A novel pathway for receptor‐mediated post‐translational activation of inducible nitric oxide synthase

Inducible nitric oxide synthase (iNOS) is a major source of nitric oxide during inflammation whose activity is thought to be controlled primarily at the expression level. The B1 kinin receptor (B1R) post‐translationally activates iNOS beyond its basal activity via extracellular signal regulated kinase (ERK)‐mediated phosphorylation of Ser⁷⁴⁵. Here we identified the signalling pathway causing iNOS activation in cytokine‐treated endothelial cells or HEK293 cells transfected with iNOS and B1R. To allow kinetic measurements of nitric oxide release, we used a sensitive porphyrinic microsensor (response time = 10 msec.; 1 nM detection limit). B1Rs signalled through Gαi coupling as ERK and iNOS activation were inhibited by pertussis toxin. Furthermore, transfection of constitutively active mutant Gαi Q204L but not Gαq Q209L resulted in high basal iNOS‐derived nitric oxide. G‐βγ subunits were also necessary as transfection with the β‐adrenergic receptor kinase C‐terminus inhibited the response. B1R‐dependent iNOS activation was also inhibited by Src family kinase inhibitor PP2 and trans‐fection with dominant negative Src. Other ERK‐MAP kinase members were involved as the response was inhibited by dominant negative H‐Ras, Raf kinase inhibitor, ERK activation inhibitor and MEK inhibitor PD98059. In contrast, PI3 kinase inhibitor LY94002, calcium chelator 1,2‐bis‐(o‐Aminophenoxy)‐ethane‐N,N,N′,N′‐tetraacetic acid, tetraacetoxymethyl ester (BAPTA‐AM), protein kinase C inhibitor calphostin C and protein kinase C activator PMA had no effect. Angiotensin converting enzyme inhibitor enalaprilat also directly activated B1Rs to generate high output nitric oxide via the same pathway. These studies reveal a new mechanism for generating receptor‐regulated high output nitric oxide in inflamed endothelium that may play an important role in the development of vascular inflammation.     

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。     

Integrin‐linked kinase is involved in TNF‐α‐induced inducible nitric‐oxide synthase expression in myoblasts

Tumor necrosis factor‐α (TNF‐α) is a pleiotropic cytokine produced by activated macrophages. Nitric oxide (NO) is a highly reactive nitrogen radical implicated in inflammatory responses. We investigated the signaling pathway involved in inducible nitric oxide synthase (iNOS) expression and NO production stimulated by TNF‐α in cultured myoblasts. TNF‐α stimulation caused iNOS expression and NO production in myoblasts (G7 cells). TNF‐α‐mediated iNOS expression was attenuated by integrin‐linked kinase (ILK) inhibitor (KP392) and siRNA. Pretreatment with Akt inhibitor, mammalian target of rapamycin (mTOR) inhibitor (rapamycin), NF‐κB inhibitor (PDTC), and IκB protease inhibitor (TPCK) also inhibited the potentiating action of TNF‐α. Stimulation of cells with TNF‐α increased ILK kinase activity. TNF‐α also increased the Akt and mTOR phosphorylation. TNF‐α mediated an increase of NF‐κB‐specific DNA–protein complex formation, p65 translocation into nucleus, NF‐κB‐luciferase activity was inhibited by KP392, Akt inhibitor, and rapamycin. Our results suggest that TNF‐α increased iNOS expression and NO production in myoblasts via the ILK/Akt/mTOR and NF‐κB signaling pathway. J. Cell. Biochem. 109: 1244–1253, 2010. © 2010 Wiley‐Liss, Inc.     

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.     

Expression of inducible nitric oxide synthase in human cutaneous leishmaniasis

Cutaneous leishmaniasis (CL) is an infectious disease caused by Leishmania parasite. The expression of inducible nitric oxide synthase (iNOS) and generation of nitric oxide in response to IFN-γ and TNF-α is important in control of infection. The aim of the study was to determine the expression of iNOS in the lesions of Leishmania tropica, and whether there was a correlation between the level of expression and the duration of the disease. Punch biopsy was performed from patients (n = 29) and iNOS immunohistochemical staining was applied. Expression of iNOS protein was detected 82.8% of patients. There was a strong expression with the duration of the disease less than 6 months (p < 0.002). These findings demonstrate that iNOS has a role in L. tropica especially during the early stages of the infection. (Mol Cell Biochem xxx: 147–149, 2005)     

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.     

Release of nitric oxide and expression of constitutive nitric oxide synthase of human endothelial cells: Enhancement by a 14-membered ring macrolide

A 14-membered ring macrolide, erythromycin, acts not only as an antibacterial but also as an anti-inflammatory agent. We have previously reported that erythromycin modulates neutrophil functions and ameliorates neutrophil-induced endothelial cell damage through the action of cyclic AMP-dependent protein kinase (PKA) and nitric oxide (NO). We investigated the effect of erythromycin on human endothelial cell functions. Erythromycin enhanced intracellular calcium ion concentration ([Ca²⁺]_i) of endothelial cells and NO release from endothelial cells. The enhancement of NO release from endothelial cells by erythromycin was abolished by addition of EGTA in the medium and was partially reduced by addition of H-89, an inhibitor of PKA. These results suggest that erythromycin enhances NO release from endothelial cells through the action of PKA and [Ca²⁺]_i. In addition, constitutive NO synthase (cNOS) protein expression of endothelial cells was dose-dependently enhanced by treatment with erythromycin, which might also contribute to the enhancement of NO release from endothelial cells by erythromycin. The effect of erythromycin as an anti-inflammatory agent might be partially mediated through the enhancement of NO release from endothelial cells and the drug might be a useful tool for the investigation of cNOS of endothelial cells.     

Antihypertensive effects of inducible nitric oxide synthase inhibition in experimental pre‐eclampsia

Upregulation of inducible nitric oxide synthase (iNOS) has been reported in both experimental and clinical hypertension. However, although pro‐inflammatory cytokines that up‐regulate iNOS contribute to pre‐eclampsia, no previous study has tested the hypothesis that a selective iNOS inhibitor (1400 W) could exert antihypertensive effects associated with decreased iNOS expression and nitrosative stress in pre‐eclampsia. This study examined the effects of 1400 W in the reduced uteroplacental perfusion pressure (RUPP) placental ischaemia animal model and in normal pregnant rats. Sham‐operated and RUPP rats were treated with daily vehicle or 1 mg/kg/day N‐[3‐(Aminomethyl) benzyl] acetamidine (1400 W) subcutaneously for 5 days. Plasma 8‐isoprostane levels, aortic reactive oxygen species (ROS) levels and nicotinamide adenine dinucleotide phosphate (NADPH)‐dependent ROS production were evaluated by ELISA, dihydroethidium fluorescence microscopy and lucigenin chemiluminescence respectively. Inducible nitric oxide synthase expression was assessed by western blotting analysis and aortic nitrotyrosine was evaluated by immunohistochemistry. Mean arterial blood pressure increased by ~30 mmHg in RUPP rats, and 1400 W attenuated this increase by ~50% (P < 0.05). While RUPP increased plasma 8‐isoprostane levels, aortic ROS levels, and NADPH‐dependent ROS production (P < 0.05), treatment with 1400 W blunted these alterations (P < 0.05). Moreover, while RUPP increased iNOS expression and aortic nitrotyrosine levels (P < 0.05), treatment with 1400 W blunted these alterations (P < 0.05). These results clearly implicate iNOS in the hypertension associated with RUPP. Our findings may suggest that iNOS inhibitors could be clinically useful in the therapy of pre‐eclampsia, especially in particular groups of patients genetically more prone to express higher levels of iNOS. This issue deserves further confirmation.     

Effects of endomorphins on human umbilical vein endothelial cells under high glucose

The endomorphin-1 (EM1) and endomorphin-2 (EM2) are endogenous opioid peptides, which modulate extensive bioactivities such as pain, cardiovascular responses, immunological responses and so on. The present study was undertaken to investigate the effects of EM1/EM2 on the primary cultured human umbilical vein endothelial cells (HUVECs) damaged by high glucose. PI AnnexinV-FITC detection was performed to evaluate the apoptosis rate. Levels of nitric oxide (NO) and nitric oxide synthase (NOS) activity were measured by the Griess reaction and the conversion of 3H-arginine to 3H-citrulline, respectively. Endothelin-1 (ET-1) was evaluated by the enzyme-linked immunosorbent assay (ELISA). Cell proliferation was determined by the MTT viability assay. mRNA expression of endothelial nitric oxide synthase (eNOS) and ET-1 were measured by real-time PCR. Our data showed that EM1/EM2 inhibited cell apoptosis. The high glucose induced increase in expression of NO, NOS and ET-1 were significantly attenuated by pretreatment with EM1/EM2 in a dose dependent manner. In addition, EM1/EM2 suppressed the mRNA eNOS and mRNA ET-1 expression in HUVECs under high glucose conditions. Naloxone, the nonselective opioid receptor antagonist, did not influence the mRNA eNOS expression when it was administrated on its own; but it could significantly antagonize the effects induced by EM1/EM2. Furthermore, in all assay systems, EM1 was more potent than EM2. The results suggest that EM1/EM2 have a beneficial effect in protecting against the endothelial dysfunction by high glucose in vitro, and these effects were mediated by the opioid receptors in HUVECs.     

Triptolide inhibits cyclooxygenase-2 and inducible nitric oxide synthase expression in human colon cancer and leukemia cells

Triptolide (TP),a traditional Chinese medicine,has been reported to be effective in thetreatment of autoimmune diseases and exerting antineoplastic activity in several human tumor cell lines.Thisstudy investigates the antitumor effect of TP in human colon cancer cells (SW114) and myelocytic leukemia(K562),and elucidates the possible molecular mechanism involved.SW114 and K562 cells were treatedwith different doses of TP (0,5,10,20,or 50 ng/ml).The cell viability was assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). Results demonstrated that TP inhibited the proliferation ofboth tumor cell lines in a dose-dependent manner.To further investigate its mechanisms,the productsprostaglandin E_2 (PGE2) and nitric oxide (NO) were measured by enzyme-linked immunosorbent assay(ELISA).Our data showed that TP strongly inhibited the production of NO and PGE_2. Consistent with theseresults,the expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) was up-regulatedboth at the mRNA level and the protein expression level,as shown by real-time RT-PCR and Westernblotting.These results indicated that the inhibition of the inflammatory factor COX-2 and iNOS activitycould be involved in the antitumor mechanisms of TP.     

Up-regulation of inducible nitric oxide synthase and nitric oxide in Helicobacter pylori-infected human gastric epithelial cells: possible role of interferon-gamma in polarized nitric oxide secretion

Background. Nitric oxide (NO) generated by nitric oxide synthase (NOS) is known to be an important modulator of the mucosal inflammatory response. In this study, we questioned whether Helicobacter pylori infection could up‐regulate the epithelial cell inducible NOS (iNOS) gene expression and whether NO production could show polarity that can be regulated by immune mediators. Materials and Methods. Human gastric epithelial cell lines were infected with H. pylori, and the iNOS mRNA expression was assessed by quantitative RT‐PCR. NO production was assayed by determining nitrite/nitrate levels in culture supernatants. To determine the polarity of NO secretion by the H. pylori‐infected epithelial cells, Caco‐2 cells were cultured as polarized monolayers in transwell chambers, and NO production was measured. Results. iNOS mRNA levels were significantly up‐regulated in the cells infected with H. pylori, and expression of iNOS protein was confirmed by Western blot analysis. Increased NO production in the gastric epithelial cells was seen as early as 18 hours postinfection, and reached maximal levels by 24 hours postinfection. The specific MAP kinase inhibitors decreased H. pylori‐induced iNOS and NO up‐regulation. After H. pylori infection of polarized epithelial cells, NO was released predominantly into the apical compartment, and IL‐8 was released predominantly into basolateral compartment. The addition of IFN‐γ to H. pylori‐infected polarized epithelial cells showed a synergistically higher apical and basolateral NO release. Conclusion. These results suggest that apical NO production mediated by MAP kinase in H. pylori‐infected gastric epithelial cells may influence the bacteria and basolateral production of NO and IL‐8 may play a role in the tissue inflammation.     

Enhancing amplification of late‐outgrowth endothelial cells by bilobalide

Transfusion of autologous late‐outgrowth endothelial cells (OECs) is a promising treatment for restenosis after revascularization. Preparing cells by in vitro amplification is a key step to implement the therapy. This study aimed to demonstrate that bilobalide, a terpenoid, enhances the OEC amplification. Human‐, rabbit‐ and rat OECs and a mouse femoral artery injury model were used. Expanding OECs used endothelial growth medium‐2 as the standard culture medium while exploring the mechanisms used endothelial basal medium‐2. Proliferation assay used MTT method and BrdU method. Migration assay used the modified Boyden chamber. Intracellular nitric oxide, superoxide anion, hydroxyl radical/peroxynitrite and H₂O₂ were quantified with DAF‐FM DA, dihydroethidium, hydroxyphenyl fluorescein and a H₂O₂ assay kit, respectively. Activated ERK1/2 and eNOS were tested with the Western blot. Bilobalide concentration‐dependently enhanced OEC number increase in vitro. Transfusion of bilobalide‐based human OECs into femoral injured athymia nude mouse reduced the intimal hyperplasia. Bilobalide promoted OEC proliferation and migration and increased the intracellular nitric oxide level. L‐NAME, a NOS inhibitor, inhibits but not abolishes OEC proliferation, migration and ERK1/2 activation. Bilobalide concentration‐dependently enhanced the eNOS Ser‐1177 phosphorylation and Thr‐495 dephosphorylation in activated OECs. Bilobalide alleviates the increase in hydroxyl radical/peroxynitrite, superoxide anion and H₂O₂ in proliferating OECs. In conclusion, nitric oxide plays a partial role in OEC proliferation and migration; bilobalide increases OEC nitric oxide production and decreases nitric oxide depletion, promoting the OEC number increase; Bilobalide‐based OECs are active in vivo. The findings may simplify the preparation of OECs, facilitating the implementation of the autologous‐OECs‐transfusion therapy.     

Anemonin attenuates osteoarthritis progression through inhibiting the activation of IL‐1β/NF‐κB pathway

The osteoarthritis (OA) progression is now considered to be related to inflammation. Anemonin (ANE) is a small natural molecule extracted from various kinds of Chinese traditional herbs and has been shown to inhibiting inflammation response. In this study, we examined whether ANE could attenuate the progression of OA via suppression of IL‐1β/NF‐κB pathway activation. Destabilization of the medial meniscus (DMM) was performed in 10‐week‐old male C57BL/6J mice. ANE was then intra‐articularly injected into joint capsule for 8 and 12 weeks. Human articular chondrocytes and cartilage explants challenged with interleukin‐1β (IL‐1β) were treated with ANE. We found that ANE delayed articular cartilage degeneration in vitro and in vivo. In particular, proteoglycan loss and chondrocyte hypertrophy were significantly decreased in ANE ‐treated mice compared with vehicle‐treated mice. ANE decreased the expressions of matrix metalloproteinase‐13 (MMP13), A disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), collagen X (Col X) while increasing Aggrecan level in murine with DMM surgery. ANE treatment also attenuated proteoglycan loss in human cartilage explants treated with IL‐1β ex vivo. ANE is a potent protective molecule for OA; it delays OA progression by suppressing ECM loss and chondrocyte hypertrophy partially by suppressing IL‐1β/NF‐κB pathway activation.     

The role of neutral sphingomyelinase produced ceramide in lipopolysaccharide-mediated expression of inducible nitric oxide synthase

Lipopolysaccharide (LPS) and interferon-gamma (IFN) treatment of C6 rat glioma cells increased the intracellular ceramide level and the expression of the inducible nitric oxide synthase (iNOS) gene. To delineate the possible role of ceramide in the induction of iNOS, we examined the source of intracellular ceramide and associated signal transduction pathway(s) with the use of inhibitors of intracellular ceramide generation. The inhibitor of neutral sphingomyelinase (3-O-methylsphingomyelin, MSM) inhibited the induction of iNOS, whereas inhibitor of acidic sphingomyelinase (SR33557) or that of ceramide de novo synthesis (fumonisin B1) had no effect on the induction of iNOS. MSM-mediated inhibition of iNOS induction was reversed by the supplementation of exogenous C8-ceramide, suggesting that ceramide production by neutral sphingomyelinase (nSMase) is a key mediator in the induction of iNOS. The MSM-mediated inhibition of iNOS gene expression correlated with the decrease in the activity of ras. Inhibition of co-transfected iNOS promoter activity by dominant negative ras supported the role of ras in the nSMase-dependent regulation of iNOS gene. NF-kappaB DNA binding activity and its transactivity were also reduced by MSM pretreatment, and were completely reversed by the supplementation of C8-ceramide. As the dominant negative ras also reduced NF-kappaB transactivity, NF-kappaB activation may be downstream of ras. Our results suggest that ceramide generated by nSMase may be a critical mediator in the regulation of iNOS gene expression via ras-mediated NF-kappaB activation under inflammatory conditions.