High Levels Of Nitric Oxide Research Articles

Induction of proinflammatory cytokines and nitric oxide by Trypanosoma cruzi in renal cells

Chagas disease is typically associated with cardiac involvement. During the acute phase of murine infection with Trypanosoma cruzi, severe acute myocarditis can develop. Prior to cardiac alteration, however, infected mice present with renal inflammatory infiltration causing acute kidney injury due to an ischemia/reperfusion lesion. Thus, the present study was undertaken in order to evaluate whether the parasites or some of their components would directly affect renal cells. As such, this study employed kidney cell lines (mesangial, epithelial, and proximal tubular) that mimic different regions of the renal system. Mesangial cells are more resistant to infection, showing reduced parasite internalization relative to epithelial and proximal tubular cells. Upon infection, mesangial cells produced more nitric oxide, tumor factor necrosis-α, and interferon-γ and showed decreased viability when compared to the other cell lines. These results indicate that the resistance of mesangial cells to infection may be related to the increased expression of nitric oxide and proinflammatory cytokines. Conversely, the high levels of nitric oxide produced by these cells caused impairment of cell integrity and viability. Higher nitric oxide concentrations promote cellular injury and can be involved in the genesis of ischemia/reperfusion lesions in acute kidney injury.

JS-K, a nitric oxide-releasing prodrug, induces breast cancer cell death while sparing normal mammary epithelial cells

Targeted therapy with reduced side effects is a major goal in cancer research. We investigated the effects of JS-K, a nitric oxide (NO) prodrug designed to release high levels of NO when suitably activated, on human breast cancer cell lines, on non-transformed human MCF-10A mammary cells, and on normal human mammary epithelial cells (HMECs). Cell viability assay, flow cytometry, electron microscopy, and Western blot analysis were used to study the effects of JS-K on breast cancer and on mammary epithelial cells. After a 3-day incubation, the IC50s of JS-K against the breast cancer cells ranged from 0.8 to 3 µM. However, JS-K decreased the viability of the MCF-10A cells by only 20% at 10-µM concentration, and HMECs were unaffected by 10 µM JS-K. Flow cytometry indicated that JS-K increased the percentages of breast cancer cells under-going apoptosis. Interestingly, flow cytometry indicated that JS-K increased acidic vesicle organelle formation in breast cancer cells, suggesting that JS-K induced autophagy in breast cancer cells. Electron microscopy confirmed that JS-K-treated breast cancer cells underwent autophagic cell death. Western blot analysis showed that JS-K induced the expression of microtubule light chain 3-II, another autophagy marker, in breast cancer cells. However, JS-K did not induce apoptosis or autophagy in normal human mammary epithelial cells. These data indicate that JS-K selectively induces programmed cell death in breast cancer cells while sparing normal mammary epithelial cells under the same conditions. The selective anti-tumor activity of JS-K warrants its further investigation in breast tumors.

Increased Inducible Nitric Oxide Synthase Expression in Organs Is Associated with a Higher Severity of H5N1 Influenza Virus Infection

BackgroundThe mechanisms of disease severity caused by H5N1 influenza virus infection remain somewhat unclear. Studies have indicated that a high viral load and an associated hyper inflammatory immune response are influential during the onset of infection. This dysregulated inflammatory response with increased levels of free radicals, such as nitric oxide (NO), appears likely to contribute to disease severity. However, enzymes of the nitric oxide synthase (NOS) family such as the inducible form of NOS (iNOS) generate NO, which serves as a potent anti-viral molecule to combat infection in combination with acute phase proteins and cytokines. Nevertheless, excessive production of iNOS and subsequent high levels of NO during H5N1 infection may have negative effects, acting with other damaging oxidants to promote excessive inflammation or induce apoptosis.Methodology/Principal FindingsThere are dramatic differences in the severity of disease between chickens and ducks following H5N1 influenza infection. Chickens show a high level of mortality and associated pathology, whilst ducks show relatively minor symptoms. It is not clear how this varying pathogenicty comes about, although it has been suggested that an overactive inflammatory immune response to infection in the chicken, compared to the duck response, may be to blame for the disparity in observed pathology. In this study, we identify and investigate iNOS gene expression in ducks and chickens during H5N1 influenza infection. Infected chickens show a marked increase in iNOS expression in a wide range of organs. Contrastingly, infected duck tissues have lower levels of tissue related iNOS expression.Conclusions/SignificanceThe differences in iNOS expression levels observed between chickens and ducks during H5N1 avian influenza infection may be important in the inflammatory response that contributes to the pathology. Understanding the regulation of iNOS expression and its role during H5N1 influenza infection may provide insights for the development of new therapeutic strategies in the treatment of avian influenza infection.

1,3,5-Trihydroxy-4-prenylxanthone represses lipopolysaccharide-induced iNOS expression via impeding posttranslational modification of IRAK-1

Both high level of nitric oxide (NO) and its generating enzyme, inducible NO synthase (iNOS), play important roles in pathophysiological conditions such as inflammatory processes. We previously found that 1,3,5-trihydroxy-4-prenylxanthone (TH-4-PX) isolated from Cudrania cochinchinensis repressed lipopolysaccharide (LPS)-induced NO production in RAW264.7 macrophages. Here we further examined the underlying mechanisms using RT-PCR and Western blot analyses. Consistent with NO inhibition, suppression of LPS-induced iNOS expression by TH-4-PX through abolishing IκB kinase (IKK) phosphorylation, IκB degradation and nuclear factor-κB (NF-κB) nuclear translocation was observed. After LPS stimulation, the increased nuclear level of c-Fos and c-Jun (major components of activator protein-1, AP-1) and the phosphorylated level of upstream signal molecules, such as c-Jun NH2-terminal kinase (JNK) and extracellular signal-regulated kinase, (ERK) were all significantly suppressed by TH-4-PX, while p38 remained unaffected. A further experiment revealed that TH-4-PX inhibited the phosphorylation of transforming growth factor-β (TGF-β)-activated kinase 1 (TAK1), an upstream signaling molecule required for IKK and mitogen-activated protein kinases (MAPKs) activation. Stimulation with LPS also triggered the modification (phosphorylation and ubiquitination) and eventually the proteasomal degradation of membrane-associated interleukin (IL)-1 receptor-associated serine/threonine kinase 1 (IRAK-1), an essential signaling component to toll-like receptor (TLR)-mediated TAK-1 activation. Interestingly, the modified pattern of IRAK-1 in the presence LPS was significantly attenuated by TH-4-PX treatment. In conclusion, TH-4-PX inhibited LPS-induced NF-κB and AP-1 activations by interfering with the posttranslational modification (phosphorylation and/or ubiquitinylation) of IRAK-1 in the cell membrane to impede TAK1-mediated activation of IKK and MAPKs signal transduction.

Uric Acid; a Possible Mediator of the Adjuvant Effect of Alum in Mice Immunized with Ovalbumin

One proposed mechanism by which alum enhances an immune response is by its ability to induce an inflammatory response that results in the release of uric acid from necrotic cells. Uric acid is thought to be a mediator in enhancing the immune response. The aim of this study was to investigate the immunopotentiating effect of uric acid. Groups of BALB/c mice were injected intraperitoneally with ovalbumin, ovalbumin + alum, ovalbumin + uric acid, uric acid, alum, or allopurinol. Two other groups were pretreated with allopurinol and were given ovalbumin + alum, or ovalbumin + uric acid 24 hours later. An additional two groups served as controls. On days 4, 7 and 10 post-injection, the numbers of Interleukin 4(IL-4) and Interferon-γ (IFN-γ) secreting spleen cells were determined by the ELISPOT assay. Serum uric acid levels were determined using an autoanalyser and nitric oxide using the Greiss reagent. The groups that received alum + ovalbumin or uric acid + ovalbumin had the highest numbers of IL-4 and INF-γ secreting cells as compared to all the groups. Allopurinol administration one day prior to alum + ovalbumin or uric acid + ovalbumin resulted in a decrease in the number of IL-4 and INF-γ secreting cells when compared to alum+ ovalbumin or uric acid + ovalbumin allopurinol - untreated groups. Groups that received alum, alum + ovalbumin, uric acid, and uric acid + ovalbumin had high serum uric acid levels as compared to all the groups. All groups that received alum had the highest levels of nitric oxide when compared to the groups that were not given alum. In conclusion, it appears that uric acid might be a mediator in the adjuvant effect of alum.

Mechanisms of nitric oxide-mediated inhibition of EMT in cancer

The role of nitric oxide (NO) in cancer has been controversial and is based on the levels of NO and the responsiveness of the tumor type. It remains unclear whether NO can inhibit the epithelial to mesenchymal transition (EMT) in cancer cells. EMT induction is mediated, in part, by the constitutive activation of the metastasis-inducer transcription factor, Snail and EMT can be inhibited by the metastasis-suppressor Raf-1 kinase inhibitor protein (RKIP) and E-cadherin. Snail is transcriptionally regulated by NF-κB and in turn, Snail represses RKIP transcription. Hence, we hypothesized that high levels of NO, that inhibit NF-κB activity, may also inhibit Snail and induce RKIP and leading to inhibition of EMT. We show that treatment of human prostate metastatic cell lines with the NO donor, DETANONOate, inhibits EMT and reverses both the mesenchymal phenotype and the cell invasive properties. Further, treatment with DETANONOate inhibits Snail expression and DNA-binding activity in parallel with the upregulation of RKIP and E-cadherin protein levels. The pivotal roles of Snail inhibition and RKIP induction in DETANONOate-mediated inhibition of EMT were corroborated by both Snail silencing by siRNA and by ectopic expression of RKIP. The in vitro findings were validated in vivo in mice bearing PC-3 xenografts and treated with DETANONOate. The present findings show, for the first time, the novel role of high subtoxic concentrations of NO in the inhibition of EMT. Thus, NO donors may exert therapeutic activities in the reversal of EMT and metastasis.

LXR Deficiency Confers Increased Protection against Visceral Leishmania Infection in Mice

BackgroundThe liver X receptors (LXRs) are a family of nuclear receptor transcription factors that are activated by oxysterols and have defined roles in both lipid metabolism and cholesterol regulation. LXRs also affect antimicrobial responses and have anti-inflammatory effects in macrophages. As mice lacking LXRs are more susceptible to infection by intracellular bacteria Listeria monocytogenes and Mycobacterium tuberculosis, we hypothesized that LXR might also influence macrophage responses to the intracellular protozoan parasite Leishmania chagasi/infantum, a causative agent of visceral leishmaniasis.Methods and FindingsSurprisingly, both LXRα knock-out and LXRα/LXRβ double-knock-out (DKO) mice were markedly resistant to systemic L. chagasi/infantum infection compared to wild-type mice. Parasite loads in the livers and spleens of these animals were significantly lower than in wild-type mice 28 days after challenge. Bone marrow-derived macrophages from LXR-DKO mice infected with L. chagasi/infantum in vitro in the presence of IFN-γ were able to kill parasites more efficiently than wild-type macrophages. This enhanced killing by LXR-deficient macrophages correlated with higher levels of nitric oxide produced, as well as increased gene expression of IL-1β. Additionally, LXR ligands abrogated nitric oxide production in wild-type macrophages in response to infection.ConclusionsThese observations suggest that LXR-deficient mice and macrophages mount antimicrobial responses to Leishmania infection that are distinct from those mounted by wild-type mice and macrophages. Furthermore, comparison of these findings to other intracellular infection models suggests that LXR signaling pathways modulate host antimicrobial responses in a complex and pathogen-specific manner. The LXR pathway thus represents a potential therapeutic target for modulating immunity against Leishmania or other intracellular parasites.

Part II. Initial molecular and cellular characterization of high nitric oxide-adapted human tongue squamous cell carcinoma cell lines

It is not understood why some head and neck squamous cell carcinomas, despite having identical morphology, demonstrate different tumor aggressiveness, including radioresistance. High levels of the free radical nitric oxide (NO) and increased expression of the NO-producing enzyme nitric oxide synthase (NOS) have been implicated in tumor progression. We previously adapted three human tongue cancer cell lines to high NO (HNO) levels by gradually exposing them to increasing concentrations of an NO donor; the HNO cells grew faster than their corresponding untreated ("parent") cells, despite being morphologically identical. Herein we initially characterize the HNO cells and compare the biological properties of the HNO and parent cells. HNO/parent cell line pairs were analyzed for cell cycle distribution, DNA damage, X-ray and ultraviolet radiation response, and expression of key cellular enzymes, including NOS, p53, glutathione S-transferase-pi (GST-pi), apurinic/apyrimidinic endonuclease-1 (APE1), and checkpoint kinases (Chk1, Chk2). While some of these properties were cell line-specific, the HNO cells typically exhibited properties associated with a more aggressive behavior profile than the parent cells (greater S-phase percentage, radioresistance, and elevated expression of GST-pi/APE1/Chk1/Chk2). To correlate these findings with conditions in primary tumors, we examined the NOS, GST-pi, and APE1 expression in human tongue squamous cell carcinomas. A majority of the clinical samples exhibited elevated expression levels of these enzymes. Together, the results herein suggest cancer cells exposed to HNO levels can develop resistance to free radicals by upregulating protective mechanisms, such as GST-pi and APE1. These upregulated defense mechanisms may contribute to their aggressive expression profile.

Bone marrow-derived mesenchymal stem cells modulate BV2 microglia responses to lipopolysaccharide

The immunoregulatory properties of mesenchymal stem cells (MSC) have been demonstrated on a wide range of cells. Here, we describe the modulatory effects of mouse bone marrow-derived MSC on BV2 microglia proliferation rate, nitric oxide (NO) production and CD40 expression. Mouse bone marrow MSC were co-cultured with BV2 cells at various seeding density ratios and activated with lipopolysaccharide (LPS). We show that MSC exert an anti-proliferative effect on microglia and are potent producers of NO when stimulated by soluble factors released by LPS-activated BV2. MSC suppressed proliferation of both untreated and LPS-treated microglia in a dose-dependent manner, significantly reducing BV2 proliferation at seeding density ratios of 1:0.2 and 1:0.1 (p < .05). Co-culturing MSC with BV2 cells at different ratios revealed interesting dynamics in NO production. A high number of MSC significantly increases NO in co-cultures whilst a lower number reduces NO. The increased NO levels in co-cultures may be MSC-derived, as we also show that activated BV2 cells stimulate MSC to produce NO. Cell–cell interaction is not a requirement for this effect as soluble factors released by activated BV2 cells alone do stimulate MSC to produce high levels of NO. Although NO is implicated as a mediator for T cell proliferation, it does not appear to play a major role in the suppression of microglia proliferation. Additionally, MSC reduced the expression of the microglial co-stimulator molecule, CD40. Collectively, these regulatory effects of MSC on microglia offer insight into the potential moderating properties of MSC on inflammatory responses within the CNS.

Involvement of Interferon-γ in Bowel Disease Pathogenesis by Nitric Oxide Pathway: A Study in Algerian Patients

The pathogenesis of inflammatory bowel disease (IBD) is complex, involving a wide range of molecules including cytokines. Abnormalities in the expression of immunoregulatory cytokines such as interferon-γ (IFN-γ) and interleukin-12 (IL-12) may indicate a dysregulation of intestinal immunity probably associated with pathogenic events. The aim of this work was to study the implication of IFN-γ and nitric oxide (NO) in bowel disease pathogenesis. In this study, we investigated the circulating IFN-γ and IL-12 production in 2 groups of Algerian patients with IBD (Crohn's disease and ulcerative colitis). Moreover, systemic NO concentrations and NO generation by colonic mucosa were determined in these patients. Finally, we examined the effect of IFN-γ on NO production by peripheral blood mononuclear cells (PBMCs) of these patients. Our results indicate that IFN-γ/IL-12 production in IBD patients was increased in comparison to healthy donors. This strong production correlates with high levels of NO in sera and colonic mucosa culture. Interestingly, NO production was related to the clinical stage of IBD patients (inactive or active stage). The relationship between IFN-γ and NO production in IBD patients were confirmed by in vitro experiments and the role of IFN-γ in NO synthase induction in patients' PBMC culture was suggested. Collectively, our results show that IFN-γ plays a pivotal role in IBD pathogenesis through NO pathway.

VEGF and nitric oxide synthase immunoexpression in Down’s syndrome amniotic fluid stem cells

It has been previously observed that the amniotic fluid obtained from Down's syndrome (DS) pregnancies showed lower levels of vascular endothelial growth factor (VEGF) and higher levels of nitric oxide (NO) with respect to the controls, suggesting the presence of an imbalance between placental vascularization and altered endothelial function. The aim of our study was to evaluate the immunohistochemical expression and localization of VEGF and nitric oxide synthase (NOS) isoforms in cultured amniotic fluid mesenchymal stem cells (AF-MSCs) isolated from normal euploid pregnancies and pregnancies complicated by trisomy 21. In addition, we measured the VEGF and NO content in cell culture supernatants to analyse their production by AF-MSCs. AF-MSCs were obtained from women with foetal DS and controls matched for age and gestation, and expanded in culture. The cells were then evaluated for the immunohistochemical expression of VEGF and NOS isoforms, as well as for the release of VEGF and NO. Our analyses showed that both the VEGF expression and production were significantly lower in DS-AF-MSCs with respect to the controls. As regards NOS, immunohistochemical expression of eNOS was significantly reduced in DS-AF-MSCs, whereas the nNOS and iNOS were similarly immunoexpressed in both groups of cells. Moreover, we observed that the NO content was significantly higher in medium derived by DS-AF-MSCs. Our study shows, for the first time, the differences between AF-MSCs isolated from control and trisomy 21 pregnancies and suggest an involvement of NO and VEGF in the physiopathological mechanisms associated with DS pregnancy.

The Effect of Statin Therapy on Stimulation of Endothelium-Derived Nitric Oxide before and after Coronary Artery Bypass Surgery

The purpose of this study was to determine the effects of statins on endothelium-derived nitric oxide (NO) levels during coronary artery bypass grafting (CABG) surgery. In a prospective study, 130 patients with coronary artery disease were randomized according to preoperative atorvastatin treatment. The patients in group 1 took 40 mg atorvastatin daily for at least 1 month preoperatively, and those in group 2 took no atorvastatin preoperatively. Plasma nitrite and nitrate were measured at baseline and after inducing reactive hyperemia, both before and after surgery. Reactive hyperemia was induced by placing a blood pressure cuff on the upper forearm, inflating it for 5 minutes at 250 mm Hg, and then rapidly deflating the cuff. Blood was collected from the radial artery on the same side 2 minutes after cuff deflation. Plasma levels of total cholesterol, triglycerides, and high- and low-density lipoproteins were measured and analyzed for correlations with NO. The mean (+/-SD) baseline plasma NO levels before operation were as follows: group 1, 33.97 +/- 18.27 nmol/L; group 2, 24.24 +/- 8.53 nmol/L (P < .001). A significant difference between the 2 groups in plasma NO levels was observed after preoperative reactive hyperemia induction: group 1, 56.43 +/- 15.03 nmol/L; group 2, 43.12 +/- 10.67 nmol/L (P < .001). Two hours after cardiopulmonary bypass (CPB), we observed no significant differences in plasma NO levels, either at baseline (group 1, 11 +/- 3.41 nmol/L; group 2, 9 +/- 5.51 nmol/L) or after reactive hyperemia (group 1, 17.98 +/- 6.77 nmol/L; group 2, 18.00 +/- 6.47 nmol/L). A correlation with preoperative nitroglycerine use was observed (P = .007; r = 0.23). Linear regression analysis (F = 1.463; R = 0.314; R2 = 0.099; P = .16) indicated that the only significant correlation was with preoperative nitroglycerine use (P = .007; t = 2.746). Preoperative atorvastatin treatment in patients with coronary artery disease increases plasma NO levels before and after reactive hyperemia prior to surgery. CABG surgery with CPB significantly impairs endothelial-derived NO levels, with or without preoperative atorvastatin treatment. Preoperative nitroglycerine use is correlated with higher NO levels after CABG.

The role of nitric oxide in the treatment of tumours with aminolaevulinic acid-induced photodynamic therapy

Photodynamic therapy (PDT) is a local cancer treatment which induces cell death by the interaction of light with a photosensitizing drug. Previous studies indicate that nitric oxide (NO) plays a role in Photofrin-PDT, but this has not been investigated in aminolaevulinic acid (ALA)-PDT. The current study determines whether inhibition of nitric oxide synthase (NOS) activity modulates treatment responses to ALA-PDT, in tumours displaying differential levels of NO. Murine tumours with low (EMT6) or high (RIF-1) NO levels were implanted into the cremaster muscle of BALB/c or C3H/HeN mice respectively. Animals were prepared for in vivo microscopy 7–14 days later. Mice received oral ALA (200 mg/kg) 4 h before PDT. l-NAME, l-NNA or 1400 W (10 mg/kg) were administered via the jugular vein 5 min before PDT. NOS inhibition ( l-NAME or l-NNA) combined with ALA-PDT in RIF-1 tumours demonstrated enhanced damage to both the tumour and normal microvasculature, with increased macromolecular leak and reduction in vessel diameter, whereas ALA-PDT alone had no effect. In contrast, EMT6 tumours responded to ALA-PDT alone but sensitivity was not enhanced in the presence of NOS inhibition. 1400 W combined with ALA-PDT induced similar microvascular effects to l-NAME in both tumours, but were less pronounced. The data demonstrates that NO has an important role in events likely to be critical for treatment response, sensitivity and therapeutic outcome of ALA-PDT.

Extracellular products from virulent strain of Edwardsiella tarda stimulate mouse macrophages (RAW264.7) to produce nitric oxide (NO) and tumor necrosis factor (TNF)-α

We have previously found that high virulent strain (NUF251) of Edwardsiella tarda, but not low virulent strain (NUF194), was able to survive and multiply within Japanese flounder ( Paralichthys olivaceus) peritoneal macrophages. Further studies demonstrated that NUF251 induced much higher levels of NO and TNF-α productions than NUF194 in both Japanese flounder peritoneal macrophages and mouse macrophage cell line RAW264.7. In this study, we examined the effects of extracellular products (ECP) from two strains of E. tarda on RAW264.7 cells in terms of the induction of NO and TNF-α. ECP from NUF251 stimulated RAW264.7 cells to induce NO production in a concentration-dependent manner. The activity of NUF251-ECP completely disappeared by heat-treatment (at 100 °C for 5 min), but it could not be removed by dialysis. Polymyxin B, an endotoxin inhibitor, had no effect on NUF251-ECP-induced NO production. These results suggest that active agents in NUF251-ECP responsible for NO induction may be heat-labile high molecular weight substances rather than the cell wall derived endotoxin like substances. Since NO synthase (NOS) inhibitor, l-NAME, suppressed NUF251-ECP-induced NO production, inducible NO synthase (iNOS) in RAW264.7 cells may be a main source of NO. Furthermore, NUF251-ECP-induced high level of TNF-α secretion from RAW264.7 cells. Both NO and TNF-α productions induced by NUF251-ECP were significantly blocked by a JNK inhibitor. In contrast to NUF251-ECP, no significant activities of NUF194-ECP to induce NO and TNF-α productions were detected. SDS-PAGE and subsequent proteomic analysis of ECP from both strains suggested that NUF251-specific protein, which has sequence homology with flagellin, is present in NUF251-ECP as a main component. Our results suggest that the high virulent strain (NUF251) of E. tarda may specifically produce an exotoxin capable of inducing high levels of NO and TNF-α from macrophages through the activation of JNK system, and most probable candidate for such exotoxin might be a flagellin-like protein.

Increase in CGRP- and nNOS-immunoreactive neurons in the rat trigeminal ganglion after infusion of an NO donor

Nitrovasodilators, such as glyceroltrinitrate (GTN), which produce nitric oxide (NO) in the organism, are known to cause delayed headaches in migraineurs, accompanied by increased plasma levels of calcitonin gene-related peptide (CGRP) in the cranial venous outflow. Increases in plasma CGRP and NO metabolites have also been found in spontaneous migraine attacks. In a rat model of meningeal nociception, infusion of NO donors induced activity of neurons in the spinal trigeminal nucleus. Isoflurane-anaesthetised rats were intravenously infused with GTN (250 µg/kg) or saline for two hours and fixed by perfusion after a further four hours. Cryosections of dissected trigeminal ganglia were immunostained for detection of CGRP and neuronal NO synthase (nNOS). The ganglion neurons showing immunofluorescence for either of these proteins were counted. The proportions of CGRP- and nNOS- as well as double-immunopositive neurons were increased after GTN infusion compared to saline treatment in all parts of the trigeminal ganglion (CGRP) or restricted to the ophthalmic region (nNOS). The size of immunopositive neurons was not significantly different compared to controls. High levels of NO may induce the expression or availability of CGRP and nNOS. Similar changes may be involved in nitrovasodilator-induced and spontaneous headache attacks in migraineurs.

Study of the nitric oxide system in the rat cerebellum during aging

BackgroundThe cerebellum is the neural structure with the highest levels of nitric oxide, a neurotransmitter that has been proposed to play a key role in the brain aging, although knowledge concerning its contribution to cerebellar senescence is still unclear, due mainly to absence of integrative studies that jointly evaluate the main factors involved in its cell production and function. Consequently, in the present study, we investigate the expression, location, and activity of nitric oxide synthase isoenzymes; the protein nitration; and the production of nitric oxide in the cerebellum of adult and old rats.ResultsOur results show no variation in the expression of nitric oxide synthase isoforms with aging, although, we have detected some changes in the cellular distribution pattern of the inducible isoform particularly in the cerebellar nuclei. There is also an increase in nitric oxide synthase activity, as well as greater protein-nitration levels, and maintenance of nitrogen oxides (NOx) levels in the senescent cerebellum.ConclusionsThe nitric oxide/nitric oxide syntahses system suffers from a number of changes, mainly in the inducible nitric oxide synthase distribution and in overall nitric oxide synthases activity in the senescent cerebellum, which result in an increase of the protein nitration. These changes might be related to the oxidative damage detected with aging in the cerebellum.

Nebulization of the acidified sodium nitrite formulation attenuates acute hypoxic pulmonary vasoconstriction

BackgroundGeneralized hypoxic pulmonary vasoconstriction (HPV) occurring during exposure to hypoxia is a detrimental process resulting in an increase in lung vascular resistance. Nebulization of sodium nitrite has been shown to inhibit HPV. The aim of this project was to investigate and compare the effects of nebulization of nitrite and different formulations of acidified sodium nitrite on acute HPV.MethodsEx vivo isolated rabbit lungs perfused with erythrocytes in Krebs-Henseleit buffer (adjusted to 10% hematocrit) and in vivo anesthetized catheterized rabbits were challenged with periods of hypoxic ventilation alternating with periods of normoxic ventilation. After baseline hypoxic challenges, vehicle, sodium nitrite or acidified sodium nitrite was delivered via nebulization. In the ex vivo model, pulmonary arterial pressure and nitric oxide concentrations in exhaled gas were monitored. Nitrite and nitrite/nitrate were measured in samples of perfusion buffer. Pulmonary arterial pressure, systemic arterial pressure, cardiac output and blood gases were monitored in the in vivo model.ResultsIn the ex vivo model, nitrite nebulization attenuated HPV and increased nitric oxide concentrations in exhaled gas and nitrite concentrations in the perfusate. The acidified forms of sodium nitrite induced higher levels of nitric oxide in exhaled gas and had longer vasodilating effects compared to nitrite alone. All nitrite formulations increased concentrations of circulating nitrite to the same degree. In the in vivo model, inhaled nitrite inhibited HPV, while pulmonary arterial pressure, cardiac output and blood gases were not affected. All nitrite formulations had similar potency to inhibit HPV. The tested concentration of appeared tolerable.ConclusionNitrite alone and in acidified forms effectively and similarly attenuates HPV. However, acidified nitrite formulations induce a more pronounced increase in nitric oxide exhalation.

Hydrogen peroxide and nitric oxide mediate K+/Na+ homeostasis and antioxidant defense in NaCl-stressed callus cells of two contrasting poplars

Using callus cells of a salt-tolerant Populus euphratica Oliver and a salt-sensitive P. popularis 35–44 (P. popularis), the effects of NaCl stress on hydrogen peroxide (H2O2) and nitric oxide (NO) production and the relevance to ionic homeostasis and antioxidant defense were investigated. Results show that P. euphratica exhibited a greater capacity to tolerate NaCl stress in terms of cell viability, membrane permeability and K+/Na+ relations. NaCl salinity (150 mM) caused a rapid increase of H2O2 and NO in P. euphratica cells, but not in P. popularis. Moreover, salinised P. euphratica cells retained a high and stable level of H2O2 and NO during the period of 24-h salt stress. Noteworthy, P. eupratica cells increased activities of superoxide dismutase, ascorbate peroxidase, catalase and glutathione reductase under salinity stress, but these antioxidant enzymes were significantly inhibited by the salt treatment in P. popularis cells. Pharmacological experiments proved that the NaCl-induced H2O2 and NO was interdependent and contributed to the mediation of K+/Na+ homeostasis and antioxidant defense in P. euphratica cells. Given these results, we conclude that the increased H2O2 and NO enable P. euphratica cells to regulate ionic and ROS (reactive oxygen species) homeostasis under salinity stress in the longer term.

Plasma angiogenic markers in patients with metastatic breast cancer treated with weekly docetaxel.

e21004 Background: The aim of the study was to evaluate angiogenic plasma markers in patients with metastatic breast cancer (MBC) treated with first-line weekly docetaxel (D), considered to be standard treatment at the time of the design of the study. Methods: Females with MBC were treated with weekly D 35 mg/m2 ± trastuzumab. Plasma levels of HER2 extracellular domain (HER2-ECD), nitric oxide (NO), interleukin-8, and VEGF-A were measured by ELISA before and after the treatment, as well as in 38 healthy female controls. Prognostic and predictive significance of the pre-treatment values and their relative change with treatment were tested by Cox-regression analysis. Results: Between February 2006 and January 2008, 159 patients were treated. 61 patients (38%) responded (CR = 5%, PR = 33%) and 52 (33%) had stable disease. 37 patients received trastuzumab. Median follow-up was 33 months (2.8-45.0), median PFS 8.8 months (95%CI: 7.2-10.3) and median survival 27.7 months (95%CI: 24.7-30.6). HER2-ECD was the only plasma marker that decreased significantly with treatment (p < 0.001), from 2.08 ng/mL (range 0.2- 13.12) to 1.4 ng/mL (range 0.1-12.72). Normal controls had significantly higher NO levels than that of MBC patients before treatment (282.5 vs 199 umol/L, respectively; p = 0.006), whereas HER2-ECD levels were marginally lower (1.76 vs 2.08 ng/mL, respectively; p = 0.054). High pretreatment HER2-ECD levels were of adverse prognostic significance for survival and PFS (p = 0.033 and p = 0.046, respectively) in patients not treated with trastuzumab. High pretreatment NO levels were also of adverse prognostic value (p < 0.001 for survival, p = 0.004 for PFS), as were high pre-treatment IL8 values (p = 0.048 for survival). Plasma VEGF-A did not appear to have prognostic value. High NO relative change was associated with better survival (p = 0.003), in contrast patients with high IL8 relative change were found to have a worse PFS (p = 0.017). Responders had a smaller relative change of IL8 (p = 0.038) than non- responders. Conclusions: HER2-ECD, NO and IL8 plasma levels appear to be of prognostic value for survival in patients with MBC treated with weekly docetaxel. Tumor angiogenic markers will also be examined by PCR in these patients. No significant financial relationships to disclose.

Abstract 4506: Elevated expression of IL-1 induces iNOS/NO production and inhibits IL-1Ra synthesis leading to progression of metastatic melanoma

Abstract Aberrant expression of proinflammatory IL-1 α and β and the overproduction of nitric oxide (NO) have been shown to promote the progression of some tumors. In previous studies, we had confirmed that inducible nitric oxide synthase (iNOS) was constitutively expressed in most advanced melanomas and highly correlated with poor patient survival. In order to determine whether the imbalance of IL-1 and its natural competitor IL-1 receptor antagonist (IL-1Ra) contributes to the upregulation of iNOS in metastatic melanoma, we initiated an investigation of the expression profile of IL-1 system genes in melanoma and the linkage between IL-1 and iNOS/NO signaling pathways. Immunohistochemistry staining of 38 biopsy samples from metastatic melanoma patients showed that 33 samples highly expressed IL-1α and/or IL-1β. Only 5 samples were negative for both IL-1α and IL-1β expression. RT-PCR confirmed that melanoma cell lines A375, WM793, WM35, and SB2 constitutively expressed high levels of IL-1, IL-1 receptor I (IL-1RI), and iNOS. However, in the same cell lines, there was no detectable IL-1Ra mRNA, which is known to maintain IL-1 balance in normal skin tissues. Our study suggests that the imbalance of IL-1 and IL-Ra is widespread in metastatic melanoma. The engagement of IL-1 and IL-1RI can induce several kinase cascades leading to the activation of SAPK/JNK and NF-κB, and it is well known that transcription of iNOS can be regulated by NF-κB. In our studies, siRNA knockdown of either IL-1α or MyD88 (an important adaptor for IL-1RI signal pathway) in WM375 and A375 led to a dramatic decrease in the phoshorylation of SAPK/JNK and IκB. Furthermore, an 80% decrease of iNOS expression in WM793 was observed after the siRNA knockdown of MyD88. We confirmed that upregulated IL-1 signal pathway can activate NF-κB and the iNOS transcription. Treatment with 200 μM 7-Nitroindazole, a NO synthase inhibitor, not only reduced NO production, but also induced over 50% inhibition of cell growth and a marked increase in IL-1Ra mRNA in WM793 and A375. This indicates that a high level of NO can inhibit the IL-1Ra expression in melanoma cells. Based on our data, we proposed an inflammatory dysfunction model to explain the development of melanoma. It proposes that the constitutive expression of IL-1 can inhibit IL-1Ra expression mediated by the upregulation of iNOS/NO production in melanoma. The constant inhibition of IL-1Ra expression by NO results in a cycle of kinase transduction cascades, which drives NF-κB and its downstream inflammatory genes (including iNOS) constitutively expressed in melanoma. Our studies cast new insight into the mechanism of the pathology of metastatic melanoma. Selective inhibition of iNOS and increasing IL-1Ra levels are promising therapeutic strategies to suppress the aberrant IL-1 inflammation pathway in melanoma. Supported by MDACC SPORE in Melanoma CA093459. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4506.