Specific iNOS Inhibitor Research Articles

Signaling-mediated Functional Activation of Inducible Nitric-oxide Synthase and Its Role in Stimulating Platelet Activation

Nitric oxide (NO) is a short lived secondary messenger, synthesized by nitric-oxide synthases (NOS). It is believed that the activity of inducible NOS (iNOS) is regulated primarily at the transcription level by inducing expression of iNOS mRNA and protein, which then continuously produces NO, until its degradation. Platelets do not have the nuclear transcriptional regulatory mechanisms of the iNOS gene and are believed to generate NO in response to agonist stimulation via endothelial NOS (eNOS). However, here we show that agonist-induced NO production is only partially eNOS-dependent and is also mediated by iNOS. Platelet agonist-induced NO production is significantly reduced in iNOS-knockout platelets. Platelet NO production occurs within seconds after agonist addition and is not accompanied by changes in iNOS protein levels, indicating a signaling-mediated functional activation mechanism of iNOS. Importantly, iNOS knock-out and iNOS inhibitors reduce agonist-induced platelet secretion and aggregation and cGMP levels, indicating that iNOS activation is important in stimulating platelets via the newly identified NO-cGMP-dependent platelet secretion pathway. Furthermore, iNOS knock-out mice have prolonged bleeding time, suggesting that this novel mode of regulation of iNOS activity plays a physiologically relevant role in hemostasis.

Nitric Oxide Involvement in TNF-α and IL-1β-Mediated Changes in Human Mesangial Cell MMP-9 and TIMP-1

Background: Mesangial cells are known to secrete metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) that are capable of disrupting the glomerular basement membrane (GBM). Disruption of the GBM appears to be an important mechanism in the renal disease process, however little is known about the mechanisms involved. Therefore we examined the potential role of nitric oxide (NO) in the regulation of MMP-9 and TIMP-1 by tumour necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) using the human mesangial cell line (HMCL). Methods: The HMCL was treated with various concentrations of cytokines and NO inhibitors. Activity of MMP-9 was examined by gelatin zymography and TIMP-1 expression was analysed by Western blotting. NO production was measured using the Greiss assay. Results: In this study, stimulation of HMCL cells with TNF-α or IL-1β, alone or in combination, led to a substantial increase in NO production, which was shown to result from an increase in the expression of the inducible form of NOS (iNOS). Treatment of cells with the specific iNOS inhibitor L- potentiated the increase in MMP-9 production induced by TNF-α, but prevented the suppression of TIMP-1 production observed following cytokine treatment. The NO donor, sodium nitroprusside, also stimulated a substantial increase in NO production in HMCL cells, which was associated with a reduction in basal and TNF-α-stimulated MMP-9 and a potentiation of the cytokine-induced decrease in TIMP-1. Conclusions: Our study provides convincing evidence of a modulatory role for NO on cytokine-induced MMP-9 and TIMP-1 production in human mesangial cells.

Involvement of inducible nitric oxide synthase in hydroxyl radical-mediated lipid peroxidation in streptozotocin-induced diabetes

Free radical production is implicated in the pathogenesis of diabetes mellitus, where several pathways and different mechanisms were suggested in the pathophysiology of the complications. In this study, we used electron paramagnetic resonance (EPR) spectroscopy combined with in vivo spin-trapping techniques to investigate the sources and mechanisms of free radical formation in streptozotocin-induced diabetic rats. Free radical production was directly detected in the diabetic bile, which correlated with lipid peroxidation in the liver and kidney. EPR spectra showed the trapping of a lipid-derived radical. Such radicals were demonstrated to be induced by hydroxyl radical through isotope-labeling experiments. Multiple enzymes and metabolic pathways were examined as the potential source of the hydroxyl radicals using specific inhibitors. No xanthine oxidase, cytochrome P450s, the Fenton reaction, or macrophage activation were required for the production of radical adducts. Interestingly, inducible nitric oxide synthase (iNOS) (apparently uncoupled) was identified as the major source of radical generation. The specific iNOS inhibitor 1400W as well as L-arginine pretreatment reduced the EPR signals to baseline levels, implicating peroxynitrite as the source of hydroxyl radical production. Applying immunological techniques, we localized iNOS overexpression in the liver and kidney of diabetic animals, which was closely correlated with the lipid radical generation and 4-hydroxynonenal-adducted protein formation, indicating lipid peroxidation. In addition, protein tyrosine nitration occurred in the diabetic target organs. Taken together, our studies support inducible nitric oxide synthase as a significant source of EPR-detectable reactive intermediates, which leads to lipid peroxidation and may contribute to disease progression as well.

Cisplatin alters nitric oxide synthase levels in human ovarian cancer cells: involvement in p53 regulation and cisplatin resistance

The present study determines if (1) basal protein levels of nitric oxide (NO) synthases (eNOS, iNOS, and nNOS) are different in cisplatin-sensitive (OV2008) and counterpart cisplatin-resistant (C13*) human ovarian cancer cells, (2) cisplatin alters NOS levels, (3) NO donor causes apoptosis and p53 upregulation, (4) NO donor sensitises C13* cells to cisplatin via p53 upregulation (determined by p53 siRNA gene-knockdown), and (5) inhibition of endogenous NOS alters cisplatin-induced apoptosis. Basal iNOS levels were higher in OV2008 cells than in C13* cells. Cisplatin upregulated iNOS, but dramatically reduced eNOS and nNOS, in OV2008 cells only. Failure of cisplatin to upregulate iNOS and downregulate eNOS/nNOS in cisplatin-resistant C13* cells may be an aetiological factor in the development of resistance. The NO donor S-nitroso-N-acetylpenicillamine (SNAP) increased p53 protein levels and induced apoptosis in both cell types, and enhanced cisplatin-induced apoptosis in C13* cells in a p53-dependent manner (i.e., enhancement blocked by p53 siRNA). Specific iNOS inhibitor 1400W partially blocked cisplatin-induced apoptosis in OV2008 cells. In cisplatin-resistant C13* cells, blocking all NOSs with NG-amino-L-arginine dramatically changed these cells from cisplatin-resistant to cisplatin-sensitive, greatly potentiating cisplatin-induced apoptosis. The data suggest important roles for the three NOSs in regulating chemoresistance to cisplatin in ovarian cancer cells.

Inducible Nitric Oxide Synthase (iNOS) is Not Required for IL-2–induced Hypotension and Vascular Leak Syndrome in Mice

Dose limiting side effects of interleukin-2 (IL-2) include severe hypotension and vascular leak syndrome (VLS). Previous studies have shown that nitric oxide (NO) synthesis is strongly induced after IL-2 treatment of C3H/HeN mice (180,000 IU b.i.d. for 5 d). We employed knockout mice (on C57BL/6 background) to test the role of the inducible NO synthase (iNOS) in mediating IL-2 toxicity. In contrast to C3H/HeN mice, which developed hypotension and VLS after 10 doses of only 180,000 IU IL-2, C57BL/6 mice were far more resistant requiring increased doses of 800,000 IU IL-2 (b.i.d., 5 d) to induce hypotension and VLS. Serum interferon-gamma levels were significantly more elevated by IL-2 treatment in C3H/HeN mice than in C57BL/6, correlating with the severity of hypotension and VLS. Urinary excretion of NO metabolites was markedly reduced in iNOS knockout mice (C57BL/6 iNOS) after IL-2 treatment. A surprising finding was that these mice still developed profound hypotension and VLS. Similar findings were observed after administration of a iNOS specific inhibitor, L-N[6]-(1-iminoethyl)lysine (L-NIL). In contrast, a general NOS inhibitor, N-monomethyl-L-arginine, prevented both hypotension and vascular leak. The superoxide dismutase mimetic, M40403, reversed IL-2-induced hypotension but not VLS in knockout mice. Thus, peroxynitrite-mediated mechanisms are likely responsible for hypotension, whereas NO-induced changes in vascular permeability result in VLS. The iNOS enzyme is not necessary for pathogenesis of IL-2-induced cardiovascular toxicity. These results imply that other NOS isoforms, such as endothelial NOS, may play a major role in the development of IL-2-induced cardiovascular toxicity.

Inhibition of inducible nitric oxide synthase activity prevents liver recovery in rat thioacetamide‐induced fibrosis reversal

Stimulation of nitric oxide (NO) synthesis similar to the application of NO donors could be of benefit in liver fibrosis. Many authors believe that activation of NO synthesis by pharmacological agents is promising in the treatment of liver fibrosis. However, there is considerable controversy in understanding the role of NO in fibrogenesis and fibrolysis. The aims of our study were to evaluate the effects of L-arginine, as an NO metabolic precursor, and those of NO synthase (NOS) inhibitors, L-nitroarginine methyl ester (L-NAME) and aminoguanidine (AG) in rats with thioacetamide (TAA)-induced liver fibrosis reversal. Male Wistar rats, 230-240 g, received TAA (200 mg kg(-1), intraperitoneally) twice a week for 3 months. Liver resolution was simulated by withdrawal of TAA administration. Thereafter the animals were subdivided into five groups and treated by intragastric intubation with: L-arginine (100 and 300 mg kg(-1)); L-NAME as an inhibitor of both constitutively expressed NOS (eNOS) and inducible NOS (iNOS) (20 mg kg(-1)), AG as a specific inhibitor of iNOS (100 mg kg(-1)) or placebo. The severity of liver fibrosis was assessed by morphometric evaluation of liver slides stained with Azan-Mallory, hydroxyproline (Hyp) determination and mRNA steady state levels of collagen I, transforming growth factor (TGF)-beta1, metalloproteinases (MMP)-13, -14, tissue inhibitor of MMP (TIMP)-1 and plasminogen activator inhibitor (PAI)-1 were quantified by real time PCR. The activities of serum marker enzyme, alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase, were measured. TAA treatment during 3 months induced micronodular liver fibrosis with a pronounced deposition of collagen fibres. L-Arginine did not affect this deposition nor did it affect both relative and total liver hydroxyproline content. Both NOS inhibitors significantly increased the square of the liver connective tissue stained by Azan-Mallory and the above parameters characterizing liver hydroxyproline content. Both NOS inhibitors up-regulated procollagen alpha1 (I), MMP-13, TIMP-1 and PAI-1 mRNA expression. The AG effects were more pronounced. than those of L-NAME. AG treatment also increased mRNA expression of TGF-beta1 and PAI-1. Both NOS inhibitors developed a clear pro-fibrotic effect in the liver. Aminoguanidine was more fibrotic than L-NAME. Our data suggest a significant anti-fibrotic role for iNOS rather than for eNOS. L-Arginine did not show any anti-fibrotic properties in the TAA-model used.

ACTIVATION OF CYTOSOLIC PHOSPHOLIPASE A2[alpha] THROUGH NITRIC OXIDE‐INDUCED S‐NITROSYLATION: INVOLVEMENT OF INDUCIBLE NITRIC OXIDE SYNTHASE AND CYCLOOXYGENASE‐2

Cytosolic phospholipase A2α (cPLA2α) is the rate‐limiting key enzyme that releases arachidonic acid (AA) for eicosanoid production. This study describes that cPLA2α protein is S‐nitrosylated and its activity is enhanced by nitric oxide (NO). Forced expression of inducible nitric oxide synthase (iNOS) in human epithelial cells induced cPLA2α S‐nitrosylation, enhanced its catalytic activity and increased the release of AA. The iNOS‐induced cPLA2α activation is blocked by the specific iNOS inhibitor, 1400W. Addition of the NO donor, GSNO, to isolated cell lysates induced S‐nitrosylation of cPLA2α, in vitro. Incubation of cultured cells with the iNOS substrate L‐Arginine and NO donor significantly increased cPLA2α activity and AA release. Furthermore, COX‐2 induction or expression markedly enhanced iNOS‐induced cPLA2α S‐nitrosylation and activation, leading to 9, 23, 20 fold increase of AA release and 100, 38, 88 fold of PGE2 production in A549, SG231, and HEK293 cells, respectively, whereas COX‐2 alone leads to less than 2 fold change. These results indicate that COX‐2 has the ability to enhance iNOS‐induced cPLA2α S‐nitrosylation and that maximal PG synthesis is achieved by the synergistic interaction among iNOS, cPLA2α and COX‐2. These findings disclose a novel link among cPLA2α, iNOS and COX‐2 which form a multiprotein complex leading to cPLA2α S‐nitrosylation and activation.

Inhibition of prolactin secretion by American ginseng is mediated by ginseng polysaccharide‐induced nitric oxide production

Rats treated with American ginseng exhibit an approximate 50% reduction in serum prolactin levels and a compensatory decrease in hypothalamic dopamine activity. To investigate whether ginseng has direct pituitary effects, anterior pituitaries collected from random cycling female rats were dispersed and cultured for 1 week prior to treatment with water soluble ginseng extract (0 – 2 mg/ml) for 24 hr. Ginseng produced a dose‐dependent decrease in prolactin with maximum suppression (∼50%) within 6 hr at ≥1mg/ml; there was no change in media LH levels. When the ginsenoside and polysaccharide components in ginseng were examined, ginseng polysaccharides (12.5μg/ml) inhibited prolactin whereas ginsenosides had no effect in the pituitary cultures. Ginseng and its polysaccharides both stimulated nitric oxide (NO) production from cultured cells at 24 hr and induced iNOS gene and protein expression at 12 and 24 hr, respectively. The specific iNOS inhibitor 1400W blocked the ginseng/polysaccharide effects on NO production and prolactin release, whereas eNOS and nNOS inhibitors had no effect. Together, these results suggest that the reduction in circulating prolactin levels following ginseng ingestion is due to polysaccharide‐induced pituitary iNOS and NO production and subsequent prolactin suppression.

Activation of Cytosolic Phospholipase A2α through Nitric Oxide-induced S-Nitrosylation: INVOLVEMENT OF INDUCIBLE NITRIC-OXIDE SYNTHASE AND CYCLOOXYGENASE-2

Cytosolic phospholipase A(2)alpha (cPLA(2)alpha) is the rate-limiting key enzyme that cleaves arachidonic acid (AA) from membrane phospholipids for the biosynthesis of eicosanoids, including prostaglandin E(2) (PGE(2)), a key lipid mediator involved in inflammation and carcinogenesis. Here we show that cPLA(2)alpha protein is S-nitrosylated, and its activity is enhanced by nitric oxide (NO). Forced expression of inducible nitric-oxide synthase (iNOS) in human epithelial cells induced cPLA(2)alpha S-nitrosylation, enhanced its catalytic activity, and increased AA release. The iNOS-induced cPLA(2)alpha activation is blocked by the specific iNOS inhibitor, 1400W. The addition of the NO donor, S-nitrosoglutathione, to isolated cell lysates or purified recombinant human cPLA(2)alpha protein induced S-nitrosylation of cPLA(2)alpha in vitro. Incubation of cultured cells with the iNOS substrate L-arginine and NO donor significantly increased cPLA(2)alpha activity and AA release. These findings demonstrate that iNOS-derived NO S-nitrosylates and activates cPLA(2)alpha in human cells. Site-directed mutagenesis revealed that Cys-152 of cPLA(2)alpha is critical for S-nitrosylation. Furthermore, COX-2 induction or expression markedly enhanced iNOS-induced cPLA(2)alpha S-nitrosylation and activation, leading to 9-, 23-, and 20-fold increase of AA release and 100-, 38-, and 88-fold of PGE(2) production in A549, SG231, and HEK293 cells, respectively, whereas COX-2 alone leads to less than 2-fold change. These results indicate that COX-2 has the ability to enhance iNOS-induced cPLA(2)alpha S-nitrosylation and that maximal PG synthesis is achieved by the synergistic interaction among iNOS, cPLA(2)alpha, and COX-2. Since COX-2 enhances the formation of cPLA(2)alpha-iNOS binding complex, it appears that COX-2-induced augmentation of cPLA(2)alpha S-nitrosylation is mediated at least in part through increased association between iNOS and cPLA(2)alpha. These findings disclose a novel link among cPLA(2)alpha, iNOS, and COX-2, which form a multiprotein complex leading to cPLA(2)alpha S-nitrosylation and activation. Therefore, therapy aimed at disrupting this interplay may represent a promising strategy to effectively inhibit PGE(2) production and prevent inflammation and carcinogenesis.

Pioglitazone inhibition of lipopolysaccharide-induced nitric oxide synthase is associated with altered activity of p38 MAP kinase and PI3K/Akt

BackgroundPrevious studies have suggested that peroxisome proliferator activated receptor-gamma (PPAR-γ)-mediated neuroprotection involves inhibition of microglial activation and decreased expression and activity of inducible nitric oxide synthase (iNOS); however, the underlying molecular mechanisms have not yet been well established. In the present study we explored: (1) the effect of the PPAR-γ agonist pioglitazone on lipopolysaccharide (LPS)-induced iNOS activity and nitric oxide (NO) generation by microglia; (2) the differential role of p38 mitogen-activated protein kinase (p38 MAPK), c-Jun NH(2)-terminal kinase (JNK), and phosphoinositide 3-kinase (PI3K) on LPS-induced NO generation; and (3) the regulation of p38 MAPK, JNK, and PI3K by pioglitazone.MethodsMesencephalic neuron-microglia mixed cultures, and microglia-enriched cultures were treated with pioglitazone and/or LPS. The protein levels of iNOS, p38 MAPK, JNK, PPAR-γ, PI3K, and protein kinase B (Akt) were measured by western blot. Different specific inhibitors of iNOS, p38MAPK, JNK, PI3K, and Akt were used in our experiment, and NO generation was measured using a nitrite oxide assay kit. Tyrosine hydroxylase (TH)-positive neurons were counted in mesencephalic neuron-microglia mixed cultures.ResultsOur results showed that pioglitazone inhibits LPS-induced iNOS expression and NO generation, and inhibition of iNOS is sufficient to protect dopaminergic neurons against LPS insult. In addition, inhibition of p38 MAPK, but not JNK, prevented LPS-induced NO generation. Further, and of interest, pioglitazone inhibited LPS-induced phosphorylation of p38 MAPK. Wortmannin, a specific PI3K inhibitor, enhanced p38 MAPK phosphorylation upon LPS stimulation of microglia. Elevations of phosphorylated PPAR-γ, PI3K, and Akt levels were observed with pioglitazone treatment, and inhibition of PI3K activity enhanced LPS-induced NO production. Furthermore, wortmannin prevented the inhibitory effect of pioglitazone on the LPS-induced NO increase.ConclusionWe demonstrate that pioglitazone protects dopaminergic neurons against LPS insult at least via inhibiting iNOS expression and NO generation, which is potentially mediated via inhibition of p38 MAPK activity. In addition, the PI3K pathway actively participates in the negative regulation of LPS-induced NO production. Our findings suggest that PPAR-γ activation may involve differential regulation of p38 MAPK and of the PI3K/Akt pathway in the regulation of the inflammatory process.

Bacterial DNA induces myocardial inflammation and reduces cardiomyocyte contractility: role of Toll-like receptor 9

Myocardial function is severely compromised during sepsis. Several underlying mechanisms have been proposed. The innate immune system, i.e. toll-like receptor (TLR) 2 and 4, significantly contributes to cardiac dysfunction. Little is known regarding TLR9 and its pathogenic ligand bacterial DNA in the myocardium. We therefore studied the role of TLR9 in myocardial inflammation and cardiac contractility. Wild-type (WT, C57BL/6) and TLR9-deficient (TLR9-D) mice and isolated cardiomyocytes were challenged with synthetic bacterial DNA (CpG-ODN). Myocardial contractility as well as markers of inflammation/signalling were determined. Isolated cardiomyocytes incorporated fluorescence-marked CpG-ODN. In WT mice, CpG-ODN caused a robust response in hearts demonstrated by increased levels of tumour necrosis factor (TNF-alpha), interleukin (IL)-1beta, IL-6, inducible nitric oxide synthase (iNOS), and nuclear factor kappaB activity. This inflammatory response was absent in TLR9-D mice. Under similar conditions, contractility measurements of isolated ventricular cardiomyocytes demonstrated a TLR9-dependent loss of sarcomeric shortening after CpG-ODN exposure. This observation was iNOS dependent as the application of a specific iNOS inhibitor reversed sarcomeric shortening to normal levels. Our data suggest that bacterial DNA contributes to myocardial cytokine production and loss of cardiomyocyte contractility via TLR9.

Liver Tissue Inducible Nitric Oxide Synthase (iNOS) Expression and Lipid Peroxidation in Experimental Hepatic Ischemia Reperfusion Injury Stimulated With Lipopolysaccharide: The Role of Aminoguanidine

Hepatic ischemia-reperfusion (HIR) is a severe condition that is seen after hepatic arterial injury and in hepatic grafts in living donor transplantation. HIR not only causes liver injury by lipid peroxidation, but also stimulates systemic and portal endotoxemia. Also, lipopolysaccharide (LPS) induces hepatic injury mediated by inducible nitric oxide synthase (iNOS). There is little knowledge on the role of specific iNOS inhibitors in prevention of HIR injury followed by LPS administration. The aim of this study on a LPS induced HIR model was to investigate the effect of aminoguanidine (AG) administration on hepatic tissue iNOS expression and lipid peroxidation when given before or after LPS. Six groups were designed; A: Sham, B: HIR, C: HIR + AG, D: HIR + LPS, E: HIR + LPS + AG, F: HIR + AG + LPS. No substance was given to the rats in Group A and B. HIR injury was induced with vascular occlusion for 45 min and reperfusion for 45 min. Drugs were given intraperitoneally 10 min before reperfusion. Serum and tissue analysis for myeloperoxidase (MPO), and malondialdehyde (MDA), and tissue NA+/K+ adenosine 5'triphosphatases (ATPase) and tissue iNOS staining were performed. Permission for this study was obtained from the local Ethics Committee. The level of MPO, MDA, and iNOS staining scores in Group B were significantly higher than Group A and ATPase was lower in Group B (P < 0.05). Contrary to results in Group C, results of MPO, MDA, and iNOS staining scores of Group D was higher than Group B (P < 0.05); however, although iNOS in Group C was lower than Group B, the difference was not significant (P > 0.05). MPO and MDA levels of Groups E and F were significantly lower than Group D. Level of ATPase in Group F was significantly different from Groups D and E. iNOS scoring was low in Group F compared with Group D (P < 0.05). MDA, MPO, and iNOS levels of Group F was lower than Group E, and ATPase of Group F was higher than Group E (P < 0.05). The results of this study in a LPS induced HIR model showed that LPS after HIR aggravated HIR injury by increasing neutrophil activation and lipid peroxidation both in serum and liver tissue and iNOS in liver, and depleting energy in liver. AG, a selective iNOS inhibitor, ameliorated the negative effects of endotoxemia induced by LPS after HIR; however, energy depletion and iNOS expression in liver tissue were attenuated only when AG was administered prior to LPS. The findings of this study supported the hypothesis that LPS after HIR would aggravate HIR injury and AG would ameliorate this aggravated injury.

Dynamic Receptor-dependent Activation of Inducible Nitric-oxide Synthase by ERK-mediated Phosphorylation of Ser745

Nitric oxide (NO) is a pleiotropic regulator of vascular function, and its overproduction by inducible nitric-oxide synthase (iNOS) in inflammatory conditions plays an important role in the pathogenesis of vascular diseases. iNOS activity is thought to be regulated primarily at the level of expression to generate "high output" NO compared with constitutive NO synthases. Here we show iNOS activity is acutely up-regulated by activation of the B1-kinin receptor (B1R) in human endothelial cells or transfected HEK293 cells to generate 2.5-5-fold higher NO than that stimulated by Arg alone. Increased iNOS activity was dependent on B1R activation of the MAPK ERK. In HEK293 cells transfected with human iNOS and B1R, ERK phosphorylated iNOS on Ser745 as determined by Western analysis using phospho-Ser antibody, in vitro kinase assays with activated ERK, and MALDI-TOF mass spectrometry. Mutation of Ser745 to Ala did not affect basal iNOS activity but eliminated iNOS phosphorylation and activation in response to B1R agonist. Mutation of Ser745 to Asp resulted in a basally hyperactive iNOS whose activity was not further increased by B1R agonist. ERK and phospho-ERK (after B1R activation) were co-localized with iNOS as determined by confocal fluorescence microscopy. Furthermore, ERK co-immunoprecipitated with iNOS. The discovery that iNOS can be phosphorylated by ERK and acutely activated by receptor-mediated signaling reveals a new level of regulation for this isoform. These findings provide a novel therapeutic target to explore in the treatment of vascular inflammatory diseases.

Postimmunization with IFN-γ-Secreting Glioma Cells Combined with the Inducible Nitric Oxide Synthase Inhibitor Mercaptoethylguanidine Prolongs Survival of Rats with Intracerebral Tumors

High-grade gliomas are one of the most aggressive human tumors with <1% of patients surviving 5 years after surgery. Immunotherapy could offer a possibility to eradicate remnant tumor cells after conventional therapy. Experimental immunotherapy can induce partial cure of established intracerebral tumors in several rodent models. One reason for the limited therapeutic effects could be immunosuppression induced by both the growing tumor and the induced immune reaction. NO has been implicated in tumor-derived immune suppression in tumor-bearing hosts, and unspecific inhibitors of NO synthase have been shown to boost antitumor immunity. In this study, we show that the inducible NO synthase (iNOS)-specific inhibitor mercaptoethylguanidine (MEG) superiorly enhanced lymphocyte reactivity after polyclonal stimulation compared with the iNOS-specific inhibitor L-NIL and the unspecific NO synthase inhibitor L-NAME. Both iNOS inhibitors increased the number and proliferation of T cells but not of B cells. When combined during postimmunization with IFN-gamma-secreting N32 rat glioma cells of rats harboring intracerebral tumors, only MEG increased the cure rate. However, this was only achieved when MEG was administered after immunizations. These findings implicate that NO has both enhancing and suppressive effects after active immunotherapy.

Pharmacological preconditioning by levosimendan is mediated by inducible nitric oxide synthase and mitochondrial K ATP channel activation in the in vivo anesthetized rabbit heart model

Background Provocation of fatal cardiac arrhythmias has limited the use of inotropic agents as heart failure therapy. Levosimendan (LEV) is a new inodilator, whose mechanism of action includes calcium sensitization of contractile proteins and the opening of ATP-dependent potassium channels. Objectives and methods The aim of this investigation was to test whether the administration of LEV has cardioprotective and antiarrhythmic effects against ischemia and reperfusion injury in a manner similar to ischemic preconditioning (IPC) in a well-standardized model of reperfusion arrhythmias in anesthetized adult male rabbits ( n = 122) subjected to 30 min occlusion of the left coronary artery followed by 120 min of reperfusion. Results Pretreatment with either 1 cycle of IPC, LEV (0.1 μmol/kg, i.v.), or IPC + LEV prior to the period of coronary occlusion offers significant infarct size reduction (21.6 ± 1.6%, 22.1 ± 2.2%, and 21.4 ± 1.4%, respectively vs 38.7 ± 3.6% in saline control group; P < 0.01) and antiarrhythmic effects. IPC, LEV and IPC + LEV treatment significantly attenuated the incidence of life-threatening arrhythmias like sustained VT (13%, 13% and 13%, respectively vs 100% in saline control group; P < 0.005) and other arrhythmias (25%, 25% and 13%, respectively vs 100% in saline control group; P < 0.005), and increased the number of surviving animals without arrhythmias. Pretreatment with 5-HD, N ω-nitro- l-arginine methyl ester ( l-NAME, a nonspecific NOS inhibitor) and the specific iNOS inhibitor 1400W [ N-(-3-(aminomethyl)benzyl) acetamidine] abolished the beneficial effects of IPC, and LEV on reperfusion induced arrhythmias and cardioprotection suggesting that benefits have been achieved via both the selective activation of cardiomyocyte mitochondrial K ATP channels and NO. One cycle of IPC and LEV pretreatment significantly preserved the level of ATP in the 30 min ischemic heart and 120 min reperfused heart. Conclusions The present study demonstrates similarities between acute LEV treatment and IPC of the rabbit myocardium in terms of survival, cardioprotection, antiarrhythmic activity, and metabolic status.

Effect of nitric oxide on phagocytic activity of lipopolysaccharide‐induced macrophages: Possible role of exogenous l‐arginine

Among the antimicrobial mechanisms associated with macrophages, NO produced by iNOS plays a major role in intracellular killing, but the relationship between NO and phagocytic activity after injection of inflammatory agents into the peritoneal cavity is not clear. The aim of the present study was to investigate the effect of nitric oxide (NO) on macrophage function after treatment with intraperitoneal lipopolysaccharide (LPS) and the role of exogenous L-arginine administration in this event. Six experimental groups and one control group, each consisting of seven Wistar rats were used: Group I: Control; Group II: LPS; Group III: LPS+L-arginine; Group IV: LPS+L-arginine+Aminoguanidine; Group V: LPS+Aminoguanidine; Group VI: L-arginine; Group VII: Aminoguanidine. Macrophage phagocytic activity and total plasma nitrite levels were increased in the LPS group. In the LPS+L-arginine group, both the phagocytic activity and total plasma nitrite levels showed large increases. Administration of aminoguanidine (AG), a specific iNOS inhibitor, abolished macrophage phagocytic activity and total plasma nitrite levels in the LPS and LPS+L-arginine groups. As a result, we showed that NO produced by macrophages has a role not only in intracellular killing, but also in phagocytic activity.

Myeloid suppressor cell‐associated immune dysfunction in CSA1M fibrosarcoma tumor‐bearing mice

CSA1M tumor-bearing mice exhibited a severe immune dysfunction but the underlying mechanism remained unclear. In this study, we demonstrated that the myeloid suppressor cell (Mac-1(+)Gr-1(+) cells)-(MSC) related T cell immunosuppression in this tumor-bearing model. In mice at the late stage of CSA1M tumor-bearing (Late TB [8-10 weeks after cell inoculation in male BALB/c mice]), the percentages for CD4(+) and CD8(+) T cells decreased but Mac-1(+) cells increased in spleens with severe splenomegaly. There was no deficit for concanavalin A-induced CD4(+) and CD8(+) T cell proliferation, interferon-gamma (IFN-gamma) and interleukin (IL)-4 production, but delayed-type hypersensitivity reaction were attenuated. Analysis of cytokine production in unfractionated spleen cells showed a significant reduction of IFN-gamma and a marked increase of IL-10 and IL-4. In Late-TB mice, splenic MSC number intensively accumulated; the mRNA expressions of the signal transducer and activator of transcription 1, interferon regulatory factor 1 (IRF-1), and inducible nitric-oxide synthase (iNOS) were enhanced in MSC; the nitric oxide production and arginase enzyme activity increased in MSC as well. Furthermore, the concanavalin A-induced T cell proliferation was inhibited in the presence of lipopolysaccharide- or IFN-gamma-activated MSC from Late-TB mice, which could be reversed by the iNOS specific inhibitor L-NMMA. iNOS seemed to be required more than arginase for the suppressive activity of MSC. Taken together, our results suggest that the immune dysfunction in tumor-bearing mice might be causally associated with the accumulation of MSC and its tumor-favoring property.

Insights into ligand selectivity in nitric oxide synthase isoforms: A molecular dynamics study

Molecular dynamics (MD) simulations were carried out for inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) isoforms complexed with substrate ( l-arginine) and the iNOS specific inhibitor GW 273629, 2 for a time period of 1.2 ns. The simulations were compared both within and across the isoforms. iNOS specificity of inhibitor 2 is attributed to water mediated interactions and cooperative hydrogen bond networks. Juxtaposition of the carboxylic and ammonium groups in the substrate and inhibitor serve as a modulating key in binding to the isoforms. Based on these investigations, molecules 3 and 4 were rationally designed to attain specificity among the isoforms. The capability of the designed ligands was theoretically tested through MD simulations to envisage binding patterns with both isoforms. A detailed analysis of the molecular recognition pattern shows molecule 4 to be more selective to iNOS when compared to eNOS.

Nitric oxide in the pathogenesis of diffuse pulmonary fibrosis

By studying the responses of nitric oxide in pulmonary fibrosis, the role of inducible nitric oxide synthase in diffuse pulmonary fibrosis as caused by lipopolysaccharide (LPS) treatment was investigated. When compared to rats treated with LPS only, the rats pretreated with 1400W (an iNOS-specific inhibitor) were found to exhibit a reduced level in: (i) NO x (nitrate/nitrite) production, (ii) collagen type I protein expression, (iv) soluble collagen production, and (iv) the loss of body weight and carotid artery PO 2. In the pulmonary fibroblast culture, exogenous NO from LPS-stimulated secretion by macrophages or from a NO donor, such as DETA NONOate, was observed to induce the expression of TIMP-1, HSP47, TGF-β1, and collagen type I as well as the phosphorylation of SMAD-2. After inhalation of NO for 24 h, an up-regulation of collagen type I protein was also noted to occur in rat pulmonary tissue. The results suggest that the NO signal pathway enhanced the expression of TGF-β1, TIMP-1, and HSP47 in pulmonary fibroblasts, which collectively demonstrate that the NO signal pathway could activate the SMAD-signal cascade, by initiating a rapid increase in TGF-β1, thereby increasing the expression of TIMP-1 and HSP47 in pulmonary fibroblasts, and play an important role in pulmonary fibrosis.

Endothelial nitric oxide synthase (NOS3) knockout decreases NOS2 induction, limiting hyperoxygenation and conferring protection in the postischemic heart

Although it has been shown that endothelial nitric oxide synthase (eNOS)-derived nitric oxide downregulates mitochondrial oxygen consumption during early reperfusion, its effects on inducible NOS (iNOS) induction and myocardial injury during late reperfusion are unknown. Wild-type (WT) and eNOS(-/-) mice were subjected to 30 min of coronary ligation followed by reperfusion. Expression of iNOS mRNA and protein levels and peroxynitrite production were lower in postischemic myocardium of eNOS(-/-) mice than levels in WT mice 48 h postreperfusion. Significantly improved hemodynamics (+/-dP/dt, left ventricular systolic pressure, mean arterial pressure), increased rate pressure product, and reduced myocardial infarct size (18 +/- 2.5% vs. 31 +/- 4.6%) were found 48 h after reperfusion in eNOS(-/-) mice compared with WT mice. Myocardial infarct size was also significantly decreased in WT mice treated with the specific iNOS inhibitor 1400W (20.5 +/- 3.4%) compared with WT mice treated with PBS (33.9 +/- 5.3%). A marked reperfusion-induced hyperoxygenation state was observed by electron paramagnetic resonance oximetry in postischemic myocardium, but Po(2) values were significantly lower from 1 to 72 h in eNOS(-/-) than in WT mice. Cytochrome c-oxidase activity and NADH dehydrogenase activity were significantly decreased in postischemic myocardium in WT and eNOS(-/-) mice compared with baseline control, respectively, and NADH dehydrogenase activity was significantly higher in eNOS(-/-) than in WT mice. Thus deficiency of eNOS exerted a sustained beneficial effect on postischemic myocardium 48 h after reperfusion with preserved mitochondrial function, which appears to be due to decreased iNOS induction and decreased iNOS-derived peroxynitrite in postischemic myocardium.