Calreticulin promotes angiogenesis via activating nitric oxide signalling pathway in rheumatoid arthritis

Calreticulin (CRT) is a multi-functional endoplasmic reticulum protein implicated in the pathogenesis of rheumatoid arthritis (RA). The present study was undertaken to determine whether CRT was involved in angiogenesis via the activating nitric oxide (NO) signalling pathway. We explored the profile of CRT expression in RA (including serum, synovial fluid and synovial tissue). In order to investigate the role of CRT on angiogenesis, human umbilical vein endothelial cells (HUVECs) were isolated and cultured in this study for in-vitro experiments. Our results showed a significantly higher concentration of CRT in serum (5·4 ± 2·2 ng/ml) of RA patients compared to that of osteoarthritis (OA, 3·6 ± 0·9 ng/ml, P < 0·05) and healthy controls (HC, 3·7 ± 0·6 ng/ml, P < 0·05); and significantly higher CRT in synovial fluid (5·8 ± 1·2 ng/ml) of RA versus OA (3·7 ± 0·3 ng/ml, P < 0·05). High levels of CRT are expressed in synovial membrane localized predominantly to inflammatory cells and synovial perivascular areas in both the lining and sublining layers of RA synovial tissue (RAST). Increased nitric oxide (NO) production and phosphorylation level of endothelial nitric oxide synthase (eNOS) were measured in HUVECs following CRT stimulation, while the total eNOS expression was not significantly changed. Furthermore, CRT promoted the proliferation, migration and tube formation of HUVECs, which were significantly inhibited by a specific eNOS inhibitor. These findings suggested that CRT may be involved in angiogenesis events in RA through NO signalling pathways, which may provide a potential therapeutic target in the treatment of RA.     

一氧化氮途径对大鼠精子发生的影响

为阐明一氧化氮途径对精子发生的影响 ,探讨一氧化氮对精子发生的调节作用 ,将雄性SD大鼠分为 4组 ,分别于腹腔内注射左旋精氨酸 (L arginine ,L ARG)、N 硝基左旋精氨酸甲酯 (N nitro L arginine mythel ester,L NAME)、L ARG +L NAME与生理盐水 ,每天一次 ,共 1 2d。于最后一次注射后 2h采血并处死动物。放免法测定血清内睾酮含量 ;GREISS法测定血清NO-x (硝酸盐 亚硝酸盐 )含量 ;常规组织学切片观察生精上皮形态 ,并对Ⅶ -Ⅷ期生精上皮横断面的各级生精细胞进行定量分析 ;扫描电镜观察生精小管内精子密度。结果表明 :L ARG组血清内NO-x 含量高于对照组 (P <0 0 1 ) ,睾酮含量低于对照组 (P <0 0 1 ) ,精子生成减少 (P <0 0 1 ) ;L NAME组血清内NO-x 含量低于对照组 (P <0 0 1 ) ,睾酮含量高于对照组 (P <0 0 1 ) ,精子生成增加 (P <0 0 1 )。L ARG +L NAME组NO-x 与睾酮含量及精子的生成无显著性变化。因此 ,加强NO途径抑制精子发生 ,抑制NO途径促进精子形成     

Role of nitric oxide during rotavirus infection

The pathophysiological mechanisms behind rotavirus-induced diarrhoea still remain incomplete. Current views suggest that the non-structural protein 4 (NSP4) of rotavirus and the enteric nervous system (ENS) participate in water secretion and diarrhoea. In the present work the role of nitric oxide (NO) in rotavirus infection and disease has been studied in vitro, mice and humans. Incubation of human intestinal epithelial cells (HT-29) with purified NSP4 but not with infectious virus produced NO2/NO3 accumulation in the incubation media. The NSP4-induced release of NO metabolites occurred within the first minutes after the addition of the toxin. Mice infected with murine rotavirus (strain EDIM) accumulated NO2/NO3 in the urine at the onset for diarrhoea. Following rotavirus infection, inducible nitric oxide synthetase (iNOS) mRNA was upregulated in ileum, but not in duodenum or jejunum of newborn pups within 5 days post-infection. A prospective clinical study including 46 children with acute rotavirus infection and age-matched controls concluded that rotavirus infection stimulates NO production during the course of the disease (P < 0.001). These observations identify NO as an important mediator of host responses during rotavirus infection.     

Ischemic damage increases nitric oxide production via inducible nitric oxide synthase in the cochlea

The present study was designed to elucidate the dynamic changes of nitric oxide (NO) production in the perilymph and to investigate the immunostaining for inducible nitric oxide synthase (iNOS) in the cochlea for 7 days after transient cochlear ischemia. Moreover, aminoguanidine, which is a selective iNOS inhibitor, was administrated immediately following ischemia and every 24h thereafter for 7 days to investigate whether the production of NO is dependent on the iNOS pathway. Significant increases in the oxidative NO metabolites, nitrite (NO(2)(-)) and nitrate (NO(3)(-)), were measured on day 1 using an in vivo microdialysis and on-line high performance liquid chromatography (HPLC) system. The immunostaining for iNOS was strongly expressed on days 1 and 4 and returned to normal on day 7 after the ischemia. The administration of aminoguanidine reduced the oxidative NO metabolites on day 1 and suppressed the expression of iNOS. These findings suggest that transient ischemia causes a remarkable increase in NO production in the perilymph, which might be attributable to the iNOS pathway.     

Inhibition of implant-associated infections via nitric oxide release

The in vivo antibacterial activity of nitric oxide (NO)-releasing xerogel coatings was evaluated against an aggressive subcutaneous Staphylococcus aureus infection in a rat model. The NO-releasing implants were created by coating a medical-grade silicone elastomer with a sol-gel-derived (xerogel) film capable of storing NO. Four of the bare or xerogel-coated silicone materials were subcutaneously implanted into male rats. Ten rats were administered 10 microl of a 10(8) cfuml(-1)S. aureus colony directly into the subcutaneous pocket with the implant prior to wound closure. Infection was quantitatively and qualitatively evaluated after 8d of implantation with microbiological and histological methods, respectively. A 82% reduction in the number of infected implants was achieved with the NO-releasing coating. Histology revealed that the capsule formation around infected bare silicone rubber controls was immunoactive and that a biofilm may have formed. Capsule formation in response to NO-releasing implants had greater vascularity in comparison with uninoculated or untreated controls. These results suggest that NO-releasing coatings may dramatically reduce the incidence of biomaterial-associated infection.     

Acute hypoxia-induced depletion of striatal nitric oxide synthase pathway

Hypoxia-induced alteration of nitric oxide (NO) production may lead to brain disease, especially in the areas most sensitive to oxygen deficiency, such as the striatum. To date, the behaviour of the striatal NO pathway under hypoxia/reoxygenation remains unknown and its elucidation constitutes the aim of this work.Wistar rats were submitted to hypoxia (20 min) and analyzed after 0 h, 24 h, and 5 days of reoxygenation. Expression, activity, and location of the NO synthase (NOS) isoforms (neuronal, endothelial, and inducible) as well as nitrated protein expression were analyzed in the rat striatum. NO levels were indirectly quantified as nitrates and nitrites (NO_x), which act as NO-generating molecules.NOS isoform mRNA levels remained unaltered in hypoxic groups vs. normoxic control. However, quantification of immunoreaction showed a significant decrease in eNOS and nNOS after hypoxia. While in situ NOS activity and NO_x levels fell, levels of nitrotyrosine-modified proteins rose throughout the reoxygenation period.Our data revealed the great complexity of the NO pathway, showing that both acute hypoxia and the successive recovery period down-regulated the NOS system in the rat striatum. However, under hypoxia/reoxygenation NO may be produced in a NOS-independent way from the NO-storage molecules, compensating for the hypoxia-reduced NOS activity.     

The nitric oxide pathway in pre-eclampsia: pathophysiological implications

Pre-eclampsia, one of the most significant health problems inhuman pregnancy, complicates 6-7% of all gestations and is theleading cause of fetal growth retardation, infant morbidityand mortality, premature birth and maternal death. Recent researchimplicates free radicals in the pathophysiology of pre-eclampsia.This review covers the biochemistry of nitric oxide (NO) andpossible interactions with other free radicals. Studies in therat show that pregnancy is associated with enhanced productionand responsiveness to NO in both reproductive tissues and bloodvessels. Rats infused with N^G-nitro-L-arginine methyl ester(L-NAME, a NO synthase inhibitor) have been used as an animalmodel of pre-eclampsia, and the effects of steroid hormoneson blood pressure in this model have been tested. Results suggestthat pre-eclampsia may be a state of NO deficiency. However,in humans there seem to be contradictions regarding the involvementof NO in maternal adaptation to pregnancy. It is suggested thatNO may be one of several systems that act in concert to maintaina symbiotic relationship between mother and fetus. However,the input of each system may be genetically determined.     

The effect of carbohydrate mouth rinse on maximal strength and strength endurance

It has been previously reported that carbohydrate (CHO) mouth rinse can improve exercise performance. The proposed mechanism involves increased activation of brain regions believed to be responsible for reward/motivation and motor control. Since strength-related performance is affected by central drive to the muscles, it seems reasonable to hypothesize that the positive CNS response to oral CHO sensing may counteract the inhibitory input from the muscle afferent pathways minimizing the drop in the central drive. The purpose of the current study was to test if CHO mouth rinse affects maximum strength and strength endurance performance. Twelve recreationally strength-trained healthy males (age 24.08 ± 2.99 years; height 178.09 ± 6.70 cm; weight 78.67 ± 8.17 kg) took part in the study. All of the tests were performed in the morning, after an 8 h overnight fasting. Subjects were submitted to a maximum strength test (1-RM) and a strength endurance test (six sets until failure at 70% of 1-RM), in separate days under three different experimental conditions (CHO mouth rinse, placebo—PLA mouth rinse and control—CON) in a randomized crossover design. The CHO mouth rinse (25 ml) occurred before every attempt in the 1-RM test, and before every set in the endurance strength test. Blood glucose and lactate were measured immediately before and 5 min post-tests. There were no significant differences in 1-RM between experimental conditions (CHO 101 ± 7.2 kg; PLA 101 ± 7.4 kg; CON 101 ± 7.2 kg; p = 0.98). Furthermore, there were no significance between trial differences in the number of repetitions performed in each set (p = 0.99) or the total exercise volume (number of repetitions × load lifted [kg]) (p = 0.98). A main effect for time (p < 0.0001) in blood lactate concentration was observed in both tests (1-RM and strength endurance). Blood glucose concentration did not differ between conditions. In conclusion, CHO mouth rinse does not affect maximum strength or strength endurance performance.     

Impact of volume-dependent alveolar diffusing capacity on exhaled nitric oxide concentration

Exhaled endogenous nitric oxide (NO) holds promise as a potential biomarker of pulmonary inflammation. Previous experimental and theoretical work has concluded that the alveolar concentration approaches a constant steady state value at end exhalation due to both a constant maximum flux or release of NO (J_max,alv) and a constant diffusing capacity (D_NO,alv) in the alveolar region. We have recently demonstrated that D_NO,alv is not constant, but increases with alveolar volume (V_A) given by the following average relationship: D_NO,alv=48^*V _A ^2/3 ml/min/mmHg (where V_A is expressed in liters, STPD). We investigated the potential impact of a variable D_NO,alv on exhaled concentration by incorporating the volume dependence into the currently accepted two-compartment model for NO exchange dynamics. Our results suggest that the mechanism underlying the plateau in exhaled concentration is a constant ratio J_max,alv/D_NO,alv.This constant ratio requires a volume dependence of J_max,alv similar to D_NO,alv, and is likely due to a decreasing alveolar surface area during exhalation. © 2001 Biomedical Engineering Society. PAC01: 8719Uv, 8710+e, 8715Vv, 8239Rt     

Human-bacteria nitric oxide cycles in HIV-1 infection

The human intestinal tract harbors a complex microbiotic environment containing commensal bacteria and immunocompetent mucosal cells. There is considerable communication between the bacteria and host cells through dietary constituents and metabolic cycles. We propose that in the pathogenesis of acquired immunodeficiency syndrome (AIDS), the human immunodeficiency virus-1 (HIV-1) triggers a change in a coupled transorganism (human-bacteria) nitric oxide interchange cycle, that may influence the biosynthesis and recycling of nitric oxide (NO) in AIDS patients. Normally, nitric oxide (NO) is produced from arginine through nitrate NO(3)(-), which is ultimately eliminated in the urine and feces. In HIV infection, however, the NO(3)(-) is converted into NO and nitrite NO(2)(-) and recirculated in the body, perhaps as a result of concomitant opportunistic bacterial infections and cellular hypoxia. Due to the efficient coupling of the human-bacteria nitric oxide cycles, persistently high levels of nitrite and the free radicals peroxynitrite (ONOO(-)) may occur in AIDS patients, contributing to the etiology of AIDS-related dementia, persistent immunosuppression and Kaposi's sarcoma.     

Serum nitric oxide in children with dengue infection

One hundred and ten patients (M/F = 67/43) from King Chulalongkorn Memorial Hospital and the provincial hospitals of Uttaradit, Ayudhaya, and Sakonnakorn, who were clinically diagnosed with dengue infection and serologically confirmed by ELISA anti-Dengue IgM and IgG were recruited. Their serum NO level was measured using commercially available assay kits to investigate its correlation with the severity of the dengue infection: dengue fever (DF), DHF I/II, and DHF III/IV or dengue shock syndrome (DSS). Serum NO levels were also measured in 38 healthy controls (M/F = 19/19). Serum NO levels in dengue patients were lower than those of the controls (control = 168.18 +/- 24.10 micromol/l, DF = 124.94 +/- 36.79 micromol/l, DHF I/II = 99.69 +/- 33.42 micromol/l, and DHF III/IV = 120.63 +/- 46.26 micromol/l; p < 0.05). Serum NO levels in patients with DHF I/II were significantly lower than in those with DHF III/IV. These preliminary data revealed that levels of serum NO in dengue patients were significantly lower than those of normal controls. Patients with DSS had higher NO levels than those with DHF I/II. The decreased NO in dengue patients could be due to endothelial damage rendering the endothelium incapable of producing NO. Endothelial function seems to play a role in the pathogenesis of dengue infection. Further studies are required to see whether serum NO levels could play a role in the course of the disease and could help predict the severity of dengue infection.     

Angiotensin-(1-7) inhibits neuronal activity of dorsolateral periaqueductal gray via a nitric oxide pathway

The midbrain periaqueductal gray (PAG) is a neural site for several physiological functions related to cardiovascular regulation, pain modulation and behavioral reactions. Recently, angiotensin-(1-7) [Ang-(1-7)] has been considered as an important biologically active component of the renin–angiotensin system in the CNS. The purpose of this study was to determine (1) existence of Ang-(1-7) receptor, Mas-R, within the dorsolateral PAG (dl-PAG), (2) the role for Ang-(1-7) in modulating activity of dl-PAG neurons, and (3) the mechanisms by which Ang-(1-7) plays a regulatory role. Western blot analysis showed that Mas-R appears within the dl-PAG. Whole cell patch-clamp recording demonstrated that the discharge rates of dl-PAG neurons were decreased from 4.35 ± 0.32 Hz of control to 1.06 ± 0.34 Hz (P < 0.05, vs. control) by 100 nM of Ang-(1-7). With pretreatment of A-779, a Mas-R inhibitor, the discharge rate was 4.66 ± 0.62 Hz (P > 0.05, vs. control) during infusion of Ang-(1-7). Additionally, neuronal nitric oxide synthase (nNOS) was largely localized within the dl-PAG among the three isoforms. The effects of Ang-(1-7) on neuronal activity of the PAG were attenuated in the presence of S-methyl-L-thiocitrulline (SMTC), a nNOS inhibitor. The discharge rates were 4.21 ± 0.39 Hz in control and 4.09 ± 0⋅47 Hz (P > 0.05, vs. control) when Ang-(1-7) was applied with pretreatment of SMTC. Those findings suggest that Ang-(1-7) plays an inhibitory role in the dl-PAG via a NO dependent signaling pathway. This offers the basis for the physiological role of Ang-(1-7) and Mas R in the regulation of various functions in the CNS.     

Expression of nitric oxide synthase and effect of substrate manipulation of the nitric oxide pathway in mouse ovarian follicles

BACKGROUND: Nitric oxide (NO) is a cell messenger with multiple actions in different biological systems, implicated in the control of follicle and oocyte function. NO is formed from L-arginine by isoforms of nitric oxide synthase (NOS) via NG-hydroxy-L-arginine, with L-citrulline as a byproduct. This study aimed to show how modulation of NO by manipulating NOS substrates would affect mouse follicle growth and ovulation in vitro, where vascular effects of NO are attenuated. METHODS: Immunohistochemistry [endothelial (eNOS) and inducible (iNOS)] and in situ hybridization (iNOS) were applied on mouse ovaries. Cultured follicles were also stained for iNOS by immunohistochemistry. For follicles cultured in the presence or absence of L-arginine, the ability of L-citrulline or NG-hydroxy-L-arginine to substitute for L-arginine was assessed in terms of follicle growth and ovulation. RESULTS: iNOS and eNOS were localized in oocytes and theca, with some staining in granulosa. iNOS mRNA occurred predominantly in granulosa and oocyte. Omission of L-arginine significantly reduced follicle survival and ovulation. Partial compensation for L-arginine withdrawal was achieved with L-citrulline and NG-hydroxy-L- arginine. Specific abnormalities of follicle growth were noted. CONCLUSIONS: NOS is present in mouse follicles, and its action is necessary at a local level for normal follicle development in vitro. Reduced growth, persistent basement membranes and reduced ovulation were associated with in vitro disruption of NO.     

内皮素—1对血管通透性的影响及一氧化氮的介导作用

应用大鼠脾，肾切除模型，观察内皮素－１对血管通透性的影响及一氧化氮的介导作用，结果发现：静脉恒流速灌注内皮素－１１ｈ平均动脉压和血球压积明显高于生理盐水对照组，血浆容量明显少于对照组，血浆蛋白漏出和＾１２５Ｉ－白蛋白在心，肺和空肠组织的分布明显高于对照组，同时灌注一氧化氮合成酶抑制剂Ｌ－ＮＭＭＡ，虽然加强内皮素－１的升压作用，但明显减少内皮素－１引起的血浆蛋白漏出率增加，恢复血浆容量和血球压积，使     

Fluoxetine increases the nitric oxide production via nuclear factor kappa B-mediated pathway in BV2 murine microglial cells

A body of recent evidence implicates that antidepressants affect the inflammatory response and immune system. The present study is focused on the effects of the most widely used antidepressant agent, fluoxetine on the production of nitric oxide (NO) in BV2 microglial cells. In this study, we observed interesting result that NO production was increased by fluoxetine. The mRNA level of nitric oxide synthase (iNos, Nos2) by RT-PCR was also stimulated by fluoxetine. We next conducted electophoretic mobility shift assay (EMSA) to determine the DNA binding activity of nuclear factor kappa B (Nfkappab), an important upstream modulator for Nos2 expression, to find that fluoxetine increased DNA binding activity of Nfkappab. By Western blot analysis, phosphorylation levels of p38 mitogen-activated protein kinase (p38 Mapk, Mapk14) and extracellular signal-related kinase (Erk)1/2 Mapk, upstream signaling mediators of Nfkappab were found to be increased by fluoxetine. In addition, the mRNA expressions of other proinflammatory cytokines, interleukin 6 (Il6) and tumor necrosis factor alpha (Tnfalpha) were examined. The expressions of both Il6 and Tnfalpha by fluoxetine treatment were similar to those of Nos2 and Nfkappab. Taken together, our results show that fluoxetine stimulates NO production via Nfkappab-mediated pathway in BV2 cells.     

束缚应激对大鼠血小板-氧化氮释放的影响

目的 观察急性应激对大鼠血小板一氧化氮(NO)释放的影响及其机制.方法 大鼠浸水-束缚应激(WRS)2、4和8 h,以胃溃疡指数(UI)作为应激损伤的标识,采用Greiss法测定血小板孵育液中亚硝酸盐(NO2-)含量;同位素法测其一氧化氮合酶(NOS)活性和L精氨酸(L-Arg)转运量.结果 WRS 2 h血小板L-Arg转运量、NOS活性和孵育液NO2-含量较对照组显著增加,但随应激时间延长,其呈下降趋势,应激8 h时均显著低于对照组,胃溃疡逐渐加重.结论 WRS应激早期阶段可上调血小板L-Arg/NO通路,促进血小板NO生成;长时间应激下调L-Arg/NO通路,减少NO释放.     

Improved haemocompatibility of cysteine-modified polymers via endogenous nitric oxide.

A novel method for improving the haemocompatibility of biomedical materials through endogenous nitric oxide (NO) is presented. L-cysteine was covalently immobilized onto two biomedical polymers: polyurethane (PU) and polyethylene terephthalate (PET). The L-cysteine content on the polymers was approximately 5-8 nmol/cm2 as quantified via a chemiluminescence-based assay. The haemocompatibility of the modified polymers was evaluated in terms of the number of adhered platelets when exposed to a platelet suspension labeled with Cr51. Platelet adherence on the L-cysteine-modified polymers was reduced more than 50% as compared to the control (glycine-modified polymers) when the platelet suspension contained plasma constituents. No difference in platelet adhesion was observed in the absence of plasma constituents. Further experiments demonstrated that NO was easily transferred to the L-cysteine-modified polymers from S-nitroso-albumin in PBS buffer. The NO was then released from the polymer. NO transfer or release was not observed for the control. The results suggest that L-cysteine-modified polymers are effective in reducing platelet adhesion via the transfer of NO from endogenous S-nitrosoproteins in plasma to the polymer followed by the subsequent release of NO. Thus, exploiting endogenous NO is a viable option for improving the haemocompatibility of biomaterials.     

Antiviral effect of nitric oxide during Japanese encephalitis virus infection

The ability of Japanese encephalitis virus (JEV) and JEV-induced macrophage derived neutrophil chemotactic factor (MDF) to produce nitric oxide (NO), and the possible antiviral effect of NO during JEV infection, was investigated. Splenic macrophages of JEV infected mice produced maximum NO in vivo at day 7 post infection, and in vitro at 24 h after JEV stimulation. MDF-induced NO production was dose dependent and maximal at 60 min after MDF treatment. The response was sensitive to anti-MDF antibody treatment and the nitric oxide synthase inhibitor NG-monomethyl-L-arginine (L-NMMA). Pretreatment of mice with L-NMMA increased the mortality to 100% in JEV infected mice in vivo and inhibited NO production in vitro, while MDF stimulated macrophages inhibited virus replication with high levels of NO production. MDF treatment increased the survival rate of JEV infected mice. The findings thus demonstrate that MDF induces production of NO during JEV infection, which has an antiviral effect. This may be one of the important mechanisms of natural immunity in controlling the initial stages of JEV infection.