Levels Of NO Production Research Articles

Effect of Cornuside on Experimental Sepsis

This study was conducted to investigate the efficacy of cornuside, a secoiridoid glucoside compound, in cultured macrophages as well as in an experimental model of sepsis induced by cecal ligation and puncture (CLP) in rats. Cornuside was added to cultured macrophages at different concentrations, and all CLP rats were randomized to receive an intravenous injection of the corresponding drug followed by observation of its antisepsis effect. Our results showed that cornuside downregulated the levels of TNF- alpha, IL-6, and NO production in a dose-dependent manner in activated macrophages, while it upregulated the level of IL-10. Intravenous injection of cornuside or imipenem alone or in combination reduced CLP-induced lethality in rats after CLP. In addition, serum levels of TNF- alpha, IL-6, triggering receptor expressed on myeloid cells, and endotoxin were downregulated. On the other hand, the serum levels of IL-10 were upregulated. Decreased bacterial counts in blood, peritoneum, spleen, liver, and mesenteric lymph nodes and decreased myeloperoxidase in lung, liver, and small intestine also were found after cornuside injection. These data indicate that the antisepsis therapeutic effect of cornuside is mediated by decreased local and systemic levels of a wide spectrum of inflammatory mediators. This work provides first evidence for the clinic use of cornuside as a new immunomodulatory drug that has the capacity to inhibit the inflammatory response in sepsis.

Vaccination Route That Induces Transforming Growth Factor β Production Fails To Elicit Protective Immunity againstLeishmania donovaniInfection

BALB/c mice immunized intraperitoneally (i.p.) and intravenously (i.v.) with Leishmania donovani promastigote membrane antigens (LAg), either free or encapsulated in liposomes, were protected against challenge infection with L. donovani, whereas mice immunized by the subcutaneous (s.c.) and intramuscular routes were not protected. Protected mice showed strong parasite resistance in both the liver and spleen, along with enhanced immunoglobulin G2a and delayed-type hypersensitivity responses. Again, mice vaccinated through the i.p. and i.v. routes showed high levels of NO production after challenge infection. s.c. vaccination resulted in an increased capacity of the spleen cells to produce prechallenge transforming growth factor beta (TGF-beta) levels during the in vitro antigen recall response, whereas i.p. immunization induced production of prechallenge gamma interferon, interleukin-12 (IL-12), and IL-4 levels, with a Th1 bias. Exposure to antigen-stimulated splenocyte supernatants of i.p. but not s.c. immunized mice activated macrophages for in vitro parasite killing. As an enhanced level of TGF-beta was detected in supernatants from unprotected s.c. immunized mice, neutralization by anti-TGF-beta antibody enhanced in vitro macrophage killing activity. The suppressive role of this cytokine was evaluated in vivo by vaccination with liposomal LAg and anti-TGF-beta antibody. Upon parasite challenge, these animals showed significant protection in both the liver and spleen. Moreover, the addition of recombinant TGF-beta in splenocyte supernatants of i.p. immunized mice in vitro as well as in vivo inhibited the protective ability of the macrophages by the i.p. route. Thus, the induction of high prechallenge TGF-beta limits the efficacy of vaccination by routes that are nonprotective.

Abstract 237: Acute Activation of eNOS by Lovastatin Involves Scavenger Receptor-B1

Statins (HMG CoA reductase inhibitors) have beneficial vascular effects independent of their ability to reduce cholesterol synthesis. We and others have previously shown that statins acutely activate endothelial cell NOS (eNOS). Our present study characterized signaling pathways by which statins activate NOS using cultured bovine aortic endothelial cells (BAEC) and COS 7 cells. It also examined involvement of scavenger receptor - B1 (SR-B1), which is expressed in endothelial cells and maintains caveolae cholesterol concentration and eNOS localization. BAEC were exposed to lovastatin (LOV, 10 −6 M) for 2–5 min after pretreatment with the end product of HMG CoA reductase (mevalonic acid, 5×10 −4 M), a Gi protein inhibitor (pertussis toxin, 10 −4 M), a PLC inhibitor (U-73122, 10 −5 M), a receptor tyrosine kinase (RTK) inhibitor (tyrphostine 51A, 2×10 −6 M) or intracellular and extracellular calcium chelators -BAPTA-AM (10 −5 M) and EGTA (10 −4 M), respectively, and levels of NO production were determined. LOV alone acutely increased NO production by 67%. NO production in response to LOV was not blocked by mevalonic acid indicating that it occurs independent of HMG CoA reductase inhibition. Inhibition of Gi protein, PLC, and RTK almost completely blocked the LOV-induced NO generation. Removal of extracellular Ca ++ prevented statin-induced NO production by 90%, while intracellular Ca ++ chelation with BAPTA-AM reduced NO production by 45%. COS 7 cells transfected with eNOS did not show increased NO production to LOV or HDL, indicating NOS activation occurs through an endothelial cell receptor. In COS 7 cells, co-transfected with eNOS and the endothelial SR-B1, LOV or HDL (10 −5 M), a known agonist of SR-B1, increased NO production by over 200%. In COS 7 cells transfected with eNOS alone or co-transfected with the bradykinin B2 receptor, neither LOV nor HDL increased NO production, indicating effects specific for the SR-B1 receptor. In conclusion, our study shows LOV acutely activates eNOS independently of HMG CoA reductase inhibition. eNOS activation appears to involve an endothelial Gi protein receptor and subsequent activation of PLC and RTK pathways, leading to entry of extracellular calcium. Furthermore, LOV can activate eNOS via the SR-B1 receptor.

Effect of elevated temperature and of hypoxia on NO activity in the central nervous system of bivalve molluscs

By light and electron microscopy methods the effect of changes of environmental conditions on the state of the nitroxidergic system has been studied in molluscs on the background of action of elevated temperature and hypoxia. Analysis is performed of biological effect of isolated and combined effects of the studied factors on dynamics of NO synthesis. A higher resistance of CNS neurons to the combined action of hyperthermia and hypoxia is revealed in molluscs with the initially high level of nitrogen oxide production. In molluscs with the initially low level of development of the nitroxidergic system, induction of NO formation in stress has been found to be accompanied by a change of morphology of nervous structures. It is suggested that nitrogen oxide participates in evolutionary established mechanisms of protection of mollusc nerve cells from hypoxia, while the initial high level of NO production reflects larger adaptational possibilities characteristic of these organisms.

Protein kinase C δ is essential for optimal macrophage-mediated phagosomal containment ofListeria monocytogenes

Activation of macrophages and subsequent "killing" effector functions against infectious pathogens are essential for the establishment of protective immunity. NF-IL6 is a transcription factor downstream of IFN-gamma and TNF in the macrophage activation pathway required for bacterial killing. Comparison of microarray expression profiles of Listeria monocytogenes (LM)-infected macrophages from WT and NF-IL6-deficient mice enabled us to identify candidate genes downstream of NF-IL6 involved in the unknown pathways of LM killing independent of reactive oxygen intermediates and reactive nitrogen intermediates. One differentially expressed gene, PKCdelta, had higher mRNA levels in the LM-infected NF-IL6-deficient macrophages as compared with WT. To define the role of PKCdelta during listeriosis, we infected PKCdelta-deficient mice with LM. PKCdelta-deficient mice were highly susceptible to LM infection with increased bacterial burden and enhanced histopathology despite enhanced NF-IL6 mRNA expression. Subsequent studies in PKCdelta-deficient macrophages demonstrated that, despite elevated levels of proinflammatory cytokines and NO production, increased escape of LM from the phagosome into the cytoplasm and uncontrolled bacterial growth occurred. Taken together these data identified PKCdelta as a critical factor for confinement of LM within macrophage phagosomes.

Heterogeneous Expression of NO-Activated Soluble Guanylyl Cyclase in Mammalian Heart: Implications for NO- and Redox-Mediated Indirect Versus Direct Regulation of Cardiac Ion Channel Function

Nitrogen oxides exert significant but diverse regulatory effects on cardiac myocytes. Many of these effects are due to modulation of voltage-sensitive ion channel function. The redox-status of NO-related compounds is a critical factor in determining whether indirect (cGMP-dependent) versus direct (cGMP-independent) effects are dominant. However, molecular mechanisms by which different cardiac myocyte types, and associated different ion channel types expressed within them, could achieve selectivity between NO-related indirect versus direct effects are unclear We have previously demonstrated heterogeneous expression gradients of Type III NO synthase (eNOS) and sarcolemmal superoxide dismutase (ECSOD) in ferret and human ventricle, with both enzymes being highly expressed in right ventricle and left ventricular subepicardium but markedly reduced in left ventricular subendocardium. In this study we extend this previous analysis by analyzing NO-activated soluble guanylyl cyclase (sGC) expression in the heart (ferret and human). We demonstrate that, at both tissue and single myocyte levels, sGC protein expression is heterogeneous, being high in sinoatrial node, right atrium, right ventricle and left ventricular subepicardium, but markedly reduced to absent in left atrium and left ventricular subendocardium. Thus, there is a significant overlap in expression gradients of sGC, eNOS, and ECSOD among distinct cardiac tissue and myocyte types. These gradients positively correlate with both: i) experimentally measured basal NO production levels; and ii) expression gradients of specific voltage-gated ion channels (particularly Kv1 and Kv4 channels). Our results provide the first demonstration in the heart of an expressed coupled multienzymatic system for selective regulation of indirect (sGC-dependent) versus direct (sGC-independent) NO- and redox-related modulation of voltage-gated ion channel function in different myocyte types. Our results also have functional implications for NO. / redox - related modulation of ion channels expressed in other cell types, including neurons, skeletal muscle and smooth muscle.

SIR2-Deficient Leishmania infantum Induces a Defined IFN-γ/IL-10 Pattern That Correlates with Protection

The ability to manipulate the Leishmania genome to create genetically modified parasites by introducing or eliminating genes is considered a powerful alternative for developing a new generation vaccine against leishmaniasis. Previously, we showed that the deletion of one allele of the Leishmania infantum silent information regulatory 2 (LiSIR2) locus was sufficient to dramatically affect amastigote axenic proliferation. Furthermore, LiSIR2 single knockout (LiSIR2(+/-)) amastigotes were unable to replicate in vitro inside macrophages. Because this L. infantum mutant persisted in BALB/c mice for up to 6 wk but failed to establish an infection, we tested its ability to provide protection toward a virulent L. infantum challenge. Strikingly, vaccination with a single i.p. injection of LiSIR2(+/-) single knockout elicits complete protection. Thus, vaccinated BALB/c mice showed a reversal of T cell anergy with specific anti-Leishmania cytotoxic activity and high levels of NO production. Moreover, vaccinated mice simultaneously generated specific anti-Leishmania IgG Ab subclasses suggestive of both type 1 and type 2 responses. A strong correlation was found between the elimination of the parasites and an increased Leishmania-specific IFN-gamma/IL-10 ratio. Therefore, we propose that the polarization to a high IFN-gamma/low IL-10 ratio after challenge is a clear indicator of vaccine success. Furthermore these mutants, which presented attenuated virulence, represent a good model to understand the correlatives of protection in visceral leishmaniasis.

Ras/myc-transformed serum-free mouse embryo cells under simulated inflammatory and infectious conditions increase levels of nitric oxide and matrix metalloproteinase-9 without a direct association between them

Inflammatory and infectious conditions were simulated in cultures of ras/myc-transformed serum-free mouse embryo (ras/myc SFME) cells, using interferon-gamma (IFN-gamma, 100 units/ml) and lipopolysaccharide (LPS, 0.5 microg/ml) co-treatment for 24 h, to investigate their effects on the expression of inducible nitric oxide synthase (iNOS) mRNA and the production of NO. Aminoguanidine (AG, 1 mM; an NOS inhibitor) along with IFN-gamma and LPS, S-nitroso-N-acetyl-DL-penicillamine (SNAP, 100 microM; an NO donor) and/or (+/-)-N-[(E)-4-Ethyl-2-[(Z)-hydroxyimino]-5-nitro-3-hexene-1-yl]-3-pyridine carboxamide (NOR4, 100 microM; an NO donor), were also added to analyze the possible association of NO with matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1). Co-treatment of cells with IFN-gamma and LPS increased iNOS mRNA expression, NO production, MMP-9 mRNA expression, and 105 kDa MMP-9 production. Additional treatment with the NOS inhibitor AG inhibited NO production, but did not down-regulate the expression of MMP-9 mRNA or 105 kDa MMP-9. The NO donors SNAP and NOR4 did not affect the expression of MMP-9 mRNA, 105 kDa MMP-9 or TIMP-1 mRNA. These results suggest that ras/myc SFME cells respond to infectious and inflammatory conditions and can enhance malignancy as cancer cells due to their increased levels of NO and MMP-9 production, but that NO is not directly associated with MMP-9 in these cells.

Bio Imaging of Intracellular NO Production in Single Bone Cells After Mechanical Stimulation

We show the intracellular upregulation of NO production after mechanical stimulation, an essential chemical signal in bone remodeling. This is done in real time using the fluorescent chromophore DAR-4M AM. Differences in cellular response to mechanical stimulation of different regions of a single cell were observed. Osteocytes are the most abundant bone cells that are believed to be the mechanosensors of bone, responding to mechanical stresses in interstitial fluid flow through the canaliculi. Under mechanical load, chemical signals such as NO play a key role in the activity of osteoblasts/osteoclasts that regulate bone remodeling. Despite the importance of NO in signaling, its real-time detection has proved challenging. This is largely because of the short NO half-life (typically approximately 0.1-5 s). Here, we show the upregulation of intracellular NO production in single osteocytes under localized mechanical stimulation. We used the chromophore DAR-4M AM for NO detection. This is loaded into surface-attached MLO-Y4 osteocyte-like and MC3T3-E1 osteoblast-like cells that are subjected to a localized mechanical stimulation using optical tweezers or a microneedle tip. DAR-4M AM is membrane-permeable and chelates NO, forming a stable, fluorescent compound, which is visible with a rhodamine filter. Nonstimulated MLO-Y4 and MC3T3-E1 cells showed basal NO production levels, as indicated by a gradual increase in their fluorescence intensity. Localized mechanical stimulation of single MC3T3-E1 cells and MLO-Y4 cells by optical tweezers (150-550 pN, 0.5-3 Hz, 1 minute) showed a nearly 15-30% increase, whereas MLO-Y4 cells stimulated by a microneedle (10-20 nN, 1 minute) showed nearly 15-16% increase relative to their nonstimulated state. Furthermore, stimulation of a single cell process by a microneedle resulted in a 2-10% increase in the fluorescence intensity. NO is essential for mechanically induced bone remodeling and is a meaningful parameter for measuring bone cell activation after mechanical loading. Here we show NO upregulation in individual bone cells after a localized mechanical stimulation. We also show that both the cell body and the cell processes might be involved in mechanosensing. This technique allows characterization of the mechanosensitivity of different parts of a single osteocyte. This opens up the possibility to uncover the complexities and function of single osteocytes in the dynamic process of bone remodeling.

Trypanosoma cruzi-Mediated IFN-γ-Inducible Nitric Oxide Output in Macrophages Is Regulated byiNOSmRNA Stability

Although the effects of activated macrophages (Muphi) on the intracellular parasite Trypanosoma cruzi are well documented, little is known about how host-Muphi functions are affected by this pathogen before activation. This study is aimed at assessing the capacity of T. cruzi infection to modulate J77.4 murine Muphi NO generation following IFN-gamma stimulation, and identifying mechanisms regulating this modulation. Results show that parasite infection potentiates Muphi to produce inducible NO synthase (iNOS) mRNA and protein as well as NO following IFN-gamma stimulation above IFN-gamma alone controls. This potentiation occurs through the concomitant activation of NF-kappaB, ERK1/ERK2 MAPK, and stress-activated protein kinase signaling pathways. Activation of the JAK/STAT pathway by IFN-gamma then leads to STAT1alpha translocation and the transcription of a stable iNOS mRNA species. A decreased rate of iNOS mRNA degradation results in elevated levels of iNOS protein and NO production. Maximal iNOS expression is likely achieved through NF-kappaB activation by T. cruzi, whereas iNOS mRNA stability results from ERK1/ERK2 MAPK and stress-activated protein kinase activation by the infection. Taken together, our data show that T. cruzi-infected Muphi NO generation is controlled at both pre- and posttranscriptional levels and relies on signaling pathway cross-talk. This is the first report of a parasite pathogen capable of heightening host mRNA stability.

Nitric oxide monocyte production levels in patients with the hepatosplenic form of Scistosoma mansoni infection who underwent splenectomy, ligature of the left gastric vein and auto implantation of spleen tissue in the major omentum

To measure the levels of NO production by monocytes in patients with the hepatosplenic form of Schistosomiasis mansoni who underwent splenectomy, ligature of the left gastric vein and auto implantation of spleen tissue in the major omentum. Four groups of volunteers were enrolled in the investigation: G1 - 12 patients with S. mansoni infection in its hepatosplenic form without any kind of treatment (SMH); G2 - 13 SMH patients who underwent medical treatment and portal hypertension decompression splenectomy and ligature of the left gastric vein (SMH/SLGV); G3 - 19 patients similar to the later group, but additionally received auto implantation of spleen morsels in the major omentum (SMH/SLGV/AI); and G4 - 15 individuals with no S. mansoni infection coming from the same geographical area and presenting similar socio-economical status (CG). Nitrite production by monocytes was determined by a standard Griess reaction adapted to microplates. The results were presented by mean +/- SD for each group. Significant differences in NO production by monocytes were determined by Tukey-Kramer multicomparisons test. Probability values of 0.05 were considered significant. Patients from G1 (SMH) showed lower level of NO production by monocytes (5.28 +/- 1.28 micromol/ml). Patients from G2 (SMH/SLGV) showed similar results (6.67 +/- 0.44 micromol/ml-q = 2.681 p > 0.05). Individuals of G4 (CG) showed higher level of NO production by monocytes (8.19 +/- 2.74 micromol/ml). Patients from G3 (SMH/SGLV/AI) showed similar NO production by PBMC as compared to individuals of G4 (CG) - (7.41 +/- 1.65 micromol/ml- q = 1.615 p > 0.05). The volunteers from G4 (CG) and G3 (SMH/SLGV/AI) showed significantly greater levels of NO production by monocytes as compared to those from G1 (SMH) - (q = 5.837 p < 0.01, and q = 4.285 p < 0.05). Collectively, the results point to a restoration of NO normal production by monocytes in SHM patients who underwent medical and surgical treatments, especially in those who had received auto implantation of spleen tissue in the major omentum after splenectomy and ligature of the left gastric vein. The data gives further support to the hypothesis that this additional procedure is important in the restoration of the immune response of these patients, since NO synthesis by the monocytes correlates with protective immunity against infection; thus, protecting them against overwhelming post splenectomy infection.

NOS expression and activity in cerebral resistance arteries: Effects of age and exercise training

We have previously shown that endothelium-dependent vasodilation to acetylcholine (ACh) is preserved in cerebral resistance arteries from aged rats; however, the role of specific isozymes of nitric oxide synthase (NOS) in mediating dilation to ACh changes with age and exercise training. In the present study, we investigated the effects of age and exercise training on expression and activity of NOS isoforms. Young (4 months) and old (24 months) male Fischer-344 rats underwent exercise training or remained sedentary for 10 weeks. Posterior communicating arteries (PCoA) were isolated for assessment of ACh-stimulated NO production and protein levels of NOS isozymes. ACh-stimulated NO production was similar in PCoA from young and old rats. Treatment with aminoguanidine, an inhibitor of iNOS significantly reduced NO production in PCoA from young but not old rats; however, iNOS protein was not detectable in PCoA from rats in any of the groups. Although protein levels for endothelial NOS (eNOS) and neuronal NOS (nNOS) were not altered by age or exercise training; calcium-free production of NO was significantly lower in PCoA from old rats. These data indicate that NOS activity differs in PCoA from young and old rats; however, alterations of NO production that occur with age and training are likely related to changes in NOS regulatory mechanisms rather than changes in enzyme expression.

Influence of Th1/Th2 Cytokines and Nitric Oxide in Murine Systemic Infection Induced by Sporothrix schenckii

In an attempt to elucidate the effects of Sporothrix schenckii infection on the immune response, our laboratory has developed a murine model of disseminated sporotrichosis. Helper T cells can be further subdivided into Th1 and Th2 phenotypes. The differentiation of two subsets of T lymphocytes is driven by IL-12 and IL-4 cytokines, respectively. Th1 cells produce IFN-gamma that activate macrophages and promote cell-mediated immunity. In addition, we found low levels of iNOS and NO production in the initial (1st and 2nd weeks) and final (9th and 10th weeks) periods of the infection, in contrast with the period of week 4 to 7 of elevated values. The determination of IFN-gamma and IL-12 are in agreement with NO/iNOS detection, showing the presence of cellular immune response throughout the infectious process. However, the production of IL-4 shows an increase in levels after the 5th and 6th weeks suggesting a participation of Th2 response in this period as well. Regarding these results, the study demonstrated that in experimental sporotrichosis infection the cellular immune response participated throughout the period analyzed as a nitric oxide dependent mechanism. In contrast, the presence of Th2 response began in the 5th week, suggesting the participation of humoral immune response in advanced stages of sporotrichosis.

Acute Hyperglycaemia Impairs Cognitive Function in Children with IDDM

The role of growth hormone (GH) in obesity is controversial. Childhood obesity is characterized by reduced GH secretion. Low, high and normal serum insulin-like growth factor-I (IGF-I) levels have been described in obese children in the face of low GH secretion. There is conflicting information on the interaction of GH and obesity upon components of the GH-IGF-I-insulin-like growth factor binding proteins (IGFBPs) system and its relationship to cardiovascular risk factors. This review highlights the role of GH modulating cardiovascular risk factors, including lipid levels, inflammatory markers, cardiac and endothelial function in children and adolescents with obesity and insulin resistance. The interplay between GH secretion, NO production, IGF-I, IGFBP-1, and IGFBP-3 levels, lipid profile, insulin sensitivity and the degree of visceral obesity necessitates further study. These metabolic parameters may be useful markers of premature atherosclerosis in children with obesity. Further studies should address the question of optimal dose and duration of GH therapy in non-GH deficient obese patients with metabolic disturbances at risk for premature atherosclerosis.

Nitric oxide production by hemocytes of larva and pharate prepupa of Galleria mellonella in response to bacterial lipopolysaccharide: Cytoprotective or cytotoxic?

Nitric oxide production by the hemocytes of the last instar larvae and sessile pharate prepupa of Galleria mellonella (Lepidoptera: Pyralidae) was demonstrated in vitro in response to preparations of bacterial lipopolysaccharide (LPS) from Escherichia coli using the Griess reaction. Augmented, dose dependent nitric oxide production was observed in the pharate prepupal hemocytes compared with larval hemocytes. This was partially reversed in a dose dependent manner with S-methyl thiourea (SMT), a specific inhibitor of inducible nitric oxide synthase (iNOS). A decrease in NO production was also observed when non-selective inhibitors such as N G-nitro- l-arginine ( l-NAME) and N-ω-nitro- l-arginine ( l-NNA) were used, albeit the inhibition was not to the extent of SMT. Challenge with the entomopathogenic Gram-negative bacterium Photorhabdus asymbiotica also enhanced NO production by hemocytes of both stages. SMT, alone or in combination with P. asymbiotica significantly decreased levels of NO production. However, it was observed that phenoloxidase activity (a cascade for innate immune responses) was independent of NO production stimulation. NO donors, S-nitroso- N-acetyl-penicillamine (SNAP) and diethylenetriamine NO adduct (DETA/NO) at various concentrations (100–500 μM) resulted in the lysis of hemocytes dose dependently. The nitrite production in these cases was however similar to LPS stimulation (10 μg/mL) and 1.5–3 fold lower than those observed upon P. asymbiotica (2.5 × 10 7 cfu/mL) stimulation. Survival analysis (Kaplan-Meier) following injection of P. asymbiotica alone or in combination with SMT revealed that only 12.5% (median survival 25.5 h) of co-injected larvae of G. mellonella survived in comparison to 28.6% (median survival 29 h) survivors in P. asymbiotica alone-injected groups till the end of the study. In contrast, co-injected pharate prepupa survived longer (median survival 28 h) than the P. asymbiotica alone-injected individuals (median survival 24 h); however, both co-injected and P. asymbiotica-injected groups showed 100% mortality at the end of the study. Based on the above, we propose that although NO production is involved in cellular immune responses of this insect to bacterial infection it does not appear to be a part of the signalling pathway that initiates the prophenoloxidase (PPO) cascade, and the extended NO production/overproduction by pharate prepupal hemocytes could result in cytotoxic rather than cytoprotective effects compared with larval hemocytes.

Diazepam effects on Ehrlich tumor growth and macrophage activity in mice

Besides the central gabaergic receptors described for benzodiazepines, peripheral type binding sites (PBR) were also identified for these molecules in endocrine steroidogenic tissues, immune organs and cells, such as macrophages and lymphocytes. PBR activation was reported to decrease innate immunity and host defense. The present experiment was designed to analyze the effects of diazepam on Ehrlich tumor growth, and on macrophage activity of Ehrlich tumor bearing mice. Results showed that diazepam (3.0 mg/kg/day, for 7 days) increased the number of Ehrlich tumor cells and the volume of tumor-induced ascitic fluid. These effects were not observed after smaller doses of diazepam, suggesting a dose-dependant effect. Furthermore, our results show that 3.0 mg/kg of diazepam, administered daily, for 2 days, decreased (1) the number of peritoneal leukocytes retrieved after injection of the Ehrlich tumor, (2) the percents of macrophage spreading, and (3) the levels of macrophage NO production. Diazepam (3.0 mg/kg/day for 2 days) had no effect on macrophage phagocytosis or on H 2O 2 production. The present data is discussed based on a direct and/or indirect action of diazepam. Particularly, our findings might be due to a direct effect of diazepam on PBRs present on macrophages and tumor cells, or could still be mediated by PBR stimulation within the hypothalamus–pituitary–adrenal (HPA) axis.

Effects of bezafibrate on the expression of endothelial nitric oxide synthase gene and its mechanisms in cultured bovine endothelial cells

Objective Peroxisome proliferator-activated receptors alpha (PPARα) is a target gene for atherosclerosis and cardiovascular diseases. However, effects of PPARα on endothelial nitric oxide synthase (eNOS) remain unknown. We investigated the eNOS regulation by bezafibrate, a ligand of PPARα, and involved signaling pathways. Methods and results Firstly, in cultured bovine aorta endothelial cells (BAEC), bezafibrate significantly upregulated eNOS at protein, mRNA levels and NO production, respectively, in a concentration-dependent fashion (50–200 μM). Next, the effects of bezafibrate on signal pathways and eNOS mRNA stability in BAEC were investigated. Results showed that bezafibrate induced phosphorylation of MAPK. Inhibitors of PPARα, PI3 kinase and MAPK, respectively, markedly attenuate bezafibrate-induced upregulation of eNOS. Bezafibrate incubation increased eNOS mRNA half-life, activated eNOS promoter, enhanced phosphorylation of eNOS ser-1179 site, and decreased phosphorylation of eNOS thr-497 site via activating ERK and Akt. Conclusions Bezafibrate can upregulate eNOS expression, enhance phosphorylation of eNOS ser-1179, increase NO production and transcription level and stability of eNOS mRNA through pathway dependent of PPARα and nongenomic effects mediated by MAPK and PI3K pathways. Hence, PPARα ligands exert direct benefits on vessel endothelial functions through an increase in eNOS expression level and phosphorylation of eNOS ser-1179. This mechanism provides additional anti-atherosclerotic and anti-hypertension benefits of bezafibrate in addition of lipid-lowering effects.

Kinetoplastid Membrane Protein-11 DNA Vaccination Induces Complete Protection against Both Pentavalent Antimonial-Sensitive and -Resistant Strains of Leishmania donovani That Correlates with Inducible Nitric Oxide Synthase Activity and IL-4 Generation: Evidence for Mixed Th1- and Th2-Like Responses in Visceral Leishmaniasis

The emergence of an increasing number of Leishmania donovani strains resistant to pentavalent antimonials (SbV), the first line of treatment for visceral leishmaniasis worldwide, accounts for decreasing efficacy of chemotherapeutic interventions. A kinetoplastid membrane protein-11 (KMP-11)-encoding construct protected extremely susceptible golden hamsters from both pentavalent antimony responsive (AG83) and antimony resistant (GE1F8R) virulent L. donovani challenge. All the KMP-11 DNA vaccinated hamsters continued to survive beyond 8 mo postinfection, with the majority showing sterile protection. Vaccinated hamsters showed reversal of T cell anergy with functional IL-2 generation along with vigorous specific anti-KMP-11 CTL-like response. Cytokines known to influence Th1- and Th2-like immune responses hinted toward a complex immune modulation in the presence of a mixed Th1/Th2 response in conferring protection against visceral leishmaniasis. KMP-11 DNA vaccinated hamsters were protected by a surge in IFN-gamma, TNF-alpha, and IL-12 levels along with extreme down-regulation of IL-10. Surprisingly the prototype candidature of IL-4, known as a disease exacerbating cytokine, was found to have a positive correlation to protection. Contrary to some previous reports, inducible NO synthase was actively synthesized by macrophages of the protected hamsters with concomitant high levels of NO production. This is the first report of a vaccine conferring protection to both antimony responsive and resistant Leishmania strains reflecting several aspects of clinical visceral leishmaniasis.

Spermine Causes Loss of Innate Immune Response to Helicobacter pylori by Inhibition of Inducible Nitric-oxide Synthase Translation

Helicobacter pylori infection of the stomach elicits a vigorous but ineffective host immune and inflammatory response, resulting in persistence of the bacterium for the life of the host. We have reported that in macrophages, H. pylori up-regulates inducible NO synthase (iNOS) and antimicrobial NO production, but in parallel there is induction of arginase II, generating ornithine, and of ornithine decarboxylase (ODC), generating polyamines. Spermine, in particular, has been shown to restrain immune response in activated macrophages by inhibiting proinflammatory gene expression. We hypothesized that spermine could prevent the antimicrobial effects of NO by inhibiting iNOS in macrophages activated by H. pylori. Spermine did not affect the up-regulation of iNOS mRNA levels but in a concentration-dependent manner significantly attenuated iNOS protein levels and NO production. Reduction in iNOS protein was due to inhibition of iNOS translation and not due to iNOS degradation. ODC knockdown with small interfering (si) RNA resulted in increased H. pylori-stimulated iNOS protein expression and NO production without altering iNOS mRNA levels. When macrophages were cocultured with H. pylori, killing of bacteria was enhanced by transfection of ODC siRNA and prevented by addition of spermine. These results identify a mechanism of immune dysregulation induced by H. pylori in which stimulated spermine synthesis by the arginase-ODC pathway inhibits iNOS translation and NO production, leading to persistence of the bacterium and risk for peptic ulcer disease and gastric cancer.

Heat shock regulates the respiration of cardiac H9c2 cells through upregulation of nitric oxide synthase.

Mild and nonlethal heat shock (i.e., hyperthermia) is known to protect the myocardium and cardiomyocytes against ischemic injury. In the present study, we have shown that heat shock regulates the respiration of cultured neonatal cardiomyocytes (cardiac H9c2 cells) through activation of nitric oxide synthase (NOS). The respiration of cultured cardiac H9c2 cells subjected to mild heat shock at 42 degrees C for 1 h was decreased compared with that of control. The O2 concentration at which the rate of O2 consumption is reduced to 50% was increased in heat-shocked cells, indicating a lowering of O2 affinity in the mitochondria. Western blot analyses showed a fourfold increase in the expression of heat shock protein (HSP) 90 and a twofold increase in endothelial NOS (eNOS) expression in the heat-shocked cells. Immunoblots of eNOS, inducible NOS (iNOS), and neuronal NOS (nNOS) in the immunoprecipitate of HSP90 of heat-shocked cells showed that there was a sevenfold increase in eNOS and no changes in iNOS and nNOS. Confocal microscopic analysis of cells stained with the NO-specific fluorescent dye 4,5-diaminofluorescein diacetate showed higher levels of NO production in the heat-shocked cells than in control cells. The results indicate that heat shock-induced HSP90 forms a complex with eNOS and activates it to increase NO concentration in the cardiac H9c2 cells. The generated NO competitively binds to the complexes of the respiratory chain of the mitochondria to downregulate O2 consumption in heat-shocked cells. On the basis of these results, we conclude that myocardial protection by hyperthermia occurs at least partly by the pathway of HSP90-mediated NO production, leading to subsequent attenuation of cellular respiration.