Expression of cGMP-dependent protein kinases (I and II) and neuronal nitric oxide synthase in the developing rat cerebellum

The expression of neuronal nitric oxide synthase (nNOS) and the cGMP-dependent protein kinases cGKI and cGKII in rat cerebellum was evaluated at different developmental stages by quantitative RT-PCR and Western blotting. mRNAs coding for these proteins were detected in the cerebella of rats aged 7, 14 and 21 days. Expression levels, nevertheless, varied significantly at each of these developmental stages. While nNOS and cGKI mRNA levels steadily increased during development, cGKII mRNA showed a different behaviour pattern, with similar levels observed on postnatal days 7 and 14 and increased levels noted on postnatal day 21. Moreover, protein expression profiles for nNOS and cGKI showed similar patterns to the mRNAs encoding these proteins. Our results reveal the developmental regulation of the expression of these proteins in the cerebellum, giving rise to higher levels as the cerebellum matures.     

Involvement of the nitric oxide/protein kinase G pathway in polychlorinated biphenyl-induced cell death in SH-SY 5Y neuroblastoma cells

Polychlorinated biphenyls (PCB) are persistent environmental contaminants whose chronic exposure can affect nervous system development and function. The cellular and molecular mechanisms underlying neuronal damage are not yet clear. In the present study, we investigated whether nitric oxide (NO) could be involved in aroclor 1254 (A1254; a PCB mixture)-induced cytotoxicity in SH-SY5Y human neuroblastoma cells. Prolonged exposure (24 hr) to A1254 (10-100 microg/ml) caused a dose-dependent reduction of cell viability that was attenuated in the presence of a calcium entry blocker, gadolinum (Gd(3+)) at 10 microM, a concentration able to block voltage-sensitive calcium channels. In addition, A1254 caused an increase of cytosolic calcium that was dependent on extracellular calcium, as measured by fura-2 videomicroscopy. A1254-induced calcium rise may stimulate NO production through an activation of neuronal NOS (nNOS). Indeed, the concomitant addition of the selective nNOS inhibitor N(omega)-propyl-L-arginine (NPLA) and A1254 prevented cell injury, suggesting that NO production plays a major role in A1254-evoked cell injury. Furthermore, the exposure (14 hr) to A1254 (30 microg/ml) produced an up-regulation of the expression of beta isoform of nNOS. This up-regulation was calcium dependent and was accompanied by an enhancement of NO production as demonstrated by an increase of nitrite formation. Moreover, A1254-induced cell injury was prevented when KT 5823, a selective cGMP/PKG inhibitor, was added concomitantly to 30 microg/ml A1254. These results suggest that PCB-induced cell death in neuroblastoma cells is mediated by an activation of the cGMP/PKG pathway triggered by NO production.     

Protective effects of lipopolysaccharide preconditioning against nitric oxide neurotoxicity

We have characterized lipopolysaccharide (LPS) preconditioning-induced neuroprotective mechanisms against nitric oxide (NO) toxicity. Pretreatment of rat cortical cultures with LPS attenuated neurotoxicity of NO donors, including sodium nitroprusside (SNP) and diethylamine NONOate (NONOate). A transiently increased expression of endothelial nitric oxide synthase (eNOS) accompanied by an increase in NO production was observed during LPS preconditioning. Application of NOS inhibitors including L-N(5)-(1-iminoethyl)-ornithine (L-NIO) and L-nitroarginine methylester (L-NAME) abolished LPS-dependent protection against SNP toxicity. The LPS effect was also blocked by KT5823, an inhibitor of cGMP-dependent protein kinase (PKG). Consistently, application of 8-bromo-cyclic GMP (8-Br-cGMP), a slowly degradable cGMP analogue capable of PKG activation, was neuroprotective. LPS preconditioning resulted in a heightened neuronal expression of Bcl-2 protein that was abolished by L-NAME and KT5823, the respective inhibitors of NOS and PKG. Together, our results reveal the signaling cascade of "LPS --> eNOS --> NO --> cGMP/PKG --> Bcl-2" that might have contributed to the LPS protective effects in cortical neurons.     

自发性高血压大鼠脑缺血后脑区一氧化氮的变化

目的：研究自发性高血压大鼠（SHR）脑缺血后大脑皮质、海马、纹状体和小脑组织中一氧化氮（NO）的变化。采用放射免疫法和荧光分光光度法检测脑组织中一氧化氮合酶（NOS）和亚硝酸盐（NO2）的含量。结果显示SHR脑缺血10min,各脑区NOS和NO2,的含量均明显高于假手术组（P<0．01或P＜005）。说明了SHR脑缺血早期脑组织NO的生成增加．提示用特异的NO生成抑制剂类药物,可能有助于脑缺血的治疗。     

Hyperlipidemia affects neuronal nitric oxide synthase expression in brains of focal cerebral ischemia rat model★

BACKGROUND: Hyperlipidemia, a risk factor for ischemic cerebrovascular disease, may mediate production of neuronal nitric oxide synthase (nNOS) to induce increased nitric oxide levels, resulting in brain neuronal injury. OBJECTIVE: To investigate effects of hyperlipidemia on brain nNOS expression, and to verify changes in infarct volume and pathology during reperfusion, as well as neuronal injury following ischemia/reperfusion in a rat model of focal cerebral ischemia. DESIGN, TIME AND SETTING: Complete, randomized grouping experiment was performed at the Laboratory of Physiology, Shanxi Medical University from March 2005 to March 2006. MATERIALS: A total of 144 eight-week-old, male, Wistar rats, weighing 160-180 g, were selected. A rat model of middle cerebral artery occlusion was established by suture method after 4 weeks of formulated diet. Nitric oxide kit and rabbit anti-rat nNOS kit were respectively purchased from Nanjing Jiancheng Bioengineering Institute, China and Wuhan Boster Biological Technology, Ltd., China. METHODS: The rats were equally and randomly divided into high-fat diet and a normal diet groups. Rats in the high-fat diet group were fed a high-fat diet, consisting of 10% egg yolk powder, 5% pork fat, and 0.5% pig bile salt combined with standard chow to create hyperlipidemia. Rats in the normal diet group were fed a standard rat chow. A total of 72 rats in both groups were randomly divided into 6 subgroups: sham-operated, 4-hour ischemia, 4-hour ischemia/2-hour reperfusion, 4-hour ischemia/4-hour reperfusion, 4-hour ischemia/6-hour reperfusion, and 4-hour ischemia/12-hour reperfusion, with 12 rats in each subgroup. MAIN OUTCOME MEASURES: nNOS expression was measured by immunohistochemistry, and pathomorphology changes were detected by hematoxylin-eosin staining. Infarct volume and nitric oxide levels were respectively measured using 2, 3, 5-triphenyltetrazolium chloride (TTC) and immunohistochemistry. RESULTS: In the ischemic region, pathology changes were significant in the 4-hour ischemia/4-hour, 4-hour ischemia/6-hour reperfusion, and 4-hour ischemia/12-hour reperfusion subgroups fed on a high-fat diet compared to the same groups fed on a normal diet. In each ischemia subgroup, nNOS expression in brain tissues was higher than in the sham-operated subgroups fed on either the high-fat diet or normal diet (P< 0.01). At each ischemia/reperfusion time point, rats fed on a high-fat diet expressed higher levels of nNOS compared to rats fed on the normal diet (P<0.05). When tissue was stained with TTC, a white infarction area was detected in the ischemic hemisphere, demonstrating that the infarct volume gradually increased with prolonged reperfusion time in each ischemia subgroup. At each ischemia/reperfusion time point, the infarct volume was larger in rats fed on a high-fat diet compared to those fed on a normal diet. CONCLUSION: nNOS expression was greater in hyperlipidemia rats following ischemia/reperfusion. Cerebral ischemia/reperfusion injury is aggravated with prolonged reperfusion time.     

神经节苷脂对不完全性脑缺血后海马CA1区NOS阳性神经元表达的影响

目的 :探讨神经节苷脂 (GM1)对不完全性脑缺血及再灌注不同时间后海马CA1区一氧化氮合酶 (NOS)的影响及对神经元的保护作用。方法 :用双侧颈总动脉夹闭加放血的方法制成大鼠不完性脑缺血及再灌注模型 ,以还原尼克酰胺腺嘌呤二核苷酸脱氢酶 (NADPH d)组织化学方法观察缺血及再灌注后海马CA1区NOS阳性神经细胞变化及GM1对其影响。结果 :海马CA1区神经细胞受损 ,在缺血 30min时NOS阳性细胞数最高 (44 .5±7.4 ) ,为对照组的 2倍 ,再灌注 2h ,12h ,2 4h ,3d后逐渐下降 ,5d时恢复正常水平。而GM1能防止脑缺血及再灌注后神经细胞受损和NOS阳性神经细胞变化。 结论 :GM1对大鼠不完全性脑缺血及再灌注不同时间后海马CA1区NOS的表达有抑制作用 ,并对神经元具有保护作用。     

癫痫患儿血清一氧化氮和一氧化氮合酶含量的变化及意义

目的　探讨癫疒间 患儿血清一氧化氮 (NO)、一氧化氮合酶 (NOS)的变化及意义。方法　利用ELISA方法 ,测定 5 8例癫疒间 患儿 (癫疒间 组 )和 2 3名健康儿童 (对照组 )血清中NO、NOS的含量 ,并分组比较不同条件下其含量的变化。结果　癫疒间 组血清NO、NOS的含量分别为 (5 .86± 1.2 1) μmol/ml和 (2 8.2 6± 8.4 9)U/ml,较对照组的 (3.78± 0 .74 ) μmol/ml及 (17.86± 4 .5 8)U/ml明显升高 (P <0 0 1) ;发作近期为 (7.31± 1.2 7)μmol/ml和 (31.2 5± 11.35 )U/ml,明显高于发作间期 (4 .2 7± 0 .6 6 ) μmol/ml和 (2 4 .15± 7.85 )U/ml(P <0 0 1) ;癫疒间 组EEG异常者为 (7.18± 1.35 ) μmol/ml和 (34.4 8± 8.5 6 )U/ml,明显高于EEG正常者 (4 .0 4± 0 .75 ) μmol/ml和 (2 2 .85± 7.4 5 )U/ml(P <0 0 1) ;但与发作类型、病程及是否接受治疗无关 (P >0 0 5 )。结论　癫疒间 发作近期血中NO、NOS生成增加 ,NO作为内源性调质参与癫疒间 发作病理生理过程     

Lipopolysaccharide regulates both serotonin- and thrombin-induced intracellular calcium mobilization in rat C6 glioma cells: Possible involvement of nitric oxide synthase-mediated pathway

To investigate the mechanisms by which lipopolysaccharide (LPS) affects Ca²⁺ signaling systems, we studied the effects of LPS on the serotonin (5-HT)- or thrombin-induced intracellular Ca²⁺ ([Ca²⁺]_i) increase in rat C6 glioma cells. Pretreatment of the cells with 1 μg/ml LPS for 24 hr significantly inhibited [Ca²⁺]_i increase induced by 10 μM 5-HT- or 0.5 U/ml thrombin. Its inhibitory effects were both dose- and time-dependent. Treatment with 1 mM dibutyryl cGMP (dbcGMP) for 30 min also significantly inhibited the 5-HT- and thrombin-induced [Ca²⁺]_i increase to approximately 60–70% of control. However, simultaneous pretreatment with LPS and dbcGMP did not show any synergistic inhibition. The simultaneous pretreatment with LPS and the potent cGMP-dependent protein kinase (PKG) inhibitors H-8 and KT5823 for 24 hr significantly antagonized the inhibitory effect of LPS. Pretreatment of the cells with 1 μg/ml LPS for 24 hr significantly enhanced cGMP accumulation, while dexamethasone and NMMA (NOS inhibitors) significantly attenuated the LPS-induced enhancement in cGMP accumulation. In addition, pretreatment of the cells with 100 nM dexamethasone for 24 hr significantly suppressed LPS-induced inducible nitric oxide synthase (iNOS; type II NOS, NOS-II) protein expression. These results indicate that LPS may inhibit both 5-HT- and thrombin-induced [Ca²⁺]_i increase via iNOS expression and PKG activation pathway in rat C6 glioma cells. J. Neurosci. Res. 51:517–525, 1998. © 1998 Wiley-Liss, Inc.     

Decreased neuronal nitric oxide synthase expression and cell migration in the peri-infarction after focal cerebral ischemia in rats

Neuronal nitric oxide synthase (nNOS) regulates neurogenesis in the normal developing brain, but the role of nNOS in neurogenesis of the adult ischemic brain remains unclear. The aim of this study was to investigate the temporal and spatial relationship between cell migration from the ependymal/subventricular zone (SVZ) to peri-infarction and nNOS expression in the rat. Ependymal/subventricular zone cells were prelabeled with fluorescence dye DiI. Focal cerebral ischemia was induced by occlusion of the left middle cerebral artery. At 1, 3, 7, 14 and 21 days after ischemia, the rats were killed in order to determine the number of migrating cells, the colocalization of DiI and nNOS as well as nNOS quantity in specific regions. Compared to non-ischemic control and 1 day post-ischemia, the number of DiI-labeled cells in the selected regions increased at 3 days and peaked 14 days following ischemia. During 3–7 days post-ischemia, none of the migrating cells expressed nNOS and decreased nNOS expression was observed in the regions where migrating cells passed through. These results suggest the possible association between ependymal/SVZ cell migration and decreased nNOS expression within the areas including the migrating routes towards the peri-infarction.     

一氧化氮与一氧化氮合酶抑制剂对脑缺血后海马神经元的保护作用

目的 研究硝普钠、7-硝基吲唑(7-NI)和AMT对急性脑缺血后海马神经元细胞是否具有保护作用.方法 将80只SD大鼠随机分组,按照不同给药时间和给药方法制作全脑缺血再灌注模型,待全脑缺血15 min后再灌注,第5天行多聚甲醛灌注,石蜡包埋,尼氏染色观察海马CA1区神经元的存活情况.结果 在脑缺血再灌注后5 d,单次应...     

依达拉奉对慢性脑缺血大鼠一氧化氮及一氧化氮合酶阳性神经元数量的影响

目的观察依达拉奉对脑缺血大鼠脑组织NO含量及NOS阳性神经元数量的影响,为临床上防治慢性脑缺血引发的疾病提供指导。方法 54只Wistar大鼠,分为NO含量组和NOS阳性神经元组,每组再分为模型组、治疗组、假手术组,利用结扎大鼠两侧颈总动脉制作大鼠慢性脑缺血模型,硝酸还原酶法测NO含量,NADPH-d组织化学方法染色NOS阳性神经元,光镜下观察。结果治疗组各时间点NO含量和NOS阳性神经元数量较模型组减少。结论依达拉奉对慢性脑缺血大鼠脑组织具有保护作用。     

脑缺血早期大脑皮质区神经元内一氧化氮合酶活性的变化

建立大鼠ＭＣＡＯ局灶脑缺血模型,利用ＮＡＤＰＨ－ｄ组织化学方法检测脑缺血早期大脑皮质缺血区神经元内一氧化氮合酶活性的变化。结果显示脑缺血早期３０ｍｉｎ神经元内一氧化氮合酶活性开始至高峰,随后下降,脑缺血后６０ｍｉｎ,降至正常。     

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。     

Role of neuronal and inducible nitric oxide synthases in the guinea pig ileum myenteric plexus during in vitro ischemia and reperfusion

Background Intestinal ischemia and reperfusion (I/R) injury leads to abnormalities in motility, namely delay of transit, caused by damage to myenteric neurons. Alterations of the nitrergic transmission may occur in these conditions. This study investigated whether an in vitro I/R injury may affect nitric oxide (NO) production from the myenteric plexus of the guinea pig ileum and which NO synthase (NOS) isoform is involved. Methods The distribution of the neuronal (n) and inducible (i) NOS was determined by immunohistochemistry during 60 min of glucose/oxygen deprivation (in vitro ischemia) followed by 60 min of reperfusion. The protein and mRNA levels of nNOS and iNOS were investigated by Western‐immunoblotting and real time RT‐PCR, respectively. NO levels were quantified as nitrite/nitrate. Key Results After in vitro I/R the proportion of nNOS‐expressing neurons and protein levels remained unchanged. nNOS mRNA levels increased 60 min after inducing ischemia and in the following 5 min of reperfusion. iNOS‐immunoreactive neurons, protein and mRNA levels were up‐regulated during the whole I/R period. A significant increase of nitrite/nitrate levels was observed in the first 5 min after inducing I/R and was significantly reduced by N^ω‐propyl‐l ‐arginine and 1400 W, selective inhibitors of nNOS and iNOS, respectively. Conclusions & Inferences Our data demonstrate that both iNOS and nNOS represent sources for NO overproduction in ileal myenteric plexus during I/R, although iNOS undergoes more consistent changes suggesting a more relevant role for this isoform in the alterations occurring in myenteric neurons following I/R.     

Involvement of endogenous nitric oxide signalling system in brain muscarinic acetylcholine receptor activation

Summary. Biochemical signalling events coupled to muscarinic cholinergic receptors (mAChR), specifically those related to nitric oxide (NO) production, were studied on rat cerebral frontal cortex. The mAChR agonist carbachol was found to exert a specific biphasic action on NO synthase (NOS) activity: low doses ranging between 10⁻⁹ M to 10⁻⁷ M lead to NOS activation while higher doses (>10⁻⁶ M) inhibited enzymatic activity. Carbachol stimulatory action was blunted by agents that interfere with calcium-calmodulin while a protein kinase (PKC) inhibitor, staurosporine was able to abrogate the inhibitory effect. Moreover, PKC activity showed maximum translocation to cerebral frontal cortex membranes with carbachol concentrations that inhibited NO production. Products from phosphoinosite (PI) hydrolysis are involved in these actions as carbachol was found to increase PI turnover in a dose dependent manner. These results would serve as an example of cross-talk between both enzymatic pathways. Accepted December 22, 1997; received October 2, 1997     

首发偏执型精神分裂症血清NO/NOS水平的研究

目的探讨首发偏执型精神分裂症血清一氧化氮/一氧化氮合成酶（NO/NOS）水平及与精神症状的关系。方法共收集首发偏执型精神分裂症患者26例（研究组）,健康对照者30例（对照组）,采用阳性与阴性症状量表（PANSS）评定患者的精神症状,同时检测血清NO/NOS水平。结果首发偏执型精神分裂症血清NO/NOS水平均显著高于健康对照组（t=2.08,P〈0.05;t=2.72,P〈0.05）,血清NO/NOS水平与精神症状无显著相关性（P〉0.05）;血清NO水平与血清NOS水平两者存在显著正相关（r=0.41,P〈0.05）。结论首发偏执型精神分裂症患者存在血清NO/NOS水平病理性增高。     

Protein tyrosine kinase inhibitors suppress the production of nitric oxide in mixed glia, microglia-enriched or astrocyte-enriched cultures

Nitric oxide (NO) produced by glial cells has been implicated in the neuropathogenesis of various diseases. However, the signaling transduction pathway(s) for the production of NO in these cells is not well understood. To test whether protein tyrosine kinases (PTKs) are required for signaling events of NO production in glial cells, this study examined the effects of genistein and tyrphostin A25, two potent inhibitors of PTKs, on the production of NO in mouse primary mixed glia, microglia-enriched or astrocyte-enriched cultures exposed to lipopolysaccharide (LPS) or a combination of LPS and interferon-γ (IFNγ). LPS induced a dose-dependent increase in NO production from the mixed glia cultures. The LPS-induced NO production was significantly enhanced by stimulating the cells with IFNγ. Genistein or tyrphostin A25 inhibited the production of NO in both LPS- and IFNγ/LPS-stimulated mixed glia cultures. The production of NO in the stimulated microglia-enriched or astrocyte-enriched cultures was also inhibited by tyrphostin A25. To verify the cellular sources of NO, immunocytochemical staining of inducible NO synthase (iNOS) was followed by staining with the microglia marker Mac-1 or the astrocyte marker glial fibrillary acid protein (GFAP) in microglia-enriched or astrocyte-enriched cultures. The expression of iNOS and the production of NO in microglia-enriched cultures were significantly higher than those in the identically stimulated astrocyte-enriched cultures. These results demonstrate that PTKs are involved in the signaling events of LPS-induced NO production in microglia and astrocytes, and that microglia are more responsive than astrocytes to stimuli which induce NO. These results may provide insights into therapeutic interventions in the pathway for NO production in the brain.     

NADPH-diaphorase/nitric oxide synthase containing neurons in normal and Parkinson's disease putamen

Summary Nitric oxide (NO) is thought to be involved in neurodegenerative processes. Concerning Parkinson's disease (PD) it remains to be elucidated, if NO contributes to pathological alterations in the striatum. The present study evaluates the post-mortem putamen of PD patients and control subjects for distribution patterns of NO-synthase containing neurons, using the NADPH-diaphorase technique. The ratio of positively stained neurons and the total number of cells (control: 1,120±69 per mm², n=5; PD: 575±164mm², n=5) shows striking differences between controls and PD patients. Our findings give reason to conclude that NADPH-diaphorase positive structures may have pathogenetic importance in degenerative processes in PD putamen.     

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。