Identification and characterization of nitric oxide synthase inSalmonella typhimurium

The presence of the nitric oxide synthase (NOS) enzyme from Salmonella typhimurium (S. typhimurium) was identified by measuring radiolabeled L-[3H]citrulline and NO, and Western blot analysis. NOS was partially purified by both Mono Q ion exchange and Superose 12HR size exclusion column chromatography, sequentially. The molecular weight of NOS was estimated to be 93.3 kDa by Western blot analysis. The enzyme showed a significant dependency on the typical NOS cofactors; an apparent Km for L-arginine of 34.7 mM and maximum activity between 37 degrees C and 43 degrees C. The activity was inhibited by NOS inhibitors such as aminoguanidine and N(G),N(G)-dimethyl-L-arginine. Taken together, partially purified NOS in S. typhimurium is assumed to be a different isoform of mammalian NOSs.     

一氧化氮及一氧化氮合酶在成年与老年大鼠前列腺中的变化

目的:研究一氧化氮(NO)及一氧化氮合酶(NOS)在成年与老年大鼠前列腺中的变化,探讨在大鼠前列腺老年化过程中的临床意义。方法:取4月龄及20月龄SD雄性大鼠各6只,分别取前列腺组织匀浆,检测其NO与NOS活性。结果:老年组大鼠的NO与NOS活性明显低于成年组(P<0.05)。结论:老年大鼠前列腺组织中NO水平及NOS活性的降低可能与前列腺良性增生及功能减退有关。     

Inhibition of neuronal nitric oxide synthase by antipsychotic drugs

There is rapidly accumulating evidence that generation of nitric oxide (NO) through a Ca²⁺ and calmodulin-dependent pathway plays various important roles in the central nervous system. In the present study, effects of several antipsychotics on the activity of NO synthase were investigated in rat cerebellum and neuroblastoma N1E-115 cells, due to the known ability of these agents to inhibit calmodulin. In cytosolic preparations of rat cerebellum, the antipsychotic drugs inhibited the conversion of [³H]l-arginine into [³H]l-citrulline by NO synthase in a concentration-dependent manner. This inhibition was noncompetitive in nature, and it exhibited an excellent correlation with blockade of calmodulin activity. Furthermore, these drugs attenuated cyclic GMP formation induced by a calcium ionophore in N1E-115 cells, a response which takes place as a consequence of NO generation. Taken together, our data demonstrate that antipsychotic drugs inhibit NO formation in vitro. It is unlikely, however, that these actions might contribute to their therapeutic and/or side effects, since they take place at relatively high concentrations.     

胃幽门螺杆菌与胃粘膜一氧化氮合酶表达的意义

目的探讨在胃癌发生过程中Hp感染与iNOS表达的关系及意义。方法采用快速尿素酶试验及Warthin-Starry银染色法检测入选病例的Hp,用免疫组织化学染色法检测iNOS。结果①在浅表性胃炎、胃溃疡、萎缩性胃炎及胃癌各组中Hp感染阳性率分别为55.2%、56.0%、43.5%、65.4%,各组间无显著性差异(P>0.05)。②iNOS表达在浅表性胃炎、胃溃疡、萎缩性胃炎和胃癌各组中阳性率分别为51.7%、52.0%、65.2%、80.8%,各组同浅表性胃炎比较:胃癌组有显著性差异(P<0.05),萎缩性胃炎组无显著性差异(P>0.05)。③Hp感染与iNOS表达的关系:各组慢性胃病中Hp感染阳性者iNOS表达率均显著高于非感染者(P<0.05)。④iNOS于胃癌中表达增强,在不同分化程度的胃癌中的表达无明显规律性。结论Hp可能参与慢性胃病的发生发展过程。iNOS高表达提示NO在炎症、溃疡乃至胃癌发生过程中有重要意义。     

Balloon angioplasty and induction of non-endothelial nitric oxide synthase in rabbit carotid arteries

The purpose of the study was to evaluate whether balloon angioplasty is associated with changes in nitric oxide synthase (NO synthase) activity. Normal rabbit carotid arteries were examined 10 min or 1, 2, 3 or 10 weeks after angioplasty with 2 or 2.5-mm balloons. Immunohistology was used to evaluate intimal thickening and endothelial cell regeneration. The NO synthase activity was studied functionally using isolated segments in organ chambers. Immunohistochemistry of the endothelial cell markers von Willebrand factor and platelet endothelial cell adhesion molecule-1 indicated that the regeneration of endothelial cells from patchy islands that remained after angioplasty was virtually complete within 2 weeks. However, the endothelium-dependent relaxations elicited by acetylcholine remained impaired up to 10 weeks after dilatation. Contractions elicited by 5-hydroxytryptamine (5-HT) were attenuated, but were significantly augmented by the NO synthase blocker, nitro- -arginine. Furthermore, in contrast to normal arteries, the balloon-treated arteries developed marked contractions in response to nitro- -arginine methyl ester ( -NAME), contractions which could be reversed by -arginine. The latter contractions and relaxations were not influenced by endothelial removal. These results suggest that although the endothelium quickly regenerates after severe balloon injury, the endothelium-dependent release of nitric oxide remains disturbed. However, the functional data also suggest that angioplasty led to a significant induction of NO synthase in ‘non-endothelial’ cells of the artery.     

血浆一氧化氮、一氧化氮合酶和基质金属蛋白酶9在子宫颈癌中的临床意义

目的 探讨血浆一氧化氮（NO）、一氧化氮合酶（NOS）和基质金属蛋白酶（MMP-9）水平在子宫颈癌中的变化及临床意义.方法 经病理活检确诊为宫颈癌患者69例,分别于手术前1d、术后15 d、30 d空腹抽取外周静脉血,健康对照组取空腹静脉血,采用化学比色法和ELISA法分别测定血浆中NO、NOS和MMP-9含量,比较上述指标的变化及相关性.结果 宫颈癌患者血浆中NO、NOS、MMP-9的水平与健康对照组相比明显增高.宫颈癌患者手术前与手术后15 d、30 d NO水平比较,手术后NO、NOS、MMP-9的水平均明显降低,但仍明显高于对照组.宫颈癌患者有淋巴结转移者NO、NOS、MMP-9水平明显高于无淋巴结转移者;相关性分析表明,血清中MMP-9与NO、NOS呈明显正相关性.结论 检测血浆中NO、NOS和MMP-9水平的变化,对宫颈癌的病情判断及临床治疗指导具有参考意义.     

Suppression of inducible nitric oxide synthase and cyclooxygenase-2 expression by tussilagone from farfarae flos in BV-2 microglial cells

Activated microglia produce diverse neurotoxic factors such as nitric oxide (NO) and prostaglandin E(2) (PGE(2)) that may cause neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. From the EtOAc soluble fraction of Farfarae flos (Tussilago farfara), we purified tussilagone as a bioactive compound by monitoring the inhibitory potential of NO production in activated microglia through the purification procedures. Tussilagone showed dose-dependent inhibition of NO and PGE(2) production in LPS-activated microglia with IC(50) values of 8.67 microM and 14.1 microM, respectively. It suppressed the expression of protein and mRNA of inducible nitric oxide synthase and cyclooxygenase-2 through the inhibition of 1-kappaBalpha degradation and nuclear translocation of p65 subunit of NF-kappaB. Therefore tussilagone from Farfarae flos may have therapeutic potential in the treatment of neuro-inflammatory diseases through the inhibition of overproduction of NO and PGE(2).     

Vigilance and EEG power in rats: effects of potent inhibitors of the neuronal nitric oxide synthase

We examined the effects of potent neuronal nitric oxide synthase inhibitors, 3-bromo-7-nitro indazole (3-Br-7-NI) and S-methyl-L-thiocitrulline (S-Me-TC) on general behaviour, vigilance stages and electro-encephalographic (EEG) power spectra in rats. In addition, we studied the effect of 7-nitro indazole (7-NI) on EEG power spectra in rats during dark and light periods. 3-Br-7-NI induced ptosis and decrease of slow wave sleep and rapid eye movement sleep in the rat. 7-NI and 3-Br-7-NI reduced the EEG power density in all frequency bands in the rat, suggesting a depression of central neuronal activity. This effect of 7-NI was more prominent during the day than during the night, indicating a circadian variation in the nitric oxide synthase (NOS) response to NOS inhibitor. EEG power was the most reduced in the 7-9Hz range of the rhythmic slow activity (theta rhythm), which is in accordance with decreased locomotion observed following administration of NOS inhibitors. Although S-Me-TC is the most potent NOS inhibitor in vitro experiments, it had less effect on vigilance and EEG power in the rat than other NOS inhibitors used in this study, probably due to its short lasting and blood pressure raising effect. The present results indicate that nitric oxide exerts an excitatory and circadian dependent effect in the central neuronal structures involved in the regulation of vigilance. Received: 16 September 1996 / Accepted: 4 April 1997     

脑复合剂对创伤性脑损伤模型大鼠脑内NO、nNOS活性及表达的影响

目的:探讨脑复合剂治疗对创伤性脑损伤(traumatic brain injury,TBI)模型大鼠脑保护作用可能的分子机制.方法:用自由落体打击(Feeney法)建立TBI模型,分别设立假手术组、TBI模型组及中药治疗组.中药治疗组给予脑复合剂10 g· kg-1·d-1,假手术组及TBI模型组给予同等剂量的等渗盐水...     

乳铁蛋白对福尔马林致痛大鼠行为学及一氧化氮合酶表达的影响

目的通过观察鞘内注射乳铁蛋白对福尔马林致痛大鼠行为学及一氧化氮合酶（NOS）表达的影响，探讨乳铁蛋白可能的抗伤害机制。方法雄性SD大鼠80只，随机分为生理盐水组（NS组）和福尔马林组（F组）；实验组分为乳铁蛋白1ug-福尔马林组（F-R1组），乳铁蛋白10ug-福尔马林组（F—R10组）和乳铁蛋白100ug-福尔马林组（F—R100组）。五组鞘内分别给予NS 20ul，NS 20ul，乳铁蛋白1ug，乳铁蛋白10ug，乳铁蛋白100ug；10min后，除NS组大鼠组底注射NS 100ul外，其余各组均给予5％福尔马林100ul。经以上处理8只动物在福尔马林处理后30min取脊髓观察NOS的表达，另8只动物在福尔马林处理后0～1h观察行为学。结果F组的缩腿舔爪的时间和脊髓NOS表达显著长于，强于NS组；预先给予乳铁蛋白能抑制以上的作用，且呈剂量依赖性。结论乳铁蛋白明显抑制福尔马林致痛大鼠痛行为及NOS表达，可能是其产生抗伤害作用机制之一。     

Enhancement of nitric oxide synthase induction in alveolar macrophages by in vivo administration of docetaxel

Effects of docetaxel, a taxane-derivative anti-cancer drug, on lipopolysaccharide (LPS)-induced nitric oxide (NO) synthesis were investigated in alveolar macrophages isolated from rats. LPS-induced NO production and inducible NO synthase (iNOS) expression were significantly enhanced in the macrophages isolated from rats injected intraperitoneally with docetaxel (4 mg/kg body weight per day for 5 consecutive days) compared with those in macrophages from control rats administrated a vehicle. In vivo administration of docetaxel augmented LPS-induced p38 activation but not extracellular signal-related kinase (ERK) activation in isolated macrophages. On the other hand, in vitro treatment of macrophages with docetaxel (5 and 10 μg/ml) inhibited LPS-induced NO production and iNOS expression. Levels of lactate dehydrogenase released from macrophages were neither affected by in vitro treatment with docetaxel (up to 10 μg/ml) nor by its in vivo administration. These results suggest that docetaxel has an enhancing action in vivo on LPS-induced iNOS expression and subsequent NO production through stimulation of p38 activity in alveolar macrophages.     

Inhibitory effect of CDP, a polysaccharide extracted from the mushroom Collybia dryophila, on nitric oxide synthase expression and nitric oxide production in macrophages

The effect of Collybia dryophila polysaccharide (CDP), a (1-->3), (1-->4)-beta-D-glucan extracted from the mushroom C. dryophila, was evaluated on nitric oxide (NO) production induced by lipopolysaccharide (LPS) and gamma interferon (IFNgamma) or by LPS alone in RAW 264.7 cells. CDP significantly inhibited NO production in a dose-dependent manner without affecting cell viability. The inhibition of NO by CDP was consistent with decreases in both inducible nitric oxide synthase (iNOS) protein and mRNA expression suggesting that CDP exerts its effect by inhibiting iNOS gene expression. In addition, CDP at concentrations of 400 and 800 microg/ml was shown to significantly increase prostaglandin E2 (PGE2) production in LPS- and IFNgamma-induced macrophages when compared to the control.     

Regulation of the inducible cyclo-oxygenase pathway in human cultured airway epithelial (A549) cells by nitric oxide

1. In airway epithelium, nitric oxide (NO) is synthesized in the setting of inflammation by inducible nitric oxide synthase (iNOS). Although the role of epithelial derived NO in the regulation of human airways is unknown, prostaglandin E₂ (PGE₂) is recognised as an important inhibitory mediator in human airways. Cyclo-oxygenase (COX) is the rate limiting enzyme in the production of prostanoids and since inflammatory pathways enhance the expression of an inducible COX (COX-2), both COX-2 and iNOS may be co-expressed in response to an inflammatory stimulus. Although regulation of the COX-2 pathway by NO has been demonstrated in animal models, its potential importance in human airway epithelium has not been investigated.
2. The effect of endogenous and exogenous NO on the COX-2 pathway was investigated in the A549 human airway epithelial cell culture model. Activity of the COX-2 pathway was assessed by PGE₂ EIA, and iNOS pathway activity by nitrite assay. A combination cytokine stimulus of interferon gamma (IFNγ) 100 u ml⁻¹, interleukin-1β (IL-1β) 1 u ml⁻¹ and lipopolysaccharide (LPS) 10 μg ml⁻¹ induced nitrite formation which could be inhibited by the competitive NOS inhibitor N^G-nitro-L-arginine-methyl-ester (L-NAME). IL-1β alone (1–50 u ml⁻¹) induced PGE₂ formation without significant nitrite formation, a response which was inhibited by the COX-2 specific inhibitor nimesulide. Submaximal stimuli used for further experiments were IFNγ 100 u ml⁻¹, IL-1β 1 u ml⁻¹ and LPS 10 μg ml⁻¹ to induce both the iNOS and COX-2 pathways, and IL-1β 3 u ml⁻¹ to induce COX-2 without iNOS activity.
3. Cells treated with IFNγ 100 u ml⁻¹, IL-1β 1 u ml⁻¹ and LPS 10 μg ml⁻¹ for 48 h either alone, or with the addition of L-NAME (0 to 10⁻² M), demonstrated inhibition by L-NAME of PGE₂ (3.61±0.55 to 0.51±0.04 pg/10⁴ cells; P<0.001) and nitrite (34.33±8.07 to 0 pmol/10⁴ cells; P<0.001) production. Restoration of the PGE₂ response (0.187±0.053 to 15.46±2.59 pg/10⁴ cells; P<0.001) was observed after treating cells with the same cytokine stimulus and L-NAME 10⁻⁶ M, but with the addition of the NOS substrate L-arginine (0 to 10⁻⁵ M).
4. Cells incubated with IL-1β 3 u ml⁻¹ for 6 h, either alone or with addition of the NO donor S-nitroso-acetyl-penicillamine (SNAP) (0 to 10⁻⁴ M), demonstrated increased PGE₂ formation (1.23±0.03 to 2.92±0.19 pg/10⁴ cells; P< 0.05). No increase in PGE₂ formation was seen when the experiment was repeated in the presence of the guanylate cyclase inhibitor methylene blue (50 μM). Cells treated with SNAP alone did not demonstrate an increased PGE₂ formation. Cells incubated with IL-1β 3 u ml⁻¹ for 6 h in the presence of dibutyryl cyclic guanylate monophosphate (0 to 10⁻³ M) also demonstrated an increased PGE₂ response (2.56±0.21 to 4.53±0.64 pg/10⁴ cells; P<0.05).
5. These data demonstrate that in a human airway epithelial cell culture system, both exogenous and endogenous NO increase the activity of the COX-2 pathway in the setting of inflammatory cytokine stimulation, and that this effect is likely to be mediated by guanylate cyclase. This suggests a role for NO in the regulation of human airway inflammation.

     

一氧化氮合酶抑制剂对七叶莲花镇痛作用的影响

目的 探讨一氧化氮合酶(NOS)抑制剂与七叶莲花镇痛作用的关系. 方法 采用小鼠热板法,将小鼠随机分为等渗盐水组(阴性对照组)、曲马多组(阳性对照组)及各实验组,观察L-精氨酸(L-Arg)和NOS抑制剂L-硝基精氨酸甲酯(L-NAME)对七叶莲花醇提物(SAHF)镇痛作用的影响. 结果 与等渗盐水组比较,L-NAME可显著增强七叶莲花的镇痛作用(P<0.01);与SAHF组比较,脑组织中NO水平显著降低(P<0.05). 结论 七叶莲花的镇痛作用和NOS抑制剂之间存在一定的相互作用,L-NAME可显著增强七叶莲花的镇痛作用,提示SAHF的镇痛作用机制与降低体内过量的NO有关.     

Differential roles of nitric oxide synthase isozymes in cardiotoxicity and mortality following chronic doxorubicin treatment in mice

The roles of individual nitric oxide synthases (NOS) in anthracycline-related cardiotoxicity are not completely understood. We investigated the effects of a chronic treatment with doxorubicin (DOX) on knockouts of the individual NOS isozymes and on transgenic mice with myocardial overexpression of eNOS. Fractional shortening (FS) was reduced in untreated homozygous nNOS and iNOS knockouts as well as in eNOS transgenics. DOX-induced FS decrease in wild-type mice was attenuated only in eNOS knockouts, which were found to overexpress nNOS. No worsening of contractility was observed in DOX-treated eNOS transgenics and iNOS knockouts. Although the surviving DOX-treated nNOS knockouts exhibited no further impairment in contractility, most (70%) animals died within 7 weeks after treatment onset. In comparison to untreated wild-type hearts, the nitric oxide (NO) level was lower in hearts from DOX-treated wild-type mice and in all three untreated knockouts. DOX treatment had no effect on NO in the knockouts. These data indicate differential roles of the individual NOS in DOX-induced cardiotoxicity. Protection against DOX effects conferred by eNOS deletion may be mediated by a compensatory overexpression of nNOS. NOS inhibition-based prevention of anthracycline-induced cardiotoxicity should be eNOS-selective, simultaneously avoiding inhibiting nNOS.     

Differential regulation by protein kinase C isoforms of nitric oxide synthase induction in RAW 264.7 macrophages and rat aortic smooth muscle cells

1. In RAW 264.7 murine macrophages and rat aortic smooth muscle (RASM) cells lipopolysaccharide (LPS) alone or in combination with interferon γ (IFNγ) or forskolin, respectively, stimulated the expression of the 130 kDa inducible isoform of nitric oxide synthase (iNOS) in both a time- and concentration-dependent manner.
2. Incubation with the direct activator of protein kinase C (PKC), phorbol 12-myristate 13-acetate (PMA) alone, did not result in detectable iNOS expression in either cell type.
3. Chronic PMA pretreatment resulted in significant down-regulation of α, β and ε isoforms of PKC in RAW 264.7 macrophages and corresponded to a 20–30% reduction in LPS-induced iNOS expression. In contrast, IFNγ alone or in combination with LPS stimulated an approximate 20% and 50% potentiation, respectively.
4. Pre-incubation with PKC inhibitors (calphostin C and H-7) showed similar effects upon stimulated induction of iNOS.
5. In RASM cells chronic PMA pretreatment resulted in down-regulation of α and ε PKC isoforms and corresponded to potentiation of iNOS expression in response to LPS alone or in combination with forskolin.
6. Co-incubation of RASM cells in the presence of PMA, angiotensin II (AII) or foetal calf serum (FCS) resulted in the inhibition of iNOS expression in response to LPS alone or in combination with forskolin.
7. Differential sensitivity to PKC inhibitors (calphostin C and H-7) was observed in RASM cells and exhibited both negative and positive modulation of stimulated induction.
8. In addition the PKC inhibitor compound Ro-31-8220 abolished stimulated induction in both cell types in response to all treatments.
9. These results suggest that PKC activation is required for induction of the 130 kDa isoform of NOS in both RAW 264.7 macrophages and RASM cells. However, individual PKC isoforms regulate iNOS expression in both a positive and negative manner.

     

Induction of vasorelaxation through activation of nitric oxide synthase in endothelial cells by brazilin

The vasorelaxant activity of Caesalpinia sappan L., a traditional Chinese medicine, and its major component brazilin were investigated in isolated rat aorta and human umbilical vein endothelial cells. In isolated rat aorta, C. sappan L. extract and brazilin relaxed phenylephrine-induced vasocontraction and increased cyclic guanosine 3',5'-monophosphate (cGMP) content. Induction of vasorelaxation of brazilin was endothelium-dependent and could be markedly blocked by pretreatment with nitric oxide synthase (NOS) inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME); N(G)-monomethyl-L-arginine acetate (L-NMMA) and guanylyl cyclase inhibitor, methylene blue; 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and nitric oxide (NO) scavenger, hemoglobin. The increasing cGMP content induced by brazilin was also blocked by pretreatment with L-NAME, methylene blue, and the removal of extracellular Ca(2+). In human umbilical vein endothelial cells, brazilin dose-dependently induced an increase in NO formation and NOS activity, which were greatly attenuated by either the removal of extracellular Ca(2+) or the chelating of intracellular Ca(2+) chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM). Moreover, brazilin dose-dependently induced the influx of extracellular Ca(2+) in human umbilical vein endothelial cells. Collectively, these results suggest that brazilin induces vasorelaxation by the increasing intracellular Ca(2+) concentration in endothelial cells of blood vessels and hence activating Ca(2+)/calmodulin-dependent NO synthesis. The NO is released and then transferred into smooth muscle cells to activate guanylyl cyclase and increase cGMP content, resulting in vasorelaxation.