Intracerebroventricular injection of neuronal and inducible nitric oxide synthase inhibitors attenuates fever due to LPS in rats.

Nitric oxide (NO) has been shown to be an important mediator of febrile response to lipopolisaccharide (LPS). To clarify the role of different isoforms of NO synthase (NOS) in febrile response to immune challenge, effects of selective iNOS and nNOS inhibitors on fever to LPS were examined in freely moving biotelemetered rats. Vinyl-L-NIO (N(5) - (1-Imino-3-butenyl) - ornithine (vL-NIO), a neuronal nitric oxide synthase (nNOS) inhibitor, and aminoguanidine hydrochloride, an inducible nitric oxide synthase (iNOS) inhibitor, were injected intracerebroventricularly at a dose of 10 microg/rat just before intraperitoneal injection of LPS at a dose of 50 microg/kg. Both inhibitors injected at a selected doses had no effect on normal day-time body temperature (T(b)) and normal night-time T(b). vinyl-L-NIO and aminoguanidine injected intracerebroventricularly at a dose of 10 microg/animal suppressed the LPS-induced fever in rats. The fever index calculated for rats pretreated with v-LNIO or with aminoguanidine and injected with LPS was reduced by 43% and 72%, respectively, compared to that calculated for water-pretreated and LPS-injected rats. Whereas vL-NIO partly attenuated both phases of febrile rise in T(b), administration of aminoguanidine into the brain completely prevented fever induced by LPS. These data indicate that activation of iNOS inside the brain is not only responsible for triggering but also for maintaining of LPS-induced fever in rats. It is, therefore, reasonable to hypothesize that, activation of iNOS inside the brain is more important in fever development than activation of nNOS.     

Preservation of neurological functions by nitric oxide synthase inhibitors in conscious rats following delayed hemorrhagic shock

Excessive production of nitric oxide (NO) as result of inducible nitric oxide synthase (iNOS) induction has been implicated in the pathophysiology of hemorrhagic shock. Our aim was to study the effects of NOS inhibitors, aminoguanidine (AG) and NG-nitro-L-arginine methyl ester (L-NAME), on survival rate, mean arterial blood pressure (MABP), temporal evolution of infarct volume, nitric oxide (NO) production and neurological deficit in a model of delayed hemorrhagic shock (DHS) in conscious rats. Our results showed that the NOS inhibitors significantly improved survival rate, MABP, and attenuated brain NO overproduction 24, 48 h and 72 h after DHS. AG reduced brain infarct volume and improved the neurological performance evaluated by the rotameric and grip strength tests while L-NAME did not show protective effect in rats following DHS. These findings suggest that NO formation via iNOS activation may contribute to organ damage and that the selective iNOS inhibitor, AG, may be of interest as a therapeutic agent for neurological recovery following DHS.     

Chronic inhibition of nitric oxide synthase activity by N(G)-nitro-L-arginine induces nitric oxide synthase expression in the developing rat cerebellum

Studies on chronic inhibition of nitric oxide synthase (NOS) in the CNS suggest a plastic change in nitric oxide (NO) synthesis in areas related to motor control, which might protect the animal from the functional and behavioral consequences of NO deficiency. In the present study, the acute and chronic effect of the substrate analogue inhibitor N(G)-nitro-l-arginine (l-NNA) was examined on NO production, NO-sensitive cyclic guanosine monophosphate (cGMP) levels and the expression of NOS isoforms in the developing rat cerebellum. Acute intraperitoneal administration of the inhibitor (5-200mg/kg) to 21-day-old rats reduced NOS activity and NO concentration dose dependently by 70-90% and the tissue cGMP level by 60-80%. By contrast, chronic application of l-NNA between postnatal days 4-21 diminished the total NOS activity and NO concentration only by 30%, and the tissue cGMP level by 10-50%. Chronic treatment of 10mg/kg l-NNA induced neuronal (n)NOS expression in granule cells, as revealed by in situ hybridization, NADPH-diaphorase histochemistry and Western-blot, but it had no significant influence on tissue cGMP level or on layer formation of the cerebellum. However, a higher concentration (50mg/kg) of l-NNA decreased the intensity of the NADPH-diaphorase reaction in granule cells, significantly reduced cGMP production, and retarded layer formation and induced inducible (i)NOS expression & activity in glial cells. Treatments did not affect endothelial (e)NOS expression. The administration of the biologically inactive isomer D-NNA (50mg/kg) or saline was ineffective. The present findings suggest the existence of a concentration-dependent compensatory mechanism against experimentally-induced cronich inhibition of NOS, including nNOS or iNOS up-regulation, which might maintain a steady-state NO level in the developing cerebellum.     

大鼠脑缺血再灌注后海马神经细胞NOS表达与细胞凋亡及复方丹参的保护作用

目的探讨大鼠局灶性脑缺血再灌注后海马神经细胞一氧化氮合酶(NOS)的表达与神经细胞凋亡的关系及中药复方丹参的保护作用。方法采用大脑中动脉内栓线阻断法(MCAO)造成局灶性脑缺血再灌注模型。用原位细胞凋亡检测方法观察海马神经细胞凋亡;用免疫组织化学方法检测大鼠海马神经细胞(nNOS、iNOS)的表达并做图像分析。结果与假手术对照组比较,脑缺血再灌注2h后缺血侧海马CA1、CA3区神经细胞nNOS、iNOS表达升高,并出现神经细胞凋亡,随着再灌注时间的延长,神经细胞iNOS的表达明显增强,凋亡神经细胞数逐渐增多,至24h达高峰,但神经细胞nNOS的表达并未见明显增强。复方丹参保护组神经细胞nNOS、iNOS的表达和凋亡神经细胞数明显低于缺血再灌组(P<0.01)。结论脑缺血再灌注后缺血侧海马CA1、CA3区神经细胞nNOS的表达增强,iNOS的表达显著升高,使NO的形成增加,这可能是介导脑缺血再灌注后神经细胞凋亡的机制之一。复方丹参具有下调神经细胞nNOS、iNOS的表达,减少NO的生成,抑制细胞凋亡,减轻缺血再灌注对大鼠海马损伤的作用。     

In vivo inhibition of inducible nitric oxide and evaluation of the brain tissue damage induced by Toxocara canis larvae in experimentally infected mice

Nitric oxide (NO) is known to be produced by macrophages, endothelial cells and neurons and synthesized by an enzyme called nitric oxide synthase (NOS). Various effector mechanisms and infections can affect the NO production. Excessive amount of NO will lead to biochemical reactions, which cause toxic effects. In this study the role of NO has been evaluated in larval toxocarosis, which is a systemic parasite infection caused by T. canis larvae. Infection was established in the Balb/c mice with or without inducible NOS (iNOS) inhibition and the effects of infection and NOS inhibition were observed according to the results of SOD and LPx measurements in brain tissue and NADPH-diaphorase (NADP-d) histochemistry. Results of NADPH-d histochemistry indicate that iNOS inhibition has protective effect on the brains of infected mice and that larval T. canis infection could be related to oxidative stress, and NO production and iNOS inhibition can protect the tissue from damage in this infection.     

Phagocytosis is regulated by nitric oxide in murine microglia.

Nitric oxide (NO) is produced by inducible nitric oxide synthase (iNOS) in activated microglia and has been shown to participate in host defense mechanisms. However, the role of NO produced by constitutive nitric oxide synthase (cNOS) in microglia is poorly understood. In this report, NO was found to regulate phagocytosis in murine BV-2 microglial cells as quantified by flow cytometry. Addition of NO-generating compounds caused impaired phagocytosis as compared to untreated microglia. The addition of nitric oxide synthase (NOS) inhibitors to microglial cells resulted in potentiation of phagocytosis, suggesting that constitutive NO was participating in the regulation of phagocytosis. The inverse correlation between NO production and phagocytosis was also observed when Alzheimer's beta-amyloid peptide was added. With beta-amyloid treatment, constitutive NO production decreased while phagocytosis increased. Cell extracts prepared from untreated microglia were found to contain both neuronal and endothelial NOS isoforms, but not the inducible form. The correlation of spontaneous NO production with attenuated phagocytosis suggests that constitutive NOS enzymes participate in microglial regulation.     

一氧化氮合酶的遗传、生理研究进展

一氧化氮具有广泛的生理功能,哺乳动物体内的NO是由NO合酶(NOS)氧化L-精氨酸而合成的,合成后的NO迅速跨膜扩散释放,NO合成失调能介导多种疾病。催化NO生物合成的NOS有三种亚型:神经元型NOS(nNOS)、内皮型NOS(eNOS)和诱导型NOS(iNOS),目前,人的三型NOS已纯化并且已分子克隆成功,对一氧化氮合酶的遗传研究确认了NOS家族的基因结构和染色体定位。     

Participation of the mouse implanting trophoblast in nitric oxide production during pregnancy

While considerable progress has been made in elucidating nitric oxide (NO) regulatory mechanisms in the later stages of gestation, much less is known about its synthesis and role during embryo implantation. Thus, to evaluate the participation of the trophoblast in the production of NO during this phase, this study focused on NADPH-diaphorase activity and the distribution of NO synthase isoforms (NOS) using immunohistochemistry in pre- and postimplantation mouse embryos in situ and in vitro, as well as on NO production itself, measured as total nitrite, in trophoblast culture supernatants (Griess reaction). No NADPH-diaphorase activity was found in preimplanting embryos except after culturing for at least 48 h, when a few trophoblastic giant cells were positive. Conversely, postimplantation trophoblast cells either lodged into the implantation chamber (in situ) or after culturing (in vitro) showed intense NADPH-diaphorase activity. Also in the postimplantation trophoblast, the endothelial and inducible NOS (eNOS and iNOS) isoforms were immunodetected, under both in situ and in vitro conditions, although in different patterns. Extracts of ectoplacental cone also revealed bands of 135 and 130 kDa on SDS-PAGE that reacted with anti-eNOS and anti-iNOS, respectively, on Western blot. Analysis of the culture supernatant demonstrated that the nitrite concentration was 1) proportional to the number of cultured trophoblast cells, 2) almost completely abolished in the presence of N(omega)-nitro-L-arginine methyl ester, and 3) increased 2-fold in cultures stimulated with gamma-interferon. These results strongly suggest the production of NO from constitutive and inducible isoforms of NOS by the implanting mouse trophoblast. They also emphasize the possibility of the participation of these cells in vasodilatation and angiogenesis, and in cytotoxic mechanisms involved in the intense phagocytosis of injured maternal cells, which occur during the implantation process.     

Amyloid-beta (25-35) increases activity of neuronal NO-synthase in rat brain

Nitric oxide (NO) is a free radical with multiple functions in the nervous system. NO plays an important role in the mechanisms of neurodegenerative diseases including Alzheimer's disease. The main source of NO in the brain is an enzymatic activity of nitric oxide synthase (NOS). The aim of the present study was to analyze the expression and activity of both neuronal (nNOS) and inducible (iNOS) isoenzymes in the cerebral cortex and hippocampus of rats after intracerebroventricular administration of amyloid-beta (A beta) peptide fragment A beta(25-35). NADPHd histochemistry as well as immunohistochemistry were also used to investigate nNOS and iNOS expression in rat brain. The data presented here show that A beta(25-35) did not influence levels of nNOS or iNOS mRNA or protein expression in both structures studied. A beta(25-35) activated nNOS in the cerebral cortex and hippocampus without effect on iNOS activity. A beta(25-35) decreased the number of NADPHd-expressing neurons in the neocortex, but it did not significantly influence the number NADPHd-positive cells in the hippocampus. The peptide had no effect on the number of nNOS containing cells. We hypothesize that increased synthesis of NO induced by A beta(25-35) is related to qualitative alterations of nNOS molecule, but not to changes in NOS protein expression.     

Down-Regulation of Neuronal Nitric Oxide Synthase by Nitric Oxide After Oxygen-Glucose Deprivation in Rat Forebrain Slices

Abstract : The precise role that nitric oxide (NO) plays in the mechanisms of ischemic brain damage remains to be established. The expression of the inducible isoform (iNOS) of NO synthase (NOS) has been demonstrated not only in blood and glial cells using in vivo models of brain ischemia-reperfusion but also in neurons in rat forebrain slices exposed to oxygen-glucose deprivation (OGD). We have used this experimental model to study the effect of OGD on the neuronal isoform of NOS (nNOS) and iNOS. In OGD-exposed rat forebrain slices, a decrease in the calcium-dependent NOS activity was found 180 min after the OGD period, which was parallel to the increase during this period in calcium-independent NOS activity. Both dexamethasone and cycloheximide, which completely inhibited the induction of the calcium-independent NOS activity, caused a 40-70% recovery in calcium-dependent NOS activity when compared with slices collected immediately after OGD. The NO scavenger oxyhemoglobin produced complete recovery of calcium-dependent NOS activity, suggesting that NO formed after OGD is responsible for this down-regulation. Consistently, exposure to the NO donor ( Z )-1-[(2-aminoethyl)- N -(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NONOate) for 180 min caused a decrease in the calcium-dependent NOS activity present in control rat forebrain slices. Furthermore, OGD and DETA-NONOate caused a decrease in level of both nNOS mRNA and protein. In summary, our results indicate that iNOS expression down-regulates nNOS activity in rat brain slices exposed to OGD. These studies suggest important and complex interactions between NOS isoforms, the elucidation of which may provide further insights into the physiological and pathophysiological events that occur during and after cerebral ischemia.     

Nitric oxide produced via neuronal NOS may impair vasodilatation in septic rat skeletal muscle

Impaired vascular responsiveness in sepsis may lead to maldistribution of blood flow in organs. We hypothesized that increased production of nitric oxide (NO) via inducible nitric oxide synthase (iNOS) mediates the impaired dilation to ACh in sepsis. Using a 24-h cecal ligation and perforation (CLP) model of sepsis, we measured changes in arteriolar diameter and in red blood cell velocity (V(RBC)) in a capillary fed by the arteriole, following application of ACh to terminal arterioles of rat hindlimb muscle. Sepsis attenuated both ACh-stimulated dilation and V(RBC) increase. In control rats, arteriolar pretreatment with the NO donors S-nitroso-N-acetylpenicillamine or sodium nitroprusside reduced diameter and V(RBC) responses to a level that mimicked sepsis. In septic rats, arteriolar pretreatment with the "selective" iNOS blockers aminoguanidine (AG) or S-methylisothiourea sulfate (SMT) restored the responses to the control level. The putative neuronal NOS (nNOS) inhibitor 7-nitroindazole also restored the response toward control. At 24-h post-CLP, muscles showed no reduction of endothelial NOS (eNOS), elevation of nNOS, and, surprisingly, no induction of iNOS protein; calcium-dependent constitutive NOS (eNOS+nNOS) enzyme activity was increased whereas calcium-independent iNOS activity was negligible. We conclude that 1) AG and SMT inhibit nNOS activity in septic skeletal muscle, 2) NO could impair vasodilative responses in control and septic rats, and 3) the source of increased endogenous NO in septic muscle is likely upregulated nNOS rather than iNOS. Thus agents released from the blood vessel milieu (e.g., NO produced by skeletal muscle nNOS) could affect vascular responsiveness.     

Role of Nitric Oxide in the Progression of Pneumoconiosis

Conflicting evidence has been reported as to whether nitric oxide (NO) possesses anti-inflammatory or inflammatory properties. Data are presented indicating that in vitro or in vivo exposure to selected occupational dusts, i.e., crystalline silica, organic dust contaminated with endotoxin, or asbestos, results in upregulation of inducible nitric oxide synthase (iNOS) and the production of NO by alveolar macrophages and pulmonary epithelial cells. Nitric oxide production is associated temporally and anatomically with pulmonary damage, inflammation, and disease progression in response to occupational dusts. Blockage of inducible nitric oxide synthase by administration of NOS inhibitors or in iNOS knockout mice decreases the magnitude of injury and inflammation following in vivo exposure to silica, endotoxin, or asbestos. Therefore, NO may play an important role in the initiation and progression of pneumoconiosis.     

Interaction between NO and COX pathways in retinal cells exposed to elevated glucose and retina of diabetic rats

A nonselective inhibitor of cyclooxygenase (COX; high-dose aspirin) and a relatively selective inhibitor of inducible nitric oxide synthase (iNOS; aminoguanidine) have been found to inhibit development of diabetic retinopathy in animals, raising a possibility that NOS and COX play important roles in the development of retinopathy. In this study, the effects of hyperglycemia on retinal nitric oxide (NO) production and the COX-2 pathway, and the interrelationship of the NOS and COX-2 pathways in retina and retinal cells, were investigated using a general inhibitor of NOS [N(G)-nitro-l-arginine methyl ester (l-NAME)], specific inhibitors of iNOS [l-N(6)-(1-iminoethyl)lysine (l-NIL)] and COX-2 (NS-398), and aspirin and aminoguanidine. In vitro studies used a transformed retinal Müller (glial) cell line (rMC-1) and primary bovine retinal endothelial cells (BREC) incubated in 5 and 25 mM glucose with and without these inhibitors, and in vivo studies utilized retinas from experimentally diabetic rats (2 mo) treated or without aminoguanidine or aspirin. Retinal rMC-1 cells cultured in high glucose increased production of NO and prostaglandin E(2) (PGE(2)) and expression of iNOS and COX-2. Inhibition of NO production with l-NAME or l-NIL inhibited all of these abnormalities, as did aminoguanidine and aspirin. In contrast, inhibition of COX-2 with NS-398 blocked PGE(2) production but had no effect on NO or iNOS. In BREC, elevated glucose increased NO and PGE(2) significantly, whereas expression of iNOS and COX-2 was unchanged. Viability of rMC-1 cells or BREC in 25 mM glucose was significantly less than at 5 mM glucose, and this cell death was inhibited by l-NAME or NS-398 in both cell types and also by l-NIL in rMC-1 cells. Retinal homogenates from diabetic animals produced significantly greater than normal amounts of NO and PGE(2) and of iNOS and COX-2. Oral aminoguanidine and aspirin significantly inhibited all of these increases. The in vitro results suggest that the hyperglycemia-induced increase in NO in retinal Müller cells and endothelial cells increases production of cytotoxic prostaglandins via COX-2. iNOS seems to account for the increased production of NO in Müller cells but not in endothelial cells. We postulate that NOS and COX-2 act together to contribute to retinal cell death in diabetes and to the development of diabetic retinopathy and that inhibition of retinopathy by aminoguanidine or aspirin is due at least in part to inhibition of this NO/COX-2 axis.     

Effect of chronic lithium administration on endothelium-dependent relaxation of rat mesenteric bed: role of nitric oxide

The mechanism of action of lithium, an effective treatment for bipolar disease, is still unknown. In this study, the mesenteric vascular beds of control rats and rats that were chronically treated with lithium were prepared by the McGregor method, and the mesenteric vascular bed vasorelaxation responses were examined. NADPH-diaphorase histochemistry was used to determine the activity of NOS (nitric oxide synthase) in mesenteric vascular beds. We demonstrated that ACh-induced vasorelaxation increased in the mesenteric vascular bed of rats treated with lithium. Acute No-nitro-L-arginine methyl ester (L-NAME) administration in the medium blocked ACh-induced vasorelaxation in the control group more effectively than in lithium-treated rats, while the vasorelaxant response to sodium nitroprusside, a NO donor, was not different between lithium-treated and control groups. Acute aminoguanidine administration blocked ACh-induced vasorelaxation of lithium-treated rats, but had no effect in the control rats. Furthermore, NOS activity, determined by NADPH-diaphorase staining, was significantly greater in the mesenteric vascular beds from chronic lithium-treated rats than in those from control rats. These data suggest that the enhanced ACh-induced endothelium-derived vasorelaxation in rat mesenteric bed from chronic lithium-treated rats might be associated with increased NOS activity, likely via iNOS. Simultaneous acute L-NAME and indomethacin administration suggests the possible upregulation of EDHF (endothelium-derived hyperpolarizing factor) in lithium-treated rats.     

新生大鼠缺血缺氧后脑内一氧化氮合酶的动态表达

实验采用生后14天Wistar大鼠缺血缺氧（HI）动物模型。用免疫组织化学方法观察HI复苏（HI/R0后前脑一氧化氮合酶动态表达。结果显示;神经元一氧化氮合酶（nNOS)阳性神经元主要分布于新生大鼠大脑皮层的Ⅲ-Ⅳ层。尾状核,隔核及嗅结节,HI/R早期其表达水平无明显变化;复苏48小时及5天后,可分别在右侧大脑顶皮层或右侧大脑顶皮层和尾状核区出现梗塞灶,该区nNOS阳性神经元明显减少,而诱导型一氧化氮合酶（iNOS)阳性细胞在HI/R后12小时始现于损伤侧的侧脑室;随时间的推移在损伤侧缰核,皮层,尾状核以及丘脑背外侧核,丘脑腹侧核可见iNOS阳性细胞逐渐增多并染色加深,用识别单核巨噬细胞的克隆ED1单克隆抗体检测可见ED1阳性细胞出现的时间和在脑区的分布与iNOS阳性细胞相似,本实验提示,在局灶性脑缺血缺氧早期,脑内NO的释放不依赖于nNOS阳性神经元或iNOS阳性细胞,而在局灶性脑缺血缺氧晚期,iNOS阳性细胞产生的NO可能参与了脑损伤的过程。     

Nitric oxide promotes germ cell necrosis in the delayed phase after experimental testicular torsion of rat

The purpose of this study is to determine whether inducible nitric oxide synthase (iNOS) is involved in the pathogenesis of testicular ischemia-reperfusion (I/R) injury in association with germ cell death, through either necrosis or apoptosis. Western blot analysis showed that iNOS expression was markedly increased 1 h after ischemia, and was accompanied by a huge nitric oxide (NO) production, as measured by the Griess method, with a peak at 48 h of reperfusion. Immunohistochemistry showed that iNOS was expressed predominantly in the macrophage-like cells infiltrated in the interstitial tissues of the testis. Intraperitoneal injection of aminoguanidine (AMG) (400 mg/day), the inhibitor of iNOS, reduced NO production by 57.7% at 96 h of reperfusion. Calpain activation and proteolysis of alpha-fodrin induced by I/R were inhibited by AMG. Germ cell apoptosis was demonstrated by in situ TUNEL and DNA fragmentation on agarose gel electrophoresis. Germ cell apoptosis was maximally induced at 24 h of reperfusion, and was not inhibited by AMG. NO produced by iNOS in the delayed phase of reperfusion promoted alpha-fodrin proteolysis, which is closely associated with necrosis. Inducible NOS inhibition combined with calpain inhibition may improve impaired spermatogenesis after testicular torsion.     

Increased production of nitrotyrosine in lung tissue of rats with radiation-induced acute lung injury

The purposes of this study were 1) to identify the nitric oxide (NO) synthase (NOS) isoform responsible for NO-mediated radiation-induced lung injury, 2) to examine the formation of nitrotyrosine, and 3) to see whether nitrotyrosine formation and lung injury are reduced by an inducible NOS (iNOS) inhibitor, aminoguanidine. The left hemithorax of rats was irradiated (20 Gy), and the degree of lung injury, the expression of NOS isoforms, and the formation of nitrotyrosine and superoxide were examined after 2 wk. iNOS mRNA was induced, and endothelial NOS mRNA was markedly increased in the irradiated lung. Nitrotyrosine was detected biochemically and immunohistochemically. Aminoguanidine prevented acute lung injury as indicated by decreased protein concentration and lactate dehydrogenase activity in bronchoalveolar lavage fluid and improved NMR parameters and histology. Furthermore, the formation of nitrotyrosine was significantly reduced in the aminoguanidine group. We conclude that iNOS induction is a major factor in radiation-induced lung injury and that nitrotyrosine formation may participate in the NO-induced pathogenesis.     

Induction of Nitric Oxide Synthase and Nitric Oxide-Mediated Apoptosis in Neuronal PC12 Cells After Stimulation with Tumor Necrosis FActor-α/Lipopolysaccharide

Abstract: Exposure of neuronal PC12 cells, differentiated by nerve growth factor, to tumor necrosis factor-α (TNF-α) and bacterial lipopolysaccharide (LPS) resulted in de novo synthesis of inducible nitric oxide synthase (iNOS) mRNA and protein with an increase up to 24 h. Brain NOS expression was unaffected. The induction of iNOS in differntiated PC12 cells was associated with cell death characterized by features of apoptosis, The NOS inhibitors N -monomethylarginine, aminoguanidine, and 2-amino-5,6-dihydro-6-methyl-4 H -1,3-thiazine HCl prevented TNF-α/LPS-induced cell death and DNA fragmentation, suggesting that the TNF-α/LPS-induced cell death is mediated by iNOS-derived NO. This hypothesis is supported by the finding that addition of l -arginine, which serves as a precursor and limiting factor of enzyme-derived NO production, potentiated TNF-α/LPS-induced loss of viability.