of Erk mitogen-activated protein kinase and NF-kappaB

Expression of inducible nitric oxide synthase (iNOS), which leads to the production of nitric oxide (NO), is stimulated by proinflammatory cytokines such as interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha). Here we report on the roles of nuclear factor-kappaB (NF-kappaB) and mitogen-activated protein (MAP) kinases in IL-1beta/TNF-alpha-induced iNOS expression in adult rat astroglia. Cytokine-induced increases in nitrite accumulation (an index of NO production) and iNOS expression were attenuated by inhibition of NF-kappaB with pyrrolidine dithiocarbamate (PDTC). Similar attenuation of these cytokine-induced responses was produced by inhibition of MAP kinase (MEK), the immediate upstream activator of Erk, using PD098,059. Combined treatment of astroglia with PDTC and PD098,059 completely abolished the cytokine-induced increases in iNOS expression and nitrite accumulation. By contrast, the selective p38 kinase inhibitor SB203,580 amplified the effects of IL-1beta/TNF-alpha on nitrite accumulation. In accordance with these findings, IL-1beta- and TNF-alpha-induced a time-dependent increase in Erk1/Erk2 activation. This cytokine action was completely abolished by PD098,059 but was not altered by PDTC. Finally, IL-1beta and TNF-alpha induced degradation of NF-kappaB's bound inhibitory protein, IkappaB-alpha, leading to translocation of NF-kappaB into the nucleus. IkappaB-alpha expression was not restored to control levels by inhibition of MEK. Furthermore, inhibition of MEK with PD098,059 did not alter IL-1beta- and TNF-alpha-induced expression of active NF-kappaB. The results demonstrate that autonomous Erk and NF-kappaB pathways mediate cytokine-induced increases in iNOS expression in astroglia.     

Spatiotemporal expression of inducible nitric oxide synthase and cyclooxygenase 2 in the spinal cord during early stage sciatic nerve crush injury

BACKGROUND: Previous studies have shown that inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) participate in inflammatory immune responses and neuropathic pain following peripheral nerve injury. However, few reports have addressed time-dependent expression of iNOS and COX-2 following peripheral nerve injury. OBJECTIVE: To investigate spatiotemporal expression of iNOS and COX-2 during early stage sciatic nerve crush injury.DESIGN, TIME AND SETTING: The randomized, controlled, animal experiment was performed at the Laboratory of Applied Anatomy, Department of Human Anatomy and Neurobiology, Central South University, China from September 2006 to September 2007.MATERIALS: Mouse anti-rat iNOS monoclonal antibody and goat anti-rat COX-2 monoclonal antibody (Transduction Laboratory, USA), as well as biotinylated rabbit anti-mouse lgG and biotinylated rabbit anti-goat IgG (Santa Cruz Biotechnology, USA) were used in the present study.METHODS: A total of 48 healthy, adult, Sprague Dawley rats were randomly assigned to three groups. In the model group (n = 32), crush injury to the right sciatic nerve was established using an artery clamp. The model group was further assigned to four subgroups according to survival time (6,12, 24, and 72 hours), respectively (n = 8). Sham surgery (n = 8) and normal control (n = 8) groups were also established.MAIN OUTCOME MEASURES: iNOS and COX-2 expression was detected in the L4-6 spinal cord with immunohistochemistry. Gray values of iNOS- and COX-2-postive cells in the anterior horn and posterior horn of spinal cord, as well as quantification of iNOS- and COX-2-positive cells in the anterior horn of spinal cord, were measured.RESULTS: iNOS and COX-2 expression gradually increased in the anterior horn and posterior horn of the spinal cord on the damaged side over time from 6 hours following sciatic nerve injury (P＜0.05) and peaked at 72 hours. Simultaneously, the number of iNOS- and COX-2-positive cells similarly increased in the anterior horn of spinal cord on the damaged side (P＜ 0.05).CONCLUSION: iNOS and COX-2 expression increased in the spinal cord during early stage sciatic nerve crush, which suggested that iNOS and COX-2 participate in occurrence and development of inflammatory immune responses following peripheral nerve injury.     

Effect of suckling on NADPH-diaphorase (Nitric oxide synthase, NOS) reactivity and NOS gene expression in the paraventricular and supraoptic nuclei of lactating rats

This study examined the effect of suckling on nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d, a histochemical marker for nitric oxide synthase, NOS) reactivity and neuronal NOS mRNA expression in the paraventricular (PVN) and supraoptic (SON) nuclei of lactating rats. Freely nursing (non-separated) dams and those separated from pups for 12 h and then reunited for 0, 15, 30, 60, 90, 120 and 180 min were used for the study. Dams separated from pups and sacrificed at time zero (without reunion) showed a significant decrease in NADPH-d staining and NADPH-d positive cells as well as in the NOS mRNA expression in the PVN and SON compared to that observed in non-separated dams. Reunion with pups and restoration of suckling significantly increased NADPH-d reactivity after 15, 30, 60 min, but not after 90, 120 and 180 min compared to non-reunited pups-deprived dams. A pattern of NADPH-d reactivity and neuronal NOS mRNA expression indistinguishable from that observed during free lactation was reinstated shortly (15 min) after the restoration of suckling stimulus, suggesting that the NADPH-d reactivity in lactation depends on the presence of the suckling stimulus. These results show that suckling stimulus may play a modulatory role in the regulation of NOS reactivity in the magnocellular neurones of the hypothalamic PVN and SON during lactation.     

The susceptibility of muscle cells to oxidative stress is independent of nitric oxide synthase expression

The free radical, nitric oxide (NO.), has been implicated in the pathogenesis of muscular dystrophies because the enzyme, nitric oxide synthase (NOS), which produces NO., binds to the dystrophin-glycoprotein complex (DGC). In various studies of tissue samples from human and animal muscular dystrophies due to DGC defects, correlations between reductions of NOS activity and disease severity have been reported. To test for any direct effect of NOS expression on muscle cell susceptibility, we examined muscle cells in vitro under conditions of experimentally altered NOS activity. There were no differences in susceptibility to oxidative stress between differentiated myotube cultures from wild-type and from neuronal NOS (nNOS)-deficient mice. Likewise, pharmacological inhibition of NOS did not alter cellular susceptibility to oxidative challenges. Overexpression of NOS neither enhanced nor diminished cellular susceptibility to oxidative stress. Finally, we assessed the effect of NOS overexpression on myotube cultures from dystrophin-deficient (mdx) mice. NOS protein was localized to both membrane and cytosolic compartments in the transduced cells. Still, no difference in susceptibility to oxidative stress was found between the NOS-overexpressing cells and control cells. These data suggest that muscle cell susceptibility to oxidative challenges is independent of the level of NOS expression. Therefore, any role NO. may play in the pathogenesis of muscular dystrophies is likely to be independent of its effect on the redox state of the cell.     

Endogenous nitric oxide modulates GABAergic transmission to granule cells in adult rat cerebellum

Nitric oxide (NO) is a gaseous neurotransmitter which plays an important role in neuronal signalling and plasticity throughout the brain. In the cerebellum, NO synthase (NOS) is expressed in parallel fibres and within the internal granule cell layer (IGL). During development there are changes in NOS concentration, distribution and activity within the IGL, suggesting NO may play a role in IGL function. Therefore, the actions of NO in the IGL were investigated. The similar actions of a range of NOS inhibitors and NO scavengers strongly suggested the presence of a tonic level of endogenous NO in the IGL. Both the neuronal and inducible forms of NOS appeared to be sources of this endogenous NO. The effects observed following a reduction in the concentration of endogenous NO were consistent with enhanced granule cell GABAA receptor activation. For example, a reduction in NO concentration led to an increase in the frequency of action potential-dependent phasic GABAergic inhibitory postsynaptic currents (IPSCs) and produced a TTX-insensitive GABAA receptor-mediated current. A direct action of NO on Golgi cell membrane potential and input resistance accounted for the increase in the frequency of phasic GABA release. The mechanism underlying the tonic GABA current is unclear but does not appear to be via the modulation of GABA uptake or the activation of nicotinic acetylcholine receptors. NO is a potentially novel mechanism for tuning GABAergic signalling to granule cells and therefore modulating the throughput of an important cerebellar circuit.     

神经型一氧化氮合酶在缺血预适应中的作用及其机制研究

目的 建立化学模拟缺血预适应的细胞模型,研究缺血预适应过程中神经型一氧化氮合酶(nNOS)的作用及其机制.方法 用300 μmol/L CoCl2预处理SK-N-SH细胞3 h.24 h后以无血清培养基培养细胞,建立缺血预适应的细胞模型,同时应用nNOS特异性抑制剂7-NI,研究nNOS在缺血预适应中的作用.在缺血预适应后不同时段以MTT法测定细胞增殖情况,流式细胞技术测定细胞凋亡情况,并与无血清刺激组进行比较;同时以RT-PCR技术检测nNOS和凋亡相关基因在缺血预适应后的表达情况.结果 300 μmol/L CoCol2预处理可以使细胞在无血清培养条件下的增殖能力较无血清刺激组增加2.5倍,同时使细胞凋亡减少70%,与无血清刺激组比较差异均有统计学意义(P<0.05).缺血预适应可在3 h和24 h上调nNOS的表达,同时上调凋亡抑制基因Bcl-2的表达,下调凋亡促进基因Apaf-1的表达.而这些调节作用可被nNOS抑制剂7-NI抑制.结论 300 μmol/LCoCl2预处理SK-N-SH细胞3 h可以模拟缺血预适应的保护作用.nNOS在缺血预适应中可调节Bel-2和Apaf-1基因的表达从而起到重要的作用.     

Noradrenergic regulation in mouse supraoptic nucleus involves a nitric oxide pathway only to regulate arginine-vasopressin expression and not oxytocin expression

Noradrenalin (NA) regulates the expression of arginine-vasopressin (AVP) and oxytocin (OT) by magnocellular neurons in the supraoptic nucleus (SON) of the hypothamalus. Nitric oxide (NO) may be one of the factors involved in the NA signaling pathway regulating AVP and OT expression. To test this possibility, we used an ex vivo experimental model of mouse hypothalamus slices. Increases in AVP and OT levels in the SON were detected by immunohistochemistry and immunoenzyme assays after 1 hr and 4 hr incubations with NA (10(-4) M). There was also an increase in the expression and activity of neuronal NOS and inducible NOS in the SON as assessed by immunohistochemical and histoenzymological analysis of NADPH-diaphorase, whereas endothelial NOS was undetectable. To specify the role of NO, the slices were treated with NA and L-arginine methyl ester (L-NAME, an NOS inhibitor; 3 microM). This treatment for 1 hr abolished the NA-induced increase in AVP. Treatment with sodium nitroprusside (SNP, an NO donor; 0.1 mM) increased AVP levels, confirming that NO regulates AVP expression. Addition of 1 mM EGTA during the incubation with NA reduced the AVP increase by half, indicating that both nNOS and iNOS activities are involved in the regulation. A 1-hr treatment with L-NAME did not prevent the increase in OT induced by NA; similarly, treatment with SNP had no effect. These findings show that NO is involved in the regulation of AVP expression by NA and that NA control of OT expression is independent of NO.