Effects of moderate hypothermia on constitutive and inducible nitric oxide synthase activities after traumatic brain injury in the rat

We investigated the effects of therapeutic hypothermia (30 degrees C) on alterations in constitutive (cNOS) and inducible (iNOS) nitric oxide synthase activities following traumatic brain injury (TBI). Male Sprague-Dawley rats were anesthetized with 0.5% halothane and underwent moderate (1.8-2.2 atm) parasagittal fluid-percussion (F-P) brain injury. In normothermic rats (37 degrees C) the enzymatic activity of cNOS was significantly increased at 5 min within the injured cerebral cortex compared with contralateral values (286.5+/-68.9% of contralateral value; mean+/-SEM). This rise in nitric oxide synthase activity was significantly reduced with pretraumatic hypothermia (138.8+/-17% of contralateral value; p < 0.05). At 3 and 7 days after normothermic TBI the enzymatic activity of cNOS was decreased significantly (30+/-8.4 and 28.6+/-20.9% of contralateral value, respectively; p < 0.05). However, immediate posttraumatic hypothermia (3 h at 30 degrees C) preserved cNOS activity at 3 and 7 days (69.5+/-23.3 and 78.6+/-7.6% of contralateral value, respectively; mean+/-SEM; p < 0.05). Posttraumatic hypothermia also significantly reduced iNOS activity at 7 days compared with normothermic rats (0.021+/-0.06 and 0.23+/-0.06 pmol/mg of protein/min, respectively; p < 0.05). The present results indicate that hypothermia (a) decreases early cNOS activation after TBI, (b) preserves cNOS activity at later periods, and (c) prevents the delayed induction of iNOS. Temperature-dependent alterations in cNOS and iNOS enzymatic activities may participate in the neuroprotective effect of hypothermia in this TBI model.     

Chronic stress induces the expression of inducible nitric oxide synthase in rat brain cortex

Long-term exposure to stress has detrimental effects on several brain functions in many species, including humans, and leads to neurodegenerative changes. However, the underlying neural mechanisms by which stress causes neurodegeneration are still unknown. We have investigated the role of endogenously released nitric oxide (NO) in this phenomenon and the possible induction of the inducible NO synthase (iNOS) isoform. In adult male rats, stress (immobilization for 6 h during 21 days) increases the activity of a calcium-independent NO synthase and induces the expression of iNOS in cortical neurons as seen by immunohistochemical and western blot analysis. Three weeks of repeated immobilization increases immunoreactivity for nitrotyrosine, a nitration product of peroxynitrite. Repeated stress causes accumulation of the NO metabolites NO2+ NO3- (NOx-) accumulation in cortex, and these changes occur in parallel with lactate dehydrogenase (LDH) release and impairment of glutamate uptake in synaptosomes. Administration of the selective iNOS inhibitor aminoguanidine (400 mg/kg i.p. daily from days 7 to 21 of stress) prevents NOx- accumulation in cortex, LDH release, and impairment of glutamate uptake in synaptosomes. Taken together, these findings indicate that a sustained overproduction of NO via iNOS expression may be responsible, at least in part, for some of the neurodegenerative changes caused by stress and support a possible neuroprotective role for specific iNOS inhibitors in this situation.     

Pharmacological inhibition of inducible nitric oxide synthase attenuates the development of seizures in mice

The present study has been designed to pharmacologically expound the significance of inducible nitric oxide synthase in the pathophysiological progression of seizures using mouse models of chemically induced kindled epilepsy and status epilepticus induced spontaneous recurrent seizures. Pentylenetetrazole (40 mg kg⁻¹) (PTZ) administration every second day for a period of 15 days was used to elicit kindled seizure activity in mice. Severity of kindled seizures was assessed in terms of a composite kindled seizure severity score (KSSS). Pilocarpine (100 mg kg⁻¹) was injected every 20 min until the onset of status epilepticus. A spontaneous recurrent seizure severity score (SRSSS) was recorded as a measure of quantitative assessment of the progressive development of spontaneous recurrent seizures induced after pilocarpine status epilepticus. Sub-acute PTZ administration induced the development of severe form of kindled seizures in mice. Further, pharmacological status epilepticus elicited a progressive evolution of spontaneous recurrent seizures in the animals. However, treatment of aminoguanidine, a relatively selective inhibitor of inducible nitric oxide synthase, markedly and dose dependently suppressed the development of both PTZ induced kindled seizures as well as pilocarpine induced spontaneous recurrent seizures. Therefore inducible nitric oxide synthase may be implicated in the development of seizures.     

诱导型一氧化氮合酶的激活与血压的关系

本实验旨在探讨诱导型一氧化氮合酶（iNOS)的激活与血压之间的关系,三组SD大鼠分别静脉输注不同浓度（0．3％,4％及8％）NaCl溶液以使其处于不同的血压水平,运用同位素标记的L－精氨酸转换成L－Citrulline 的转换率变化及Greiss反应,分别测定不同血压时iNOS的活性及NO的生成量,另四组大鼠包括正常Wistar,正常SD,高盐诱导的高血压（NaHR)及自发性高血压大鼠（SHR）,经测定血压后,取主动脉血管并以Western印迹印交法测定其iNOS蛋白水平,结果表明,血压较低时,SD大鼠iNOS活基本没有改变,而在输入4％和8％NaCl并处于较高血压水平的SD大鼠,其iNOS活性及NO生存均明显升高,。此外Western 印迹表明,两种高血压大鼠主动脉组织iNOS蛋白水平均较正常Wistar及正常SD大鼠高,密度扫描表明,NaHR及SHR主动脉组织iNOS蛋白分别较正常SD大鼠及正常Wistar大鼠升高149％及261％,这一结果提示,诱导型一氧化氮合酶是血液动力学调控的重要组成部分,尤其是在血压处于较高水平时,iNOS具有重要的代偿调节作用,除细胞因子,细菌产物等之外,血压也是调节iNOS表达及活性的重要因素之一。     

Inhibition of inducible nitric oxide synthase lowers the cochlear damage by lipopolysaccharide in guinea pigs

Endotoxin-treated cochleas of the guinea pig were examined electrophysiologically and immunohistochemically concerning the expression of inducible nitric oxide synthase (iNOS/NOS II). One mg of bacterial lipopolysaccharide (LPS, 5 mg/ml) or mixed solution of 1 mg of LPS plus 1 mg of N^G-nitro-L-arginine methyl ester (L-NAME, 5 mg/ml) (L-NAME/LPS) was injected into the middle ear of guinea pigs transtympanically. The electrocochleograms were measured prior to, immediately and 48 h after the injection. Immunohistological studies for iNOS followed after fixation, embedding and sectioning of the temporal bones.

The threshold and amplitude of the compound action potential (CAP) became significantly worse in the LPS treated group. In contrast, the changes of the threshold and amplitude of CAP were decreased in the L-NAME/LPS group. iNOS was expressed in the stria vascularis, the spiral ligament, the organ of Corti and the spiral ganglion in the LPS group. These immunore-activities in the L-NAME/LPS group were less intense than that in the LPS group. These results indicate that LPS has an ototoxic effect on the cochlea and that this effect could be mediated by iNOS produced high nitric oxide under inflammatory conditions.     

Neisseria meningitidis-induced death of cerebrovascular endothelium: mechanisms triggering transcriptional activation of inducible nitric oxide synthase

The intense host response to meningococcus reflects marked functional and morphological alterations in blood-brain barriers. We showed previously that mouse-derived cerebrovascular endothelium responded to meningococcal lysates with a robust nitric oxide (NO) response, resulting in the loss of cell viability. To understand how the NO synthase-2 gene in endothelium is activated by meningococcus, we investigated upstream roles for specific protein kinases. Using known kinase inhibitors, and measuring both mRNA expression and nitrite release, we found MAPK/ERK kinase (MEK)2, p38 kinase and phosphoinositide 3-kinase (but not MEK1 or phospholipase C) to be implicated in the NO synthase-2 response. Recruitment of these kinases by meningococcus did not depend on the prior release of the proinflammatory cytokines tumour necrosis factor alpha or interleukin-1beta from endothelium. These endothelial cells were found to express toll-like receptors (TLR) 2, 4 and 9 and antibodies directed against TLR 2 and 4 (but not TLR 9) blocked the NO synthase-2 response to meningococcus. Both meningococcus-induced translocation of nuclear factor-kB (NF-kB) and endothelial cell death were blocked by a known inhibitor of p38 kinase. Calpain inhibitor-1 blocked the NO synthase-2 response to meningococcus, which is further evidence of a role for NF-kB.     

Engagement of inducible nitric oxide synthase at the rostral ventrolateral medulla during mevinphos intoxication in the rat

We evaluated the relationship between the toxicity induced by the organophosphate mevinphos (Mev) and inducible nitric oxide synthase (iNOS) in the rostral ventrolateral medulla (RVLM), the medullary origin of sympathetic neurogenic vasomotor tone. Adult Sprague-Dawley rats that were anesthetized and maintained with propofol were used. Laser scanning confocal microscopic analysis revealed colocalization of the M2 subtype of muscarinic receptors (M(2)R) and iNOS immunoreactivity in RVLM neurons. Comicroinjection bilaterally of Mev (10 nmol) and artificial cerebrospinal fluid (aCSF) into the RVLM elicited a progressive decline in systemic arterial pressure (SAP) and heart rate. This was accompanied during phase 1 Mev intoxication by an increase in the power density of the very high-frequency (VHF; 5-9 Hz), high-frequency (HF; 0.8-2.4 Hz), low-frequency (LF; 0.25- 0.8 Hz) and very low-frequency (VLF; 0-0.25 Hz) components of SAP signals. Phase 2 exhibited a reversal of the VHF and VLF power to control levels and a further reduction in the power density of both HF and LF components to below baseline. Hypotension and bradycardia promoted by Mev were significantly blunted on coadministration into the RVLM of the selective iNOS inhibitors S-methylisothiourea (250 pmol) or aminoguanidine (250 pmol). Not only was the augmented power density of HF and LF components during phase 1 Mev intoxication further enhanced, the reduced power of these two spectral components during phase 2 was appreciably antagonized. On the other hand, the temporal changes in VHF and VLF power were essentially the same as with coadministration of Mev and aCSF. We conclude that, as a cholinesterase inhibitor, Mev may induce toxicity via nitric oxide produced by iNOS on activation of the M(2)R by the accumulated acetylcholine in the RVLM.     

Oxidized phospatidylcholine but not native phosphatidylcholine inhibits inducible nitric oxide synthase in RAW 264.7 macrophages

This study was designed to compare the effects of oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (PAPC) and native PAPC on the inducible nitric oxide synthase (iNOS) in the macrophage cell line RAW 264.7. Macrophages stimulated by bacterial lipopolysaccharide (1 microg/ml) were incubated with increasing amounts of native or oxidized PAPC (oxPAPC, 10-20 microg/ml). Cells incubated with oxPAPC showed a dose-dependent inhibition of inducible nitric oxide synthesis, as well as reduced iNOS protein expression and mRNA levels. Additionally, chromatin immunoprecipitation assay revealed that oxPAPC reduced the interaction of the active NF-kappaB subunit p65 with the iNOS promoter region when compared to native PAPC.     

Cobalt-60 gamma radiation increased the nitric oxide generation in cultured rat vascular smooth muscle cells

Radiation is a promising and new treatment for restenosis following angioplasty. Nitric oxide has been proposed as a potential "anti-restenotic" molecule. We radiated the cultured rat vascular smooth muscle cells with Cobalt-60 gamma radiation at doses of 14 and 25Gy and observed nitrite production, cGMP content, L-arginine uptake, inducible nitric oxide synthase (iNOS) activity, and the gene expression of iNOS. Results showed that radiation at doses of 14 and 25Gy increased cGMP content by 92.4% and 86.4%, respectively. Radiation at the dose of 25Gy increased the iNOS activity and nitrite content, but radiation at the dose of 14Gy had no significant effect on iNOS activity and NO production. Both doses of radiation significantly decreased the L-arginine transport. Radiation at the doses of 14 and 25Gy increased iNOS gene expression significantly, which was consistent with the effect of radiation on iNOS activity. In conclusion, radiation induces the NO generation by up-regulating the iNOS activity.     

Involvement of intestinal inducible nitric oxide synthase (iNOS) in the early stages of murine salmonellosis

Local induction of inducible nitric oxide synthase (iNOS) and apoptosis was examined in the intestine of mice infected with virulent Salmonella enterica serovar Enteritidis 5694 (S. enteritidis) and its attenuated derivative mutant E/1/3. Both, intestinal iNOS mRNA expression and iNOS activity showed a peak at 4 h only in animals receiving the virulent S. enteritidis. Aminoguanidine treatment abrogated intestinal epithelial damage produced by virulent S. enteritidis and diminished apoptosis at the tips of the villi. Unlike the virulent strain, mutant E/1/3 induced massive iNOS expression in Peyer's patches, these findings may be related to its protective capacity. Our results suggest that intestinal iNOS participates in the early response to intestinal infection and that the final effect depends on the nature of the insult.     

Inhibition of inducible nitric oxide synthesis by azathioprine in a macrophage cell line

Azathioprine is used as an anti-inflammatory agent. Although there are numerous data demonstrating cytotoxic and immunosuppressive properties of azathioprine and its metabolite 6-mercaptopurine, the mechanism of the anti-inflammatory action of azathioprine has not yet been fully clarified. During our study, we investigated the effects of azathioprine on the inducible nitric oxide synthase (iNOS) in lipopolysaccharide stimulated murine macrophages (RAW 264.7) by measurement of iNOS protein (immunoblotting), iNOS mRNA (semiquantitative competitive RT-PCR), and NO production (nitrite levels). Azathioprine (0-210 muM) induces a concentration dependent inhibition of inducible nitric oxide synthesis (IC50: 33.5 muM). iNOS protein expression showed a concentration dependent reduction as revealed by immunoblotting when cells were incubated with increasing amounts of azathioprine. Azathioprine decreases iNOS mRNA levels as shown by semiquantitative competitive RT-PCR. In contrast, 6-mercaptopurine showed no inhibition of inducible nitric oxide synthesis. Azathioprine did not reduce iNOS mRNA stability after the addition of actinomycin D. Enzymatic activity assays with increasing concentrations of azathioprine (0-210 muM) showed no statistically significant inhibition of iNOS enzyme activity compared to cell lysates without azathioprine. Nuclear translocation of NF-kappaB p65 subunit and binding of NF-kappaB p50 subunit from nuclear extracts to a biotinylated-consensus sequence was unaffected by azathioprine treatment. iNOS inhibition by azathioprine was associated with a decreased expression of IRF-1 (interferon regulatory factor 1) and IFN-beta (beta-interferon) mRNA. Azathioprine induced iNOS inhibition seems to be associated with an action of the methylnitroimidazolyl substituent. This suggests a route to the rational design of nontoxic anti-inflammatory agents by replacing the 6-mercaptopurine component of azathioprine with other substituents. The inhibition of inducible nitric oxide synthesis might contribute to the anti-inflammatory activities of azathioprine.     

Reduced neuronal nitric oxide synthase is involved in ischemia-induced hippocampal neurogenesis by up-regulating inducible nitric oxide synthase expression

Nitric oxide (NO), a free radical with signaling functions in the CNS, is implicated in some developmental processes, including neuronal survival, precursor proliferation, and differentiation. However, neuronal nitric oxide synthase (nNOS) -derived NO and inducible nitric oxide synthase (iNOS) -derived NO play opposite role in regulating neurogenesis in the dentate gyrus after cerebral ischemia. In this study, we show that focal cerebral ischemia reduced nNOS expression and enzymatic activity in the hippocampus. Ischemia-induced cell proliferation in the dentate gyrus was augmented in the null mutant mice lacking nNOS gene (nNOS−/−) and in the rats receiving 7-nitroindazole, a selective nNOS inhibitor, after stroke. Inhibition of nNOS ameliorated ischemic injury, up-regulated iNOS expression, and enzymatic activity in the ischemic hippocampus. Inhibition of nNOS increased and iNOS inhibitor decreased cAMP response element-binding protein phosphorylation in the ipsilateral hippocampus in the late stage of stroke. Moreover, the effects of 7-nitroindazole on neurogenesis after ischemia disappeared in the null mutant mice lacking iNOS gene (iNOS−/−). These results suggest that reduced nNOS is involved in ischemia-induced hippocampal neurogenesis by up-regulating iNOS expression and cAMP response element-binding protein phosphorylation.     

甲醛炎性痛增强大鼠海马NOS表达和NO生成

目的：观察甲醛炎性痛过程中大鼠痛行为、海马一氧化氮合酶（NOS）活性及一氧化氮（NO）含量的变化以及变化的时程及区域特征。方法：采用辐射热甩尾法测定大鼠痛阈变化;采用NADPH—d组织化学法和硝酸还原酶法分别测定大鼠海马NOS表达和No含量。结果：皮下注射甲醛溶液后,大鼠出现伤害性感受反应及痛阈降低。注射甲醛后6h,海马CA1、CA2～3区及DG区NOS阳性细胞数目、阳性细胞染色深度均显著增加。海马NO含量亦显著增加;注射甲醛后12h时这些改变最为显著,48h时恢复至对照组水平。结论：甲醛炎性痛可诱导海马NOS活性增强及NO生成增多．这种改变可发生在海马各区．并具有一定的时程特征。     

Involvement of various combinations of endogenous inflammatory cytokines in Listeria monocytogenes-induced expression of inducible nitric oxide synthase in mice

Abstract Using an in vitro infection of spleen cells with Listeria monocytogenes , the relationship between endogenous cytokines and the expression of inducible nitric oxide synthase (iNOS) was examined. When all interferon (IFN)-γ, tumor necrosis factor (TNF)-α and interleukin (IL)-1 α, or the combination of IFN-γ with either TNF-α or IL-1 α were neutralized by antibodies, there was a significant reduction of iNOS expression and nitrite production in culture. However, there was no reduction of iNOS expression and nitrite production when these cytokines were individually neutralized. After the depletion of natural killer cells, there was no change in the expression of Listeria -induced iNOS and nitrite production although the IFN-γ production was abrogated. Neutralization of TNF-α and IL-1 α in natural killer cell-depleted culture resulted in the reduction of iNOS expression. Thus, various combinations of cytokines appeared to play an important role in iNOS induction by L. monocytogenes .     

昆虫一氧化氮及其合酶的研究进展

一氧化氮作为一种重要的信息分子 ,参与调节昆虫嗅觉、视觉、机械感受、发育、机体防御及学习行为。该文从生理、生化、形态定位以及信号转导几方面综述了有关昆虫一氧化氮及其合酶的最新研究进展。     

Mice lacking inducible nitric oxide synthase develop exacerbated hepatic inflammatory responses induced by Plasmodium berghei NK65 infection

Infection of mice with Plasmodium berghei NK65 represents a well-recognized malaria model in which infection is accompanied by an intense hepatic inflammatory response. Enzyme-inducible nitric oxide synthase is an important regulator of inflammation and leukocyte recruitment in microvessels, but these functions have yet to be evaluated in experimental malaria. In this study, we assessed the involvement of inducible nitric oxide synthase in inflammatory responses to murine experimental malaria induced by P. berghei NK65. We observed that wild type (WT) and nitric oxide synthase (iNOS)-deficient mice (iNOS^−/−) mice showed similar levels of parasitemia following P. berghei NK65 infection, although infected iNOS^−/− mice presented early mortality. Inducible nitric oxide synthase deficiency led to increased leukocyte rolling and adhesion to the liver in iNOS^−/− mice relative to the WT animals, as observed via intravital microscopy. Infected iNOS^−/− mice also exhibited increased hepatic leukocyte migration and subsequent liver damage, which was associated with high serum levels of the cytokines TNF-α, IL-6 and IL-10. Our data suggest potential role for the iNOS enzyme as a regulator of hepatic inflammatory response induced by P. berghei NK65-infection, and its absence leads to exacerbated inflammation and sequential associated-hepatic damage in the animals.     

Development of immune cellular biosensing system for assessing chemicals on inducible nitric oxide synthase signaling activator

An immune cellular biosensing system has been constructed to assess immunomodulating effects of chemicals. Production of nitric oxide in the immune cellular biosensing system was used as readout of an immune cellular response for assessing the immunomodulating effects of chemicals. The macrophage-like cell line RAW264.7, which has signaling pathways of inducible nitric oxide synthase, was employed in the cellular biosensing system. The immune cellular biosensing system consisted of a Pt counter electrode, an Ag/AgCl reference electrode, and a gold electrode onto which a polyion complex layer was coated to allow adherence of the RAW264.7 cells. As the results of evaluating effects of a polyion complex layer on cell viabilities by using WST-8 assay, the polyion complex layer did not affect RAW264.7 cells. The polyion-coated gold electrode could measure NO without the drawback of electrochemical interference that occurs with differential pulse voltammetry. The detection limit of the immune cellular biosensing system was 4.2 nM released NO as measured by double potential step chronoamperometry. The potent immune activating abilities of lipopolysaccharide and interferon-gamma could be assessed by the cellular biosensing system; NO release from cells was detected within 600 ms.