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Sample records for induced inducible nitric

  1. Inducible nitric oxide synthase in the myocard.

    PubMed

    Buchwalow, I B; Schulze, W; Karczewski, P; Kostic, M M; Wallukat, G; Morwinski, R; Krause, E G; Müller, J; Paul, M; Slezak, J; Luft, F C; Haller, H

    2001-01-01

    Recognition of significance of nitric oxide synthases (NOS) in cardiovascular regulations has led to intensive research and development of therapies focused on NOS as potential therapeutic targets. However, the NOS isoform profile of cardiac tissue and subcellular localization of NOS isoforms remain a matter of debate. The aim of this study was to investigate the localization of an inducible NOS isoform (NOS2) in cardiomyocytes. Employing a novel immunocytochemical technique of a catalyzed reporter deposition system with tyramide and electron microscopical immunocytochemistry complemented with Western blotting and RT-PCR, we detected NOS2 both in rat neonatal and adult cultured cardiomyocytes and in the normal myocard of adult rats as well as in the human myocard of patients with dilative cardiomyopathy. NOS2 was targeted predominantly to a particulate component of the cardiomyocyte--along contractile fibers, in the plasma membrane including T-tubules, as well as in the nuclear envelope, mitochondria and Golgi complex. Our results point to an involvement of NOS2 in maintaining cardiac homeostasis and contradict to the notion that NOS2 is expressed in cardiac tissue only in response to various physiological and pathogenic factors. NOS2 targeting to mitochondria and contractile fibers suggests a relationship of NO with contractile function and energy production in the cardiac muscle.

  2. Nitric oxide inhibition sustains vasopressin-induced vasoconstriction.

    PubMed Central

    Dworkin, M. J.; Carnochan, P.; Allen-Mersh, T. G.

    1995-01-01

    Hepatic parenchymal vasoconstriction increases cytotoxic drug uptake into hepatic metastases by increasing the tumour to liver blood flow ratio. Prolonged infusion of the vasoconstrictor vasopressin does not result in sustained vasoconstriction, and this may limit the benefit of vasopressin in infusional chemotherapy. We have assessed whether loss of vasopressin-induced vasoconstriction is mediated by nitric oxide. Hepatic and tumour blood flow were continuously monitored, in an animal hepatic tumour model, by laser Doppler flowmetry. The response to regionally infused vasopressin and the nitric oxide inhibitor N-nitro-L-arginine methyl ester (L-NAME) were assessed over a 30 min infusion period. The vasopressin-induced vasoconstrictor effect diminished after 15 min despite continued infusion. Vasoconstriction was significantly prolonged when L-NAME was infused in addition to vasopressin. The increase in tumour to normal blood flow ratio was greater over the infusion period when L-NAME was co-administered with vasopressin. Our results suggest that the loss of vasopressin-induced vasoconstriction seen in liver parenchyma after regional infusion is prevented by the nitric oxide synthase inhibitor L-name and may be mediated by nitric oxide. PMID:7734317

  3. UV Induced Oxidation of Nitric Oxide

    NASA Technical Reports Server (NTRS)

    Parrish, Clyde, F. (Inventor); Luecke, Dale E. (Inventor)

    2007-01-01

    Nitric oxide in a gaseous stream is converted to nitrogen dioxide using oxidizing species generated at least in part using in situ UV radiation sources. The sources of the oxidizing species include oxygen and/or hydrogen peroxide. The oxygen may be a component of the gaseous stream or added to the gaseous stream, preferably near a UV radiation source, and is converted to ozone by the UV irradiation. The hydrogen peroxide is decomposed through a combination of vaporization and UV irradiation. The hydrogen peroxide is preferably stored at stable concentration levels, i.e., approximately 50% by volume and increased in concentration in a continuous process preceding vaporization within the flow channel of the gaseous stream and in the presence of the UV radiation sources.

  4. Nitric oxide-donor SNAP induces Xenopus eggs activation.

    PubMed

    Jeseta, Michal; Marin, Matthieu; Tichovska, Hana; Melicharova, Petra; Cailliau-Maggio, Katia; Martoriati, Alain; Lescuyer-Rousseau, Arlette; Beaujois, Rémy; Petr, Jaroslav; Sedmikova, Marketa; Bodart, Jean-François

    2012-01-01

    Nitric oxide (NO) is identified as a signaling molecule involved in many cellular or physiological functions including meiotic maturation and parthenogenetic activation of mammalian oocytes. We observed that nitric oxide donor SNAP was potent to induce parthenogenetic activation in Xenopus eggs. NO-scavenger CPTIO impaired the effects of SNAP, providing evidence for the effects of the latter to be specific upon NO release. In Xenopus eggs, SNAP treatment induced pigment rearrangement, pronucleus formation and exocytosis of cortical granules. At a biochemical level, SNAP exposure lead to MAPK and Rsk inactivation within 30 minutes whereas MPF remained active, in contrast to calcium ionophore control where MPF activity dropped rapidly. MAPK inactivation could be correlated to pronuclear envelope reformation observed. In SNAP-treated eggs, a strong increase in intracellular calcium level was observed. NO effects were impaired in calcium-free or calcium limited medium, suggesting that that parthenogenetic activation of Xenopus oocytes with a NO donor was mainly calcium-dependent.

  5. Nitric oxide-induced calcium release: activation of type 1 ryanodine receptor by endogenous nitric oxide.

    PubMed

    Kakizawa, Sho; Yamazawa, Toshiko; Iino, Masamitsu

    2013-01-01

    Ryanodine receptors (RyRs), located in the sarcoplasmic/endoplasmic reticulum (SR/ER) membrane, are required for intracellular Ca2+ release that is involved in a wide range of cellular functions. In addition to Ca2+-induced Ca2+ release in cardiac cells and voltage-induced Ca2+ release in skeletal muscle cells, we recently identified another mode of intracellular Ca2+ mobilization mediated by RyR, i.e., nitric oxide-induced Ca2+ release (NICR), in cerebellar Purkinje cells. NICR is evoked by neuronal activity, is dependent on S-nitrosylation of type 1 RyR (RyR1) and is involved in the induction of long-term potentiation (LTP) of cerebellar synapses. In this addendum, we examined whether peroxynitrite, which is produced by the reaction of nitric oxide with superoxide, may also have an effect on the Ca2+ release via RyR1 and the cerebellar LTP. We found that scavengers of peroxynitrite have no significant effect either on the Ca2+ release via RyR1 or on the cerebellar LTP. We also found that an application of a high concentration of peroxynitrite does not reproduce neuronal activity-dependent Ca2+ release in Purkinje cells. These results support that NICR is induced by endogenous nitric oxide produced by neuronal activity through S-nitrosylation of RyR1.

  6. Nitric oxide synthases and cyclophosphamide-induced cystitis in rats.

    PubMed

    Alfieri, A B; Malave, A; Cubeddu, L X

    2001-03-01

    The role of inducible (iNOS) and neuronal nitric oxide (nNOS) synthases and of tachykinin NK1 receptors on the pathogenesis of cyclophosphamide (CYP)-induced cystitis was investigated, in rats. CYP-induced cystitis was characterized by large increases in bladder-protein plasma extravasation (PPE), increases in the urinary excretion of nitric oxide (NO) metabolites and histological evidences of urothelial damage, edema, extensive white blood cell infiltrates and vascular congestion of the bladder. The specific iNOS inhibitor, S-methylthiourea (MITU), produced marked inhibition (>90%) of CYP-induced increases in PPE associated with amelioration of tissue inflammatory changes. Treatment with 7-nitroindazole (7-NI; 20, 40 and 80 mg/kg), a selective nNOS inhibitor, did not significantly reduce CYP-induced increases in PPE and failed to produce histological improvement. In addition, treatment with MITU, but not with 7-NI, inhibited the increases in the urinary excretion of NO metabolites induced by CYP treatment. WIN 51,708 (17-beta-hydroxy-17-alpha-ethynyl-androstano[3,2-b]pyrimido[1,2-a]benzimidazole; WIN), a selective NK1-receptor antagonist, reduced the increases in EPP and ameliorated the inflammatory changes in the bladder induced by CYP. However, the maximal degree of protection achieved with WIN was significantly less than that produced by MITU. Combined treatment with the iNOS inhibitor and the NK1 antagonist produced no greater effect than that produced by the iNOS inhibitor alone. Our results suggest that NO plays a fundamental role in the production of the cystitis associated with CYP treatment. The iNOS, and not nNOS, seems responsible for the inflammatory changes. Part of the increases in NO may due to activation of NK1 receptors by neuropeptides such as substance P possibly released from primary afferent fibers.

  7. Expression of inducible nitric oxide in human lung epithelial cells.

    PubMed

    Robbins, R A; Barnes, P J; Springall, D R; Warren, J B; Kwon, O J; Buttery, L D; Wilson, A J; Geller, D A; Polak, J M

    1994-08-30

    Nitric oxide (NO) is increased in the exhaled air of subjects with several airway disorders. To determine if cytokines could stimulate epithelial cells accounting for the increased NO, the capacity of the proinflammatory cytokines (cytomix: tumor necrosis factor-alpha, interleukin-1 beta, and interferon-gamma) to increase inducible nitric oxide synthase (iNOS) was investigated in A549 and primary cultures of human bronchial epithelial cells. Cytomix induced a time-dependent increase in nitrite levels in culture supernatant fluids (p < 0.05). Increased numbers of cells stained for iNOS and increased iNOS mRNA was detected in the cytokine-stimulated cells compared to control (p < 0.05). Dexamethasone diminished the cytokine-induced increase in nitrite, iNOS by immunocytochemistry, and iNOS mRNA. These data demonstrate that cytokines, such as those released by mononuclear cells, can induce lung epithelial iNOS expression and NO release, and that this is attenuated by dexamethasone.

  8. A Comparison of the Effects of Neuronal Nitric Oxide Synthase and Inducible Nitric Oxide Synthase Inhibition on Cartilage Damage.

    PubMed

    Gokay, Nevzat Selim; Yilmaz, Ibrahim; Komur, Baran; Demiroz, Ahu Senem; Gokce, Alper; Dervisoglu, Sergülen; Gokay, Banu Vural

    2016-01-01

    The objective of this study was to investigate the effects of selective inducible nitric oxide synthase and neuronal nitric oxide synthase inhibitors on cartilage regeneration. The study involved 27 Wistar rats that were divided into five groups. On Day 1, both knees of 3 rats were resected and placed in a formalin solution as a control group. The remaining 24 rats were separated into 4 groups, and their right knees were surgically damaged. Depending on the groups, the rats were injected with intra-articular normal saline solution, neuronal nitric oxide synthase inhibitor 7-nitroindazole (50 mg/kg), inducible nitric oxide synthase inhibitor amino-guanidine (30 mg/kg), or nitric oxide precursor L-arginine (200 mg/kg). After 21 days, the right and left knees of the rats were resected and placed in formalin solution. The samples were histopathologically examined by a blinded evaluator and scored on 8 parameters. Although selective neuronal nitric oxide synthase inhibition exhibited significant (P = 0.044) positive effects on cartilage regeneration following cartilage damage, it was determined that inducible nitric oxide synthase inhibition had no statistically significant effect on cartilage regeneration. It was observed that the nitric oxide synthase activation triggered advanced arthrosis symptoms, such as osteophyte formation. The fact that selective neuronal nitric oxide synthase inhibitors were observed to have mitigating effects on the severity of the damage may, in the future, influence the development of new agents to be used in the treatment of cartilage disorders.

  9. A Comparison of the Effects of Neuronal Nitric Oxide Synthase and Inducible Nitric Oxide Synthase Inhibition on Cartilage Damage

    PubMed Central

    Gokay, Nevzat Selim; Yilmaz, Ibrahim; Demiroz, Ahu Senem; Gokce, Alper; Dervisoglu, Sergülen; Gokay, Banu Vural

    2016-01-01

    The objective of this study was to investigate the effects of selective inducible nitric oxide synthase and neuronal nitric oxide synthase inhibitors on cartilage regeneration. The study involved 27 Wistar rats that were divided into five groups. On Day 1, both knees of 3 rats were resected and placed in a formalin solution as a control group. The remaining 24 rats were separated into 4 groups, and their right knees were surgically damaged. Depending on the groups, the rats were injected with intra-articular normal saline solution, neuronal nitric oxide synthase inhibitor 7-nitroindazole (50 mg/kg), inducible nitric oxide synthase inhibitor amino-guanidine (30 mg/kg), or nitric oxide precursor L-arginine (200 mg/kg). After 21 days, the right and left knees of the rats were resected and placed in formalin solution. The samples were histopathologically examined by a blinded evaluator and scored on 8 parameters. Although selective neuronal nitric oxide synthase inhibition exhibited significant (P = 0.044) positive effects on cartilage regeneration following cartilage damage, it was determined that inducible nitric oxide synthase inhibition had no statistically significant effect on cartilage regeneration. It was observed that the nitric oxide synthase activation triggered advanced arthrosis symptoms, such as osteophyte formation. The fact that selective neuronal nitric oxide synthase inhibitors were observed to have mitigating effects on the severity of the damage may, in the future, influence the development of new agents to be used in the treatment of cartilage disorders. PMID:27382570

  10. The role of nitric oxide in experimental cerulein induced pancreatitis.

    PubMed

    Um, Soon Ho; Kwon, Yong Dae; Kim, Chang Duck; Lee, Hong Sik; Jeen, Yoon Tae; Chun, Hoon Jai; Lee, Sang Woo; Choi, Jae Hyun; Ryu, Ho Sang; Hyun, Jin Hai

    2003-08-01

    An enhanced formation of nitric oxide (NO), due to the induction of inducible nitric oxide synthase (iNOS), has been implicated in the pathogenesis of shock and inflammation, but its role in acute pancreatitis still remains controversial. To clarify the role of NO in acute pancreatitis, the present experiment investigated the expression of iNOS and the effect of NOS inhibition on cerulein-induced pancreatitis in rats. Group I received intraperitoneal (ip) injection of normal saline. Group II received two ip injections of cerulein (20 microgram/kg). Group III received injections of N(G)-nitro-L-arginine methyl ester (L-NAME) (30 mg/kg) with cerulein. Group IV received L-arginine (250 mg/kg) with cerulein and L-NAME. The expression of iNOS in the pancreas was examined by western blot analysis. The plasma concentration of NO metabolites was measured. The severity of pancreatitis was assessed by measuring serum amylase, pancreas water content and histopathological examination. Compared with controls, the cerulein group displayed significantly increased expression of iNOS and raised plasma NO metabolites. Treatment with L-NAME significantly decreased hyperamylasemia, plasma NO level, and the extent of pancreatic injury. Treatment with L-arginine reversed the effects of L-NAME. These findings suggest that an enhanced formation of NO by iNOS plays an important role in the development of acute pancreatitis, and inhibition of NO production has the beneficial effects in reducing pancreas injury.

  11. In vivo Expression of Inducible Nitric Oxide Synthase in Experimentally Induced Neurologic Diseases

    NASA Astrophysics Data System (ADS)

    Koprowski, Hilary; Zheng, Yong Mu; Heber-Katz, Ellen; Fraser, Nigel; Rorke, Lucy; Fu, Zhen Fang; Hanlon, Cathleen; Dietzschold, Bernhard

    1993-04-01

    The purpose of this study was to investigate the induction of inducible nitric oxide synthase (iNOS) mRNA in the brain tissue of rats and mice under the following experimental conditions: in rats infected with borna disease virus and rabies virus, in mice infected with herpes simplex virus, and in rats after the induction of experimental allergic encephalitis. The results showed that iNOS mRNA, normally nondetectable in the brain, was present in animals after viral infection or after induction of experimental allergic encephalitis. The induction of iNOS mRNA coincided with the severity of clinical signs and in some cases with the presence of inflammatory cells in the brain. The results indicate that nitric oxide produced by cells induced by iNOS may be the toxic factor accounting for cell damage and this may open the door to approaches to the study of the pathogenesis of neurological diseases.

  12. Expression and activity of inducible nitric oxide synthase and endothelial nitric oxide synthase correlate with ethanol-induced liver injury

    PubMed Central

    Yuan, Guang-Jin; Zhou, Xiao-Rong; Gong, Zuo-Jiong; Zhang, Pin; Sun, Xiao-Mei; Zheng, Shi-Hua

    2006-01-01

    AIM: To study the expression and activity of inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) in rats with ethanol-induced liver injury and their relation with liver damage, activation of nuclear factor-κB (NF-κB) and tumor necrosis factor-α (TNF-α) expression in the liver. METHODS: Female Sprague-Dawley rats were given fish oil (0.5 mL) along with ethanol or isocaloric dextrose daily via gastrogavage for 4 or 6 wk. Liver injury was assessed using serum alanine aminotransferase (ALT) activity and pathological analysis. Liver malondialdehyde (MDA), nitric oxide contents, iNOS and eNOS activity were determined. NF-κB p65,iNOS, eNOS and TNF-α protein or mRNA expression in the liver were detected by immunohistochemistry or reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: Chronic ethanol gavage for 4 wk caused steatosis, inflammation and necrosis in the liver, and elevated serum ALT activity. Prolonged ethanol administration (6 wk) enhanced the liver damage. These responses were accompanied with increased lipid peroxidation, NO contents, iNOS activity and reduced eNOS activity. NF-κB p65, iNOS and TNF-α protein or mRNA expression were markedly induced after chronic ethanol gavage, whereas eNOS mRNA expression remained unchanged. The enhanced iNOS activity and expression were positively correlated with the liver damage, especially the necro-inflammation, activation of NF-κB, and TNF-α mRNA expression. CONCLUSION: iNOS expression and activity are induced in the liver after chronic ethanol exposure in rats, which are correlated with the liver damage, especially the necro-inflammation, activation of NF-κB and TNF-α expression. eNOS activity is reduced, but its mRNA expression is not affected. PMID:16688828

  13. Nitric Oxide-Induced Conformational Changes in Soluble Guanylate Cyclase

    PubMed Central

    Underbakke, Eric S.; Iavarone, Anthony T.; Chalmers, Michael J.; Pascal, Bruce D.; Novick, Scott; Griffin, Patrick R.; Marletta, Michael A.

    2014-01-01

    SUMMARY Soluble guanylate cyclase (sGC) is the primary mediator of nitric oxide (NO) signaling. NO binds the sGC heme cofactor stimulating synthesis of the second messenger cyclic-GMP (cGMP). As the central hub of NO/cGMP signaling pathways, sGC is important in diverse physiological processes such as vasodilation and neurotransmission. Nevertheless, the mechanisms underlying NO-induced cyclase activation in sGC remain unclear. Here, hydrogen/deuterium exchange mass spectrometry (HDX-MS) was employed to probe the NO-induced conformational changes of sGC. HDX-MS revealed NO-induced effects in several discrete regions. NO binding to the heme-NO/O2-binding (H-NOX) domain perturbs a signaling surface implicated in Per/Arnt/Sim (PAS) domain interactions. Furthermore, NO elicits striking conformational changes in the junction between the PAS and helical domains that propagate as perturbations throughout the adjoining helices. Ultimately, NO-binding stimulates the catalytic domain by contracting the active site pocket. Together, these conformational changes delineate an allosteric pathway linking NO-binding to activation of the catalytic domain. PMID:24560804

  14. Nitric oxide synthase is induced in sporulation of Physarum polycephalum

    PubMed Central

    Golderer, Georg; Werner, Ernst R.; Leitner, Stefan; Gröbner, Peter; Werner-Felmayer, Gabriele

    2001-01-01

    The myxomycete Physarum polycephalum expresses a calcium-independent nitric oxide (NO) synthase (NOS) resembling the inducible NOS isoenzyme in mammals. We have now cloned and sequenced this, the first nonanimal NOS to be identified, showing that it shares < 39% amino acid identity with known NOSs but contains conserved binding motifs for all NOS cofactors. It lacks the sequence insert responsible for calcium dependence in the calcium-dependent NOS isoenzymes. NOS expression was strongly up-regulated in Physarum macroplasmodia during the 5-day starvation period needed to induce sporulation competence. Induction of both NOS and sporulation competence were inhibited by glucose, a growth signal and known repressor of sporulation, and by l-N6–(1-iminoethyl)-lysine (NIL), an inhibitor of inducible NOS. Sporulation, which is triggered after the starvation period by light exposure, was also prevented by 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of NO-sensitive guanylate cyclase. In addition, also expression of lig1, a sporulation-specific gene, was strongly attenuated by NIL or ODQ. 8-Bromo-cGMP, added 2 h before the light exposure, restored the capacity of NIL-treated macroplasmodia to express lig1 and to sporulate. This indicates that the second messenger used for NO signaling in sporulation of Physarum is cGMP and links this signaling pathway to expression of lig1. PMID:11358872

  15. Nitric Oxide Signaling in Hypergravity-Induced Neuronal Plasticity

    NASA Technical Reports Server (NTRS)

    Holstein, Gay R.

    2003-01-01

    The goal of this research project was to identify the neurons and circuits in the vestibular nuclei and nucleus prepositus hypoglossi that utilize nitric oxide (NO) for intercellular signaling during gravity-induced plasticity. This objective was pursued using histochemical and immunocytochemical approaches to localize NO-producing neurons and characterize the fine morphology of the cells in ground-based studies of normal rats, rats adapted to hypergravity, and rats adapted to hypergravity and then re-adapted to the 1G environment. NO-producing neurons were identified and studied using four methodologies: i) immunocytochemistry employing polyclonal antibodies directed against neuronal nitric oxide synthase (nNOS), to provide an indication of the capacity of a cell for NO production; ii) immunocytochemistry employing a monoclonal antibody directed against L-citrulline, to provide an indirect index of the enzyme's activity; iii) histochemistry based on the NADPH-diaphorase reaction, for fuI1 cytological visualization of neurons; and iv) double immunofluorescence to co-localize nNOS and L-citrulline in individual vestibular nuclei (VN) and neurons.

  16. Oxalomalate affects the inducible nitric oxide synthase expression and activity.

    PubMed

    Irace, Carlo; Esposito, Giuseppe; Maffettone, Carmen; Rossi, Antonietta; Festa, Michela; Iuvone, Teresa; Santamaria, Rita; Sautebin, Lidia; Carnuccio, Rosa; Colonna, Alfredo

    2007-03-13

    Inducible nitric oxide synthase (iNOS) is an homodimeric enzyme which produces large amounts of nitric oxide (NO) in response to inflammatory stimuli. Several factors affect the synthesis and catalytic activity of iNOS. Particularly, dimerization of NOS monomers is promoted by heme, whereas an intracellular depletion of heme and/or L-arginine considerably decreases NOS resistance to proteolysis. In this study, we found that oxalomalate (OMA, oxalomalic acid, alpha-hydroxy-beta-oxalosuccinic acid), an inhibitor of both aconitase and NADP-dependent isocitrate dehydrogenase, inhibited nitrite production and iNOS protein expression in lipopolysaccharide (LPS)-activated J774 macrophages, without affecting iNOS mRNA content. Furthermore, injection of OMA precursors to LPS-stimulated rats also decreased nitrite production and iNOS expression in isolated peritoneal macrophages. Interestingly, alpha-ketoglutarate or succinyl-CoA administration reversed OMA effect on NO production, thus correlating NO biosynthesis with the anabolic capacity of Krebs cycle. When protein synthesis was blocked by cycloheximide in LPS-activated J774 cells treated with OMA, iNOS protein levels, evaluated by Western blot analysis and (35)S-metabolic labelling, were decreased, suggesting that OMA reduces iNOS biosynthesis and induces an increase in the degradation rate of iNOS protein. Moreover, we showed that OMA inhibits the activity of the iNOS from lung of LPS-treated rats by enzymatic assay. Our results, demonstrating that OMA acts regulating synthesis, catalytic activity and degradation of iNOS, suggest that this compound might have a potential role in reducing the NO overproduction occurring in some pathological conditions.

  17. Tyrosol attenuates ischemia-reperfusion-induced kidney injury via inhibition of inducible nitric oxide synthase.

    PubMed

    Wang, Pengqi; Zhu, Qingjun; Wu, Nan; Siow, Yaw L; Aukema, Harold; O, Karmin

    2013-04-17

    Tyrosol is a natural phenolic antioxidant compound. Oxidative stress represents one of the important mechanisms underlying ischemia-reperfusion-induced kidney injury. The aim of this study was to investigate the effect of tyrosol against ischemia-reperfusion-induced acute kidney injury. The left kidney of Sprague-Dawley rats was subjected to 45 min of ischemia followed by reperfusion for 6 h. Ischemia-reperfusion caused an increase in peroxynitrite formation and lipid peroxidation. The level of nitric oxide (NO) metabolites and the mRNA of inducible nitric oxide synthase (iNOS) were elevated in ischemia-reperfused kidneys. Administration of tyrosol (100 mg/kg body weight) to rats prior to the induction of ischemia significantly reduced peroxynitrite formation, lipid peroxidation, and the level of NO metabolites. Tyrosol administration also attenuated ischemia-reperfusion-induced NF-κB activation and iNOS expression. Such a treatment improved kidney function. Results suggest that tyrosol may have a protective effect against acute kidney injury through inhibition of iNOS-mediated oxidative stress.

  18. Susceptibility to cerulein-induced pancreatitis in inducible nitric oxide synthase-deficient mice.

    PubMed

    Qui, B; Mei, Q B; Ma, J J; Korsten, M A

    2001-07-01

    Production of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) has been proposed as a pathogenic factor in acute pancreatitis, but its role has still not been fully examined. The present study explored the role of iNOS in cerulein-induced acute pancreatitis using iNOS-deficient mice. Twelve- to 14-week-old male mice (C57B1/6 and iNOS-deficient) were administered cerulein by intraperitoneal (i.p.) injection at hourly intervals for 7 hours and killed 24 hours later after the first dose. Pancreatic wet weight, pancreatic myeloperoxidase (MPO) activity, and levels of plasma nitrite and serum amylase were measured. In another experiment isosorbide dinitrate (an NO donor) was given by oral gavage every 6 hours for 24 hours beginning simultaneously with cerulein injections in iNOS-deficient mice. Cerulein administration dose-dependently increased pancreatic wet weight, myeloperoxidase activity, and levels of nitrite and amylase in C57B1/6 mice. These parameters (except nitrite levels) were significantly intensified in iNOS-deficient mice. At the dose employed, cerulein failed to increase nitrite levels in iNOS-deficient mice. The susceptibility to cerulein toxicity in iNOS-deficient mice was abolished by NO donor treatment. NO release from an iNOS source appears to play a protective role in cerulein-induced pancreatitis. At least in part, NO may prevent neutrophil accumulation after cerulein administration.

  19. Hypergravity upregulates renal inducible nitric oxide synthase expression and nitric oxide production

    PubMed Central

    Yoon, Gun; Oh, Choong Sik; Kim, Hyun-Soo

    2016-01-01

    Exposure to hypergravity severely decreases renal blood flow, potentially causing renal dysfunction. Nitric oxide (NO), which is endogenously synthesized by inducible NO synthase (iNOS), plays an important role in the regulation of renal function. The purpose of this study was to examine the effect of hypergravity exposure on the production of NO in kidneys. To determine whether hypergravity induces renal hypoxia and alters renal iNOS expression and NO production, mice were exposed to short-term hypergravity at +3Gz for 1 h. The time course of iNOS mRNA expression, hypoxia-inducible factor (HIF)-1α expression, and NO production was examined. Renal HIF-1α levels were significantly elevated immediately after centrifugation, and this increase was sustained for 3 h post-exposure. iNOS mRNA levels were also significantly increased immediately after exposure and were maintained during the reoxygenation period. Immunohistochemical staining for iNOS revealed that the cortical tubular epithelium exhibited moderate to strong cytoplasmic iNOS immunoreactivity immediately after hypergravity exposure and during the reoxygenation period. The time course of NO production was similar to that of iNOS expression. Our results suggest that both hypoxia and reoxygenation might be involved in the upregulation of HIF-1α in the kidneys of mice exposed to hypergravity. Significant increases in renocortical iNOS expression immediately after centrifugation and during the reoxygenation period suggest that iNOS expression induced by hypergravity exposure might play a protective role against hypoxia/reoxygenation injury in the renal cortex. Further investigations are necessary to clarify the role of iNOS and NO in kidneys exposed to hypergravity. PMID:27174912

  20. Methylmalonate-induced seizures are attenuated in inducible nitric oxide synthase knockout mice.

    PubMed

    Ribeiro, Leandro Rodrigo; Fighera, Michele Rechia; Oliveira, Mauro Schneider; Furian, Ana Flávia; Rambo, Leonardo Magno; Ferreira, Ana Paula de Oliveira; Saraiva, André Luiz Lopes; Souza, Mauren Assis; Lima, Frederico Diniz; Magni, Danieli Valnes; Dezengrini, Renata; Flores, Eduardo Furtado; Butterfield, D Allan; Ferreira, Juliano; dos Santos, Adair Roberto Soares; Mello, Carlos Fernando; Royes, Luiz Fernando Freire

    2009-04-01

    Methylmalonic acidemias consist of a group of inherited neurometabolic disorders caused by deficiency of methylmalonyl-CoA mutase activity clinically and biochemically characterized by neurological dysfunction, methylmalonic acid (MMA) accumulation, mitochondrial failure and increased reactive species production. Although previous studies have suggested that nitric oxide (NO) plays a role in the neurotoxicity of MMA, the involvement of NO-induced nitrosative damage from inducible nitric oxide synthase (iNOS) in MMA-induced seizures are poorly understood. In the present study, we showed a decrease of time spent convulsing induced by intracerebroventricular administration of MMA (2 micromol/2 microL; i.c.v.) in iNOS knockout (iNOS(-/-)) mice when compared with wild-type (iNOS(+/+)) littermates. Visual analysis of electroencephalographic recordings (EEG) showed that MMA injection induced the appearance of high-voltage synchronic spike activity in the ipsilateral cortex which spreads to the contralateral cortex while quantitative electroencephalographic analysis showed larger wave amplitude during MMA-induced seizures in wild-type mice when compared with iNOS knockout mice. We also report that administration of MMA increases NOx (NO(2) plus NO(3) content) and 3-nitrotyrosine (3-NT) levels in a greater extend in iNOS(+/+) mice than in iNOS(-/-) mice, indicating that NO overproduction and NO-mediated damage to proteins are attenuated in iNOS knockout mice. In addition, the MMA-induced decrease in Na(+), K(+)-ATPase activity, but not in succinate dehydrogenase (SDH) activity, was less pronounced in iNOS(-/-) when compared with iNOS(+/+) mice. These results reinforce the assumption that metabolic collapse contributes for the secondary toxicity elicited by MMA and suggest that oxidative attack by NO derived from iNOS on selected target such as Na(+), K(+)-ATPase enzyme might represent an important role in this excitotoxicity induced by MMA. Therefore, these results may be

  1. Mice lacking inducible nitric oxide synthase are not resistant to lipopolysaccharide-induced death.

    PubMed Central

    Laubach, V E; Shesely, E G; Smithies, O; Sherman, P A

    1995-01-01

    Nitric oxide produced by cytokine-inducible nitric oxide synthase (iNOS) is thought to be important in the pathogenesis of septic shock. To further our understanding of the role of iNOS in normal biology and in a variety of inflammatory disorders, including septic shock, we have used gene targeting to generate a mouse strain that lacks iNOS. Mice lacking iNOS were indistinguishable from wild-type mice in appearance and histology. Upon treatment with lipopolysaccharide and interferon gamma, peritoneal macrophages from the mutant mice did not produce nitric oxide measured as nitrite in the culture medium. In addition, lysates of these cells did not contain iNOS protein by immunoblot analysis or iNOS enzyme activity. In a Northern analysis of total RNA, no iNOS transcript of the correct size was detected. No increases in serum nitrite plus nitrate levels were observed in homozygous mutant mice treated with a lethal dose of lipopolysaccharide, but the mutant mice exhibited no significant survival advantage over wild-type mice. These results show that lack of iNOS activity does not prevent mortality in this murine model for septic shock. Images Fig. 2 Fig. 3 PMID:7479866

  2. Inducible nitric oxide synthase suppresses the development of allograft arteriosclerosis.

    PubMed Central

    Shears, L L; Kawaharada, N; Tzeng, E; Billiar, T R; Watkins, S C; Kovesdi, I; Lizonova, A; Pham, S M

    1997-01-01

    In cardiac transplantation, chronic rejection takes the form of an occlusive vasculopathy. The mechanism underlying this disorder remains unclear. The purpose of this study was to investigate the role nitric oxide (NO) may play in the development of allograft arteriosclerosis. Rat aortic allografts from ACI donors to Wistar Furth recipients with a strong genetic disparity in both major and minor histocompatibility antigens were used for transplantation. Allografts collected at 28 d were found to have significant increases in both inducible NO synthase (iNOS) mRNA and protein as well as in intimal thickness when compared with isografts. Inhibiting NO production with an iNOS inhibitor increased the intimal thickening by 57.2%, indicating that NO suppresses the development of allograft arteriosclerosis. Next, we evaluated the effect of cyclosporine (CsA) on iNOS expression and allograft arteriosclerosis. CsA (10 mg/kg/d) suppressed the expression of iNOS in response to balloon-induced aortic injury. Similarly, CsA inhibited iNOS expression in the aortic allografts, associated with a 65% increase in intimal thickening. Finally, we investigated the effect of adenoviral-mediated iNOS gene transfer on allograft arteriosclerosis. Transduction with iNOS using an adenoviral vector suppressed completely the development of allograft arteriosclerosis in both untreated recipients and recipients treated with CsA. These results suggest that the early immune-mediated upregulation in iNOS expression partially protects aortic allografts from the development of allograft arteriosclerosis, and that iNOS gene transfer strategies may prove useful in preventing the development of this otherwise untreatable disease process. PMID:9329968

  3. Inducible nitric oxide synthase is expressed in synovial fluid granulocytes.

    PubMed

    Cedergren, J; Forslund, T; Sundqvist, T; Skogh, T

    2002-10-01

    The objective of the study was to evaluate the NO-producing potential of synovial fluid (SF) cells. SF from 15 patients with arthritis was compared with blood from the same individuals and with blood from 10 healthy controls. Cellular expression of inducible nitric oxide synthase (iNOS) was analysed by flow cytometry. High-performance liquid chromatography was used to measure l-arginine and l-citrulline. Nitrite and nitrate were measured colourimetrically utilizing the Griess' reaction. Compared to whole blood granulocytes in patients with chronic arthritis, a prominent iNOS expression was observed in SF granulocytes (P < 0.001). A slight, but statistically significant, increase in iNOS expression was also recorded in lymphocytes and monocytes from SF. l-arginine was elevated in SF compared to serum (257 +/- 78 versus 176 +/- 65 micro mol/l, P = 0.008), whereas a slight increase in l-citrulline (33 +/- 11 versus 26 +/- 9 micro mol/l), did not reach statistical significance. Great variations but no significant differences were observed comparing serum and SF levels of nitrite and nitrate, respectively, although the sum of nitrite and nitrate tended to be elevated in SF (19.2 +/- 20.7 versus 8.6 +/- 6.5 micro mol/l, P = 0.054). Synovial fluid leucocytes, in particular granulocytes, express iNOS and may thus contribute to intra-articular NO production in arthritis.

  4. Cytokines induce nitric oxide production in mouse osteoblasts.

    PubMed

    Damoulis, P D; Hauschka, P V

    1994-06-15

    MC3T3-E1 mouse clonal osteogenic cells were incubated with interferon-gamma, interleukin-1 beta, tumor necrosis factor-alpha, and E. coli lipopolysaccharide. TNF alpha, IL-1 beta, and LPS caused a dose- and time-dependent increase of nitrite (NO2-), the stable metabolite of nitric oxide (NO), in conditioned media over 48 hours, while IFN gamma had a minimal effect. Different combinations of the same factors caused a synergistic enhancement of NO2- accumulation, except for IL-1 beta with LPS. The earliest detectable NO2- production was at 6-9 hours, with continued accumulation over 48 hours. NO2- production was inhibited dose-dependently by three arginine analogs known to be specific inhibitors of NO synthase, as well as by actinomycin D, cycloheximide, and dexamethasone; EGTA or indomethacin had a small inhibitory effect. It is concluded that osteoblast-like cells can be induced by proinflammatory cytokines and bacterial endotoxin to produce NO, which can play an important role in bone pathophysiology.

  5. Inducible nitric oxide synthase as a possible target in hypertension.

    PubMed

    Oliveira-Paula, Gustavo H; Lacchini, Riccardo; Tanus-Santos, Jose E

    2014-02-01

    Nitric oxide (NO) is an important vasodilator produced by vascular endothelium. Its enzymatic formation is derived from three different synthases: neuronal (nNOS), endothelial (eNOS) and inducible (iNOS) synthases. While relatively small amounts of NO produced by eNOS are important to cardiovascular homeostasis, high NO levels produced associated with iNOS activity may have detrimental consequences to the cardiovascular system and contribute to hypertension. In this article, we reviewed current literature and found mounting evidence indicating that increased iNOS expression and activity contribute to the pathogenesis of hypertension and its complications. Excessive amounts of NO produced by iNOS up-regulation can react with superoxide anions forming peroxynitrite, thereby promoting nitrosative stress and endothelial dysfunction. In addition, abnormal iNOS activity can up-regulate arginase activity, allowing it to compete with eNOS for L-arginine, thereby resulting in reduced NO bioavailability. This may also lead to eNOS uncoupling with enhanced production of superoxide anions instead of NO. All these alterations mediated by iNOS apparently contribute to hypertension and its complications. We also reviewed current evidence showing the effects of iNOS inhibitors on different animal models of hypertension. iNOS inhibition apparently exerts antihypertensive effects, decreases oxidative and nitrosative stress, and improves vascular function. Together, these studies highlight the possibility that iNOS is a potential pharmacological target in hypertension.

  6. Lycopene inhibits LPS-induced proinflammatory mediator inducible nitric oxide synthase in mouse macrophage cells.

    PubMed

    Rafi, Mohamed M; Yadav, Prem Narayan; Reyes, Marynell

    2007-01-01

    Lycopene is a fat-soluble red-orange carotenoid found primarily in tomatoes and tomato-derived products, including tomato sauce, tomato paste, and ketchup, and other dietary sources, including dried apricots, guava, watermelon, papaya, and pink grapefruit. In this study, we have demonstrated the molecular mechanism underlying the anti-inflammatory properties of lycopene using a mouse macrophage cell line (RAW 264.7). Treatment with lycopene (10 microM) inhibited lipopolysaccharide (LPS)-stimulated nitric oxide (NO) production (40% compared with the control). Western blotting and reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that lycopene treatment decreased LPS-induced inducible nitric oxide synthase (iNOS) protein and mRNA expression in RAW 264.7 cells, respectively. These results suggest that lycopene has anti-inflammatory activity by inhibiting iNOS proteins and mRNA expressions in mouse macrophage cell lines. Furthermore, cyclooxygenase-2 (COX-2) protein and mRNA expression were not affected by treatment with lycopene.

  7. Castration induces Parkinson disease pathologies in young male mice via inducible nitric-oxide synthase.

    PubMed

    Khasnavis, Saurabh; Ghosh, Anamitra; Roy, Avik; Pahan, Kalipada

    2013-07-19

    Although Parkinson disease (PD) is a progressive neurodegenerative disorder, available animal models do not exhibit irreversible neurodegeneration, and this is a major obstacle in finding out an effective drug against this disease. Here we delineate a new irreversible model to study PD pathogenesis. The model is based on simple castration of young male mice. Levels of inducible nitric-oxide synthase (iNOS), glial markers (glial fibrillary acidic protein and CD11b), and α-synuclein were higher in nigra of castrated male mice than normal male mice. On the other hand, after castration, the level of glial-derived neurotrophic factor (GDNF) markedly decreased in the nigra of male mice. Accordingly, castration also induced the loss of tyrosine hydroxylase-positive neurons in the nigra and decrease in tyrosine hydroxylase-positive fibers and neurotransmitters in the striatum. Reversal of nigrostriatal pathologies in castrated male mice by subcutaneous implantation of 5α-dihydrotestosterone pellets validates an important role of male sex hormone in castration-induced nigrostriatal pathology. Interestingly, castration was unable to cause glial activation, decrease nigral GDNF, augment the death of nigral dopaminergic neurons, induce the loss of striatal fibers, and impair neurotransmitters in iNOS(-/-) male mice. Furthermore, we demonstrate that iNOS-derived NO is responsible for decreased expression of GDNF in activated astrocytes. Together, our results suggest that castration induces nigrostriatal pathologies via iNOS-mediated decrease in GDNF. These results are important because castrated young male mice may be used as a simple, toxin-free, and nontransgenic animal model to study PD-related nigrostriatal pathologies, paving the way for easy drug screening against PD.

  8. Inducible nitric oxide synthase haplotype associated with migraine and aura.

    PubMed

    de O S Mansur, Thiago; Gonçalves, Flavia M; Martins-Oliveira, Alisson; Speciali, Jose G; Dach, Fabiola; Lacchini, Riccardo; Tanus-Santos, Jose E

    2012-05-01

    Migraine is a complex neurological disorder with a clear neurogenic inflammatory component apparently including enhanced nitric oxide (NO) formation. Excessive NO amounts possibly contributing to migraine are derived from increased expression and activity of inducible NO synthase (iNOS). We tested the hypothesis that two functional, clinically relevant iNOS genetic polymorphisms (C(-1026)A-rs2779249 and G2087A-rs2297518) are associated with migraine with or without aura. We studied 142 healthy women without migraine (control group) and 200 women with migraine divided into two groups: 148 with migraine without aura (MWA) and 52 with aura (MA). Genotypes were determined by real-time polymerase chain reaction using the Taqman(®) allele discrimination assays. The PHASE 2.1 software was used to estimate the haplotypes. The A allele for the G2087A polymorphism was more commonly found in the MA group than in the MWA group (28 vs. 18%; P < 0.05). No other significant differences in the alleles or genotypes distributions were found (P > 0.05). The haplotype combining both A alleles for the two polymorphisms was more commonly found in the MA group than in the control group or in the MWA group (19 vs. 10 or 8%; P = 0.0245 or 0.0027, respectively). Our findings indicate that the G2087A and the C(-1026)A polymorphism in the iNOS gene affect the susceptibility to migraine with aura when their effects are combined within haplotypes, whereas the G2087A affects the susceptibility to aura in migraine patients. These finding may have therapeutic implications when examining the effects of selective iNOS inhibitors.

  9. Inducible nitric oxide synthase in Theiler's murine encephalomyelitis virus infection.

    PubMed Central

    Oleszak, E L; Katsetos, C D; Kuzmak, J; Varadhachary, A

    1997-01-01

    We investigated the role of inducible nitric oxide synthase (iNOS) in Theiler's murine encephalomyelitis virus (TMEV) infection of susceptible (SJL) and resistant (C57BL/6 [B6]) strains of mice. TMEV is an excellent model of virus-induced demyelinating disease, such as multiple sclerosis (MS). Previous studies of others have suggested that NO may play a role in the pathogenesis of demyelinating disease. The presence and level of iNOS were determined in the brains and spinal cords of SJL and B6 TMEV-infected mice by the following methods: (i) PCR amplification of iNOS transcripts, followed by Southern blotting with an iNOS-specific probe, and (ii) immunohistochemical staining with an anti-iNOS-specific affinity-purified rabbit antibody. iNOS-specific transcripts were determined in the brains and spinal cord of both SJL and B6 TMEV-infected mice on days 0 (control), days 3, 6, and 10 (encephalitic stage of disease), and days 39 to 42, 66, and 180 (demyelinating phase) postinfection (p.i.). iNOS-specific transcripts were found in the brains and spinal cords of both SJL and B6 TMEV-infected mice at 6, 10, and 39 (SJL) days p.i., but they were absent in mock-infected mice and in TMEV-infected SJL and B6 mice at 0, 3, 66, and 180 days p.i. Immunohistochemical staining confirmed the presence of iNOS protein in both TMEV-infected SJL and B6 mice at days 6 and 10 p.i., but not at days 0, 3, 66, and 180 days p.i. Weak iNOS staining was also observed in TMEV-infected SJL mice at 42 days p.i. iNOS-positive staining was found in reactive astrocytes surrounding areas of necrotizing inflammation, particularly in the midbrain. Weak iNOS staining was also observed in cells of the monocyte/macrophage lineage in areas of parenchymal inflammation and necrosis (mesencephalon) and in leptomeningeal and white matter perivascular infiltrates of the spinal cord. Rod-shaped microglia-like cells and foamy macrophages (myelin-laden) were iNOS negative. These results suggest that NO does not

  10. Progesterone modulates the LPS-induced nitric oxide production by a progesterone-receptor independent mechanism.

    PubMed

    Wolfson, Manuel Luis; Schander, Julieta Aylen; Bariani, María Victoria; Correa, Fernando; Franchi, Ana María

    2015-12-15

    Genital tract infections caused by Gram-negative bacteria induce miscarriage and are one of the most common complications of human pregnancy. LPS administration to 7-day pregnant mice induces embryo resorption after 24h, with nitric oxide playing a fundamental role in this process. We have previously shown that progesterone exerts protective effects on the embryo by modulating the inflammatory reaction triggered by LPS. Here we sought to investigate whether the in vivo administration of progesterone modulated the LPS-induced nitric oxide production from peripheral blood mononuclear cells from pregnant and non-pregnant mice. We found that progesterone downregulated LPS-induced nitric oxide production by a progesterone receptor-independent mechanism. Moreover, our results suggest a possible participation of glucocorticoid receptors in at least some of the anti-inflammatory effects of progesterone.

  11. p38 MAP kinase mediates nitric oxide-induced apoptosis of neural progenitor cells.

    PubMed

    Cheng, A; Chan, S L; Milhavet, O; Wang, S; Mattson, M P

    2001-11-16

    Neural progenitor cells (NPC) can proliferate, differentiate into neurons or glial cells, or undergo a form of programmed cell death called apoptosis. Although death of NPC occurs during development of the nervous system and in the adult, the underlying mechanisms are unknown. Here we show that nitric oxide (NO) can induce death of C17.2 NPC by a mechanism requiring activation of p38 MAP kinase, poly(ADP-ribose) polymerase, and caspase-3. Nitric oxide causes release of cytochrome c from mitochondria, and Bcl-2 protects the neural progenitor cells against nitric oxide-induced death, consistent with a pivotal role for mitochondrial changes in controlling the cell death process. Inhibition of p38 MAP kinase by SB203580 abolished NO-induced cell death, cytochrome c release, and activation of caspase-3, indicating that p38 activation serves as an upstream mediator in the cell death process. The anti-apoptotic protein Bcl-2 protected NPC against nitric oxide-induced apoptosis and suppressed activation of p38 MAP kinase. The ability of nitric oxide to trigger death of NPC by a mechanism involving p38 MAP kinase suggests that this diffusible gas may regulate NPC fate in physiological and pathological settings in which NO is produced.

  12. Characterization and functional analysis of the human inducible nitric oxide synthase gene promoter.

    PubMed Central

    Spitsin, S. V.; Koprowski, H.; Michaels, F. H.

    1996-01-01

    BACKGROUND: Nitric oxide has a wide variety of homeostatic and pathological effects. Control of the production of nitric oxide by the inducible form of the enzyme resides in the 5' promoter region of the gene. Although control of the murine isoform has been investigated, little is known about the functional aspects of the human analog. MATERIALS AND METHODS: A 3.9-kb 5' nontranslated region of the human gene was cloned, sequenced, and several reporter constructs prepared. The promoter-reporter constructs were transfected into human or murine monocytoid cells and reporter expression quantified following cytokine activation of the cells. The production of nitric oxide was also monitored. RESULTS: Although a murine promoter-reporter functioned efficiently in both human and mouse cells, the human constructs functioned only in human cells. The activity of the mouse construct increased progressively with the addition of activating cytokines, but the human promoter-reporter did not. Although interleukin 1 beta drove expression of the human inducible nitric oxide synthase reporter, actual expression of nitric oxide required both interleukin 1 beta and interferon-gamma. CONCLUSIONS: The data indicate that despite the significant homology between the human and mouse inducible nitric oxide synthase promoter sequence, control of the two genes is quite different. In addition to being more efficient in promoter activity, the murine promoter responds increasingly to cytokines that are not effective for the human analog. It is also apparent that human inducible nitric oxide synthase is controlled at both the level of transcription and post-translationally. PMID:8726465

  13. Energetic particle-induced enhancements of stratospheric nitric acid

    NASA Technical Reports Server (NTRS)

    Aikin, Arthur C.

    1994-01-01

    Inclusion of complete ion chemistry in the calculation of minor species production during energetic particle deposition events leads to significant enhancement in the calculated nitric acid concentration during precipitation. An ionization rate of 1.2 x 10(exp 3)/cu cm/s imposed for 1 day increases HNO3 from 3 x 10(exp 5) to 6 x 10(exp 7)/cu cm at 50 km. With an ionization rate of 600 cu cm/s, the maximum HNO3 is 3 x 10(exp 7)/cu cm. Calculations which neglect negative ions predict the nitric acid will fall during precipitation events. The decay time for converting HNO3 into odd nitrogen and hydrogen is more than 1 day for equinoctial periods at 70 deg latitude. Examination of nitric acid data should yield important information on the magnitude and frequency of charged particle events.

  14. Arsenic toxicity induced endothelial dysfunction and dementia: Pharmacological interdiction by histone deacetylase and inducible nitric oxide synthase inhibitors

    SciTech Connect

    Sharma, Bhupesh Sharma, P.M.

    2013-11-15

    Arsenic toxicity has been reported to damage all the major organs including the brain and vasculature. Dementia including Alzheimer's disease (AD) and vascular dementia (VaD) are posing greater risk to the world population as it is now increasing at a faster rate. We have investigated the role of sodium butyrate, a selective histone deacetylase (HDAC) inhibitor and aminoguanidine, a selective inducible nitric oxide synthase (iNOS) inhibitor in pharmacological interdiction of arsenic toxicity induced vascular endothelial dysfunction and dementia in rats. Arsenic toxicity was done by administering arsenic drinking water to rats. Morris water-maze (MWM) test was used for assessment of learning and memory. Endothelial function was assessed using student physiograph. Oxidative stress (aortic superoxide anion, serum and brain thiobarbituric acid reactive species, brain glutathione) and nitric oxide levels (serum nitrite/nitrate) were also measured. Arsenic treated rats have shown impairment of endothelial function, learning and memory, reduction in serum nitrite/nitrate and brain GSH levels along with increase in serum and brain TBARS. Sodium butyrate as well as aminoguanidine significantly convalesce arsenic induced impairment of learning, memory, endothelial function, and alterations in various biochemical parameters. It may be concluded that arsenic induces endothelial dysfunction and dementia, whereas, sodium butyrate, a HDAC inhibitor as well as aminoguanidine, a selective iNOS inhibitor may be considered as potential agents for the management of arsenic induced endothelial dysfunction and dementia. - Highlights: • As has induced endothelial dysfunction (Edf) and vascular dementia (VaD). • As has increased oxidative stress, AChE activity and decreased serum NO. • Inhibitors of HDAC and iNOS have attenuated As induced Edf and VaD. • Both the inhibitors have attenuated As induced biochemical changes. • Inhibitor of HDAC and iNOS has shown good potential in

  15. Molecular Mechanism of Silver Nanoparticles-Induced Human Osteoblast Cell Death: Protective Effect of Inducible Nitric Oxide Synthase Inhibitor

    PubMed Central

    Zielinska, Ewelina; Tukaj, Cecylia; Radomski, Marek Witold; Inkielewicz-Stepniak, Iwona

    2016-01-01

    Background Silver nanoparticles (AgNPs) show strong antibacterial properties, making them excellent candidates to be used in orthopaedic repair and regeneration. However, there are concerns regarding the cytotoxicity of AgNPs and molecular mechanisms underlying AgNPs-induced bone cells toxicity have not been elucidated. Therefore, the aim of our study was to explore mechanisms of AgNPs-induced osteoblast cell death with particular emphasis on the role of nitric oxide (NO) generated by inducible nitric oxide synthase (iNOS). Methods and Result Silver nanoparticles used in this study were 18.3±2.6 nm in size, uncoated, spherical, regular shape and their zeta potential was -29.1±2.4 mV as measured by transmission electron microscopy (TEM) and zetasizer. The release of silver (Ag) from AgNPs was measured in cell culture medium by atomic absorption spectroscopy (AAS). The exposure of human osteoblast cells (hFOB 1.19) to AgNPs at concentration of 30 or 60 μg/mL for 24 or 48 hours, respectively resulted in cellular uptake of AgNPs and changes in cell ultrastructure. These changes were associated with apoptosis and necrosis as shown by flow cytometry and lactate dehydrogenase (LDH) assay as well as increased levels of pro-apoptotic Bax and decreased levels of anti-apoptotic Bcl-2 mRNA and protein. Importantly, we have found that AgNPs elevated the levels of nitric oxide (NO) with concomitant upregulation of inducible nitric oxide synthase (iNOS) mRNA and protein. A significant positive correlation was observed between the concentration of AgNPs and iNOS at protein and mRNA level (r = 0.837, r = 0.721, respectively; p<0.001). Finally, preincubation of osteoblast cells with N-iminoethyl-l-lysine (L-NIL), a selective iNOS inhibitor, as well as treating cells with iNOS small interfering RNAs (siRNA) significantly attenuated AgNPs-induced apoptosis and necrosis. Moreover, we have found that AgNPs-induced cells death is not related to Ag dissolution is cell culture medium

  16. Nitric oxide-induced p53 accumulation and regulation of inducible nitric oxide synthase expression by wild-type p53.

    PubMed Central

    Forrester, K; Ambs, S; Lupold, S E; Kapust, R B; Spillare, E A; Weinberg, W C; Felley-Bosco, E; Wang, X W; Geller, D A; Tzeng, E; Billiar, T R; Harris, C C

    1996-01-01

    The tumor suppressor gene product p53 plays an important role in the cellular response to DNA damage from exogenous chemical and physical mutagens. Therefore, we hypothesized that p53 performs a similar role in response to putative endogenous mutagens, such as nitric oxide (NO). We report here that exposure of human cells to NO generated from an NO donor or from overexpression of inducible nitric oxide synthase (NOS2) results in p53 protein accumulation. In addition, expression of wild-type (WT) p53 in a variety of human tumor cell lines, as well as murine fibroblasts, results in down-regulation of NOS2 expression through inhibition of the NOS2 promoter. These data are consistent with the hypothesis of a negative feedback loop in which endogenous NO-induced DNA damage results in WT p53 accumulation and provides a novel mechanism by which p53 safeguards against DNA damage through p53-mediated transrepression of NOS2 gene expression, thus reducing the potential for NO-induced DNA damage. Images Fig. 1 Fig. 2 Fig. 3 PMID:8637893

  17. Inducible nitric oxide synthase (iNOS) and nitric oxide (NO) are important mediators of reflux-induced cell signalling in esophageal cells.

    PubMed

    McAdam, E; Haboubi, H N; Forrester, G; Eltahir, Z; Spencer-Harty, S; Davies, C; Griffiths, A P; Baxter, J N; Jenkins, G J S

    2012-11-01

    Nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) has been implicated in both DNA damage induction and aberrant cell signalling in various tissue and cell backgrounds. We investigated here the role of iNOS and NO in DNA damage induction and nuclear factor-kappa B (NF-κB) signalling in esophageal cells in vitro. As esophageal adenocarcinoma develops in a background of Barrett's esophagus secondary to reflux disease, it is possible that inflammatory mediators like NO may be important in esophageal cancer development. We show that reflux components like stomach acid and bile acids [deoxycholic acid (DCA)] can induce iNOS gene and protein expression and produce NO generation in esophageal cells, using real-time PCR, western blotting and NO sensitive fluorescent probes, respectively. This up-regulation of iNOS expression was not dependent on NF-κB activity. DCA-induced DNA damage was independent of NF-κB and only partially dependent on iNOS and NO, as measured by the micronucleus assay. These same reflux constituents also activated the oncogenic transcription factor NF-κB, as measured by transcription factor enzyme-linked immunosorbent assay and gene expression studies with NF-κB linked genes (e.g. interleukin-8). Importantly, we show here for the first time that basal levels of NF-κB activity (and possibly acid and DCA-induced NF-κB) are dependent on iNOS/NO and this may lead to a positive feedback loop whereby induced iNOS is upstream of NF-κB, hence prolonging and potentially amplifying this signalling, presumably through NO activation of NF-κB. Furthermore, we confirm increased protein levels of iNOS in esophageal adenocarcinoma and, therefore, in neoplastic development in the esophagus.

  18. Nitric oxide mediates glial-induced neurodegeneration in Alexander disease.

    PubMed

    Wang, Liqun; Hagemann, Tracy L; Kalwa, Hermann; Michel, Thomas; Messing, Albee; Feany, Mel B

    2015-11-26

    Glia play critical roles in maintaining the structure and function of the nervous system; however, the specific contribution that astroglia make to neurodegeneration in human disease states remains largely undefined. Here we use Alexander disease, a serious degenerative neurological disorder caused by astrocyte dysfunction, to identify glial-derived NO as a signalling molecule triggering astrocyte-mediated neuronal degeneration. We further find that NO acts through cGMP signalling in neurons to promote cell death. Glial cells themselves also degenerate, via the DNA damage response and p53. Our findings thus define a specific mechanism for glial-induced non-cell autonomous neuronal cell death, and identify a potential therapeutic target for reducing cellular toxicity in Alexander disease, and possibly other neurodegenerative disorders with glial dysfunction.

  19. Regulation of Injury-Induced Neurogenesis by Nitric Oxide

    PubMed Central

    Carreira, Bruno P.; Carvalho, Caetana M.; Araújo, Inês M.

    2012-01-01

    The finding that neural stem cells (NSCs) are able to divide, migrate, and differentiate into several cellular types in the adult brain raised a new hope for restorative neurology. Nitric oxide (NO), a pleiotropic signaling molecule in the central nervous system (CNS), has been described to be able to modulate neurogenesis, acting as a pro- or antineurogenic agent. Some authors suggest that NO is a physiological inhibitor of neurogenesis, while others described NO to favor neurogenesis, particularly under inflammatory conditions. Thus, targeting the NO system may be a powerful strategy to control the formation of new neurons. However, the exact mechanisms by which NO regulates neural proliferation and differentiation are not yet completely clarified. In this paper we will discuss the potential interest of the modulation of the NO system for the treatment of neurodegenerative diseases or other pathological conditions that may affect the CNS. PMID:22997523

  20. Inhaled nitric oxide induces cerebrovascular effects in anesthetized pigs.

    PubMed

    Kuebler, W M; Kisch-Wedel, H; Kemming, G I; Meisner, F; Bruhn, S; Koehler, C; Flondor, M; Messmer, K; Zwissler, B

    2003-09-11

    Although inhaled nitric oxide (NO(i)) is considered to act selectively on pulmonary vessels, EEG abnormalities and even occasional neurotoxic effects of NO(i) have been proposed. Here, we investigated cerebrovascular effects of increasing concentrations of 5, 10 and 50 ppm NO(i) in seven anesthetized pigs. Cerebral hemodynamics were assessed non-invasively by use of near-infared spectroscopy and indicator dilution techniques. NO(i) increased cerebral blood volume significantly and reversibly. This effect was not attributable to changes of macrohemodynamic parameters or arterial blood gases. Simultaneously, cerebral transit time increased while cerebral blood flow remained unchanged. These data demonstrate a vasodilatory action of NO(i) in the cerebral vasculature, which may occur preferentially in the venous compartment.

  1. Nitric oxide ameliorates the damaging effects of oxidative stress induced by iron deficiency in cyanobacterium Anabaena 7120.

    PubMed

    Kaushik, Manish Singh; Srivastava, Meenakshi; Srivastava, Alka; Singh, Anumeha; Mishra, Arun Kumar

    2016-11-01

    In cyanobacterium Anabaena 7120, iron deficiency leads to oxidative stress with unavoidable consequences. Nitric oxide reduces pigment damage and supported the growth of Anabaena 7120 in iron-deficient conditions. Elevation in nitric oxide accumulation and reduced superoxide radical production justified the role of nitric oxide in alleviating oxidative stress in iron deficiency. Increased activities of antioxidative enzymes and higher levels of ROS scavengers (ascorbate, glutathione and thiol) in iron deficiency were also observed in the presence of nitric oxide. Nitric oxide also supported the membrane integrity of Anabaena cells and reduces protein and DNA damage caused by oxidative stress induced by iron deficiency. Results suggested that nitric oxide alleviates the damaging effects of oxidative stress induced by iron deficiency in cyanobacterium Anabaena 7120.

  2. Contribution of radiation-induced, nitric oxide-mediated bystander effect to radiation-induced adaptive response.

    NASA Astrophysics Data System (ADS)

    Matsumoto, H.; Ohnishi, T.

    There has been a recent upsurge of interest in radiation-induced adaptive response and bystander effect which are specific modes in stress response to low-dose low-dose rate radiation Recently we found that the accumulation of inducible nitric oxide NO synthase iNOS in wt p53 cells was induced by chronic irradiation with gamma rays followed by acute irradiation with X-rays but not by each one resulting in an increase in nitrite concentrations of medium It is suggested that the accumulation of iNOS may be due to the depression of acute irradiation-induced p53 functions by pre-chronic irradiation In addition we found that the radiosensitivity of wt p53 cells against acute irradiation with X-rays was reduced after chronic irradiation with gamma rays This reduction of radiosensitivity of wt p53 cells was nearly completely suppressed by the addition of NO scavenger carboxy-PTIO to the medium This reduction of radiosensitivity of wt p53 cells is just radiation-induced adaptive response suggesting that NO-mediated bystander effect may considerably contribute to adaptive response induced by radiation

  3. Verticillium dahliae toxins-induced nitric oxide production in Arabidopsis is major dependent on nitrate reductase.

    PubMed

    Shi, Fu-Mei; Li, Ying-Zhang

    2008-01-31

    The source of nitric oxide (NO) in plants is unclear and it has been reported NO can be produced by nitric oxide synthase (NOS) like enzymes and by nitrate reductase (NR). Here we used wild-type, Atnos1 mutant and nia1, nia2 NR-deficient mutant plants of Arabidopsis thaliana to investigate the potential source of NO production in response to Verticillium dahliae toxins (VD-toxins). The results revealed that NO production is much higher in wild-type and Atnos1 mutant than in nia1, nia2 NR-deficient mutants. The NR inhibitor had a significant effect on VD-toxins-induced NO production; whereas NOS inhibitor had a slight effect. NR activity was significantly implicated in NO production. The results indicated that as NO was induced in response to VD-toxins in Arabidopsis, the major source was the NR pathway. The production of NOS-system appeared to be secondary.

  4. Role of nitric oxide in PGF2 alpha-induced ocular hyperemia.

    PubMed

    Astin, M; Stjernschantz, J; Selén, G

    1994-10-01

    The effect of nitric oxide synthase inhibition on prostaglandin F2 alpha (PGF2 alpha)-induced ocular hyperemia in the rabbit has been studied. PGF2 alpha was administered topically as the isopropyl ester (PGF2 alpha-IE) unilaterally, with the other eye serving as a control. The regional blood flow in the eye was determined with radioactively-labelled microspheres in conscious animals. Synthesis of nitric oxide (NO) was blocked by L-NMMA (200 mg kg-1 b.w., i.v.). PGF2 alpha-IE induced marked hyperemia of the surface structures of the eye (conjunctiva, eye lids, nictitating membrane, anterior sclera), as well as increased blood flow of the anterior uvea. L-NMMA blocked the hyperemia of the surface structures but not completely the increase in blood flow of the anterior uvea. PhXA41 (13,14-dihydro-17-phenyl-18,19,20-trinor-PGF2 alpha-isopropyl ester), a selective prostaglandin FP-receptor agonist, had no significant effect on the ocular blood flow. These results indicate that PGF2 alpha causes surface hyperemia of the eye by activating nitric oxide synthase, but this mechanism seems to be only partly involved in the increase in blood flow of the ciliary processes and the iris. The PGF2 alpha-induced ocular hyperemia is unlikely to be mediated by FP receptors.

  5. Methamphetamine-induced nitric oxide promotes vesicular transport in blood-brain barrier endothelial cells.

    PubMed

    Martins, Tânia; Burgoyne, Thomas; Kenny, Bridget-Ann; Hudson, Natalie; Futter, Clare E; Ambrósio, António F; Silva, Ana P; Greenwood, John; Turowski, Patric

    2013-02-01

    Methamphetamine's (METH) neurotoxicity is thought to be in part due to its ability to induce blood-brain barrier (BBB) dysfunction. Here, we investigated the effect of METH on barrier properties of cultured rat primary brain microvascular endothelial cells (BMVECs). Transendothelial flux doubled in response to METH, irrespective of the size of tracer used. At the same time, transendothelial electrical resistance was unchanged as was the ultrastructural appearance of inter-endothelial junctions and the distribution of key junction proteins, suggesting that METH promoted vesicular but not junctional transport. Indeed, METH significantly increased uptake of horseradish peroxidase into vesicular structures. METH also enhanced transendothelial migration of lymphocytes indicating that the endothelial barrier against both molecules and cells was compromised. Barrier breakdown was only observed in response to METH at low micromolar concentrations, with enhanced vesicular uptake peaking at 1 μM METH. The BMVEC response to METH also involved rapid activation of endothelial nitric oxide synthase and its inhibition abrogated METH-induced permeability and lymphocyte migration, indicating that nitric oxide was a key mediator of BBB disruption in response to METH. This study underlines the key role of nitric oxide in BBB function and describes a novel mechanism of drug-induced fluid-phase transcytosis at the BBB.

  6. Bifunctional effects of fucoidan on the expression of inducible nitric oxide synthase

    SciTech Connect

    Yang, Jin Won; Yoon, Se Young; Oh, Soo Jin; Kim, Sang Kyum; Kang, Keon Wook . E-mail: kwkang@chosun.ac.kr

    2006-07-21

    Algal fucoidan is a marine sulfated polysaccharide with a wide variety of biological activities including anti-thrombotic and anti-inflammatory effects. This study evaluated the effect of fucoidan on the expression of inducible nitric oxide synthase (iNOS) in a macrophage cell line, RAW264.7. Low concentration range of fucoidan (10 {mu}g/ml) increased the basal expression level of iNOS in quiescent macrophages. However, we found for the first time that fucoidan inhibited the release of nitric oxide (NO) in RAW264.7 cells stimulated with lipopolysaccharide (LPS). Western blot analysis revealed that fucoidan suppressed the LPS-induced expression of the inducible nitric oxide synthase (iNOS) gene. Moreover, the activation of both nuclear factor-{kappa}B (NF-{kappa}B) and activator protein 1 (AP-1) are key steps in the transcriptional activation of the iNOS gene. Here, it was revealed that fucoidan selectively suppressed AP-1 activation, and that the activation of AP-1 appears to be essential for the induction of iNOS in activated macrophages. This inhibitory effect on AP-1 activation by fucoidan might be associated with its NO blocking and anti-inflammatory effects.

  7. Auxin-induced nitric oxide, cGMP and gibberellins were involved in the gravitropism

    NASA Astrophysics Data System (ADS)

    Cai, Weiming; Hu, Liwei; Hu, Xiangyang; Cui, Dayong; Cai, Weiming

    Gravitropism is the asymmetric growth or curvature of plant organs in response to gravistimulation. There is a complex signal transduction cascade which involved in the differential growth of plants in response to changes in the gravity vector. The role of auxin in gravitropism has been demonstrated by many experiments, but little is known regarding the molecular details of such effects. In our studies before, mediation of the gravitropic bending of soybean roots and rice leaf sheath bases by nitric oxide, cGMP and gibberellins, are induced by auxin. The asymmetrical distribution of nitric oxide, cGMP and gibberellins resulted from the asymmetrical synthesis of them in bending sites. In soybean roots, inhibitions of NO and cGMP synthesis reduced differential NO and cGMP accumulation respectively, which both of these effects can lead to the reduction of gravitropic bending. Gibberellin-induced OsXET, OsEXPA4 and OsRWC3 were also found involved in the gravitropic bending. These data indicated that auxin-induced nitric oxide, cGMP and gibberellins were involved in the gravitropism. More experiments need to prove the more detailed mechanism of them.

  8. The role of photolabile dermal nitric oxide derivates in ultraviolet radiation (UVR)-induced cell death.

    PubMed

    Opländer, Christian; Suschek, Christoph V

    2012-12-21

    Human skin is exposed to solar ultraviolet radiation comprising UVB (280-315 nm) and UVA (315-400 nm) on a daily basis. Within the last two decades, the molecular and cellular response to UVA/UVB and the possible effects on human health have been investigated extensively. It is generally accepted that the mutagenic and carcinogenic properties of UVB is due to the direct interaction with DNA. On the other hand, by interaction with non-DNA chromophores as endogenous photosensitizers, UVA induces formation of reactive oxygen species (ROS), which play a pivotal role as mediators of UVA-induced injuries in human skin. This review gives a short overview about relevant findings concerning the molecular mechanisms underlying UVA/UVB-induced cell death. Furthermore, we will highlight the potential role of cutaneous antioxidants and photolabile nitric oxide derivates (NODs) in skin physiology. UVA-induced decomposition of the NODs, like nitrite, leads not only to non-enzymatic formation of nitric oxide (NO), but also to toxic reactive nitrogen species (RNS), like peroxynitrite. Whereas under antioxidative conditions the generation of protective amounts of NO is favored, under oxidative conditions, less injurious reactive nitrogen species are generated, which may enhance UVA-induced cell death.

  9. Use of aminoguanidine, a selective inducible nitric oxide synthase inhibitor, to evaluate the role of nitric oxide in periapical inflammation.

    PubMed

    Farhad, Ali R; Razavi, Seyedmohammad; Jahadi, Sanaz; Saatchi, Masoud

    2011-06-01

    The purpose of this study was to evaluate the effects of aminoguanidine (AG) as a selective inhibitor of inducible nitric oxide synthase (iNOS) on the degree of inflammatory response in periapical lesions in the canine teeth of cats. Root canals from 52 cat canine teeth were exposed to the oral cavity and sealed after 7 days. One day before pulp exposure, cats were administered either AG (experimental group) or normal saline (control group), which was continued on a daily basis until the day of sacrifice. Animals were sacrificed at 28 days after pulp exposure. Inflammatory response in the periapical zones was analyzed histologically. The degree of periapical inflammation in the AG group was significantly lower than that in the control group (P < 0.05). Selective iNOS inhibitors such as AG thus reduce the intensity of inflammatory responses in periapical lesions.

  10. Amomum tsao-ko fruit extract suppresses lipopolysaccharide-induced inducible nitric oxide synthase by inducing heme oxygenase-1 in macrophages and in septic mice.

    PubMed

    Shin, Ji-Sun; Ryu, Suran; Jang, Dae Sik; Cho, Young-Wuk; Chung, Eun Kyung; Lee, Kyung-Tae

    2015-12-01

    Amomum tsao-ko Crevost et Lemarié (Zingiberaceae) has traditionally been used to treat inflammatory and infectious diseases, such as throat infections, malaria, abdominal pain and diarrhoea. This study was designed to assess the anti-inflammatory effects and the molecular mechanisms of the methanol extract of A. tsao-ko (AOM) in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages and in a murine model of sepsis. In LPS-induced RAW 264.7 macrophages, AOM reduced the production of nitric oxide (NO) by inhibiting inducible nitric oxide synthase (iNOS) expression, and increased heme oxygenase-1 (HO-1) expression at the protein and mRNA levels. Pretreatment with SnPP (a selective inhibitor of HO-1) and silencing HO-1 using siRNA prevented the AOM-mediated inhibition of NO production and iNOS expression. Furthermore, AOM increased the expression and nuclear accumulation of NF-E2-related factor 2 (Nrf2), which enhanced Nrf2 binding to antioxidant response element (ARE). In addition, AOM induced the phosphorylation of extracellular regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) and generated reactive oxygen species (ROS). Furthermore, pretreatment with N-acetyl-l-cysteine (NAC; a ROS scavenger) diminished the AOM-induced phosphorylation of ERK and JNK and AOM-induced HO-1 expression, suggesting that ERK and JNK are downstream mediators of ROS during the AOM-induced signalling of HO-1 expression. In LPS-induced endotoxaemic mice, pretreatment with AOM reduced NO serum levels and liver iNOS expression and increased HO-1 expression and survival rates. These results indicate that AOM strongly inhibits LPS-induced NO production by activating the ROS/MAPKs/Nrf2-mediated HO-1 signalling pathway, and supports its pharmacological effects on inflammatory diseases.

  11. autoregulatory role of endothelium-derived nitric oxide (NO) on Lipopolysaccharide-induced vascular inducible NO synthase expression and function.

    PubMed

    Vo, Phuong A; Lad, Bhavini; Tomlinson, James A P; Francis, Stephanie; Ahluwalia, Amrita

    2005-02-25

    Nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) is responsible for sepsis-induced hypotension and plays a major contributory role in the ensuing multiorgan failure. The present study aimed to elucidate the role of endothelial NO in lipopolysaccharide (LPS)-induced iNOS expression, in isolated rat aortic rings. Exposure to LPS (1 mug/ml, 5 h) resulted in a reversal of phenylephrine precontracted tone in aortic rings (70.7 +/- 3.2%). This relaxation was associated with iNOS expression and NF-kappaB activation. Positive immunoreactivity for iNOS protein was localized in medial and adventitial layers of LPS-treated aortic rings. Removal of the endothelium rendered aortic rings resistant to LPS-induced relaxation (8.9 +/- 4.5%). Western blotting of these rings demonstrated an absence of iNOS expression. However, treatment of endothelium-denuded rings with the NO donor, diethylamine-NONOate (0.1 mum), restored LPS-induced relaxation (61.6 +/- 6.6%) and iNOS expression to levels comparable with arteries with intact endothelium. Blockade of endothelial NOS (eNOS) activation using geldanamycin and radicicol, inhibitors of heat shock protein 90, in endothelium-intact arteries suppressed both LPS-induced relaxation and LPS-induced iNOS expression (9.0 +/- 8.0% and 2.0 +/- 6.2%, respectively). Moreover, LPS treatment (12.5 mg/kg, intravenous, 15 h) of wild-type mice resulted in profound elevation of plasma [NO(x)] measurements that were reduced by approximately 50% in eNOS knock-out animals. Furthermore, LPS-induced changes in vascular reactivity and iNOS expression evident in wild-type tissues were profoundly suppressed in tissues taken from eNOS knockout animals. Together, these data suggest that eNOS-derived NO, in part via activation of NF-kappaB, regulates iNOS-induction by LPS. This study provides the first demonstration of a proinflammatory role of vascular eNOS in sepsis.

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

    PubMed Central

    Stadler, Krisztian; Bonini, Marcelo G.; Dallas, Shannon; Jiang, JinJie; Radi, Rafael; Mason, Ronald P.; Kadiiska, Maria B.

    2008-01-01

    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. Neither xanthine oxidase, cytochrome P450s, the Fenton reaction, nor macrophage activation were required for the production of radical adducts. Interestingly, inducible nitric oxide synthase (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 oxidation to protein free radicals 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. PMID:18620046

  13. Comparison between the role of the neuronal and inducible nitric oxide synthase in methamphetamine-induced neurotoxicity and sensitization.

    PubMed

    Itzhak, Y; Martin, J L; Ali, S F

    2000-09-01

    The involvement of the neuronal and inducible nitric oxide synthase (nNOS and iNOS, respectively) in methamphetamine (METH)-induced dopaminergic neurotoxicity and behavioral sensitization was investigated. To determine METH-induced neurotoxicity, mice deficient in the nNOS and iNOS genes, nNOS(-/-) and iNOS(-/-) mice, and wild-type controls received either saline or METH (5 mg/kg x 3). After 72 h the level of striatal dopaminergic markers were measured. Administration of METH to nNOS(-/-) mice had no significant effect on the level of striatal dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), or dopamine transporter (DAT) binding sites. However, METH caused 25-40% depletion of dopaminergic markers in iNOS(-/-) mice and 63-69% depletion in the wild-type mice. METH-induced locomotor activity was measured following the administration of a low dose (1 mg/kg) on day 1. Subsequently animals received the high dose of METH (5 mg/kg x 3). On day 4, after a 68-72 h drug free period, animals were challenged with 1 mg/kg METH, and locomotor activity was recorded. The intensity of METH-induced locomotion in nNOS(-/-) mice on day 1 and 4 was similar, suggesting that locomotor sensitization did not develop. However, the intensity of METH-induced locomotion in the iNOS(-/-) and wild-type mice on day 4 was doubled compared to day 1, suggesting the development of sensitization. The present findings indicate that nNOS(-/-) mice are more resistant to METH-induced neurotoxicity and behavioral sensitization than iNOS(-/-) mice. These results suggest a major role for nNOS rather than iNOS in the effects of METH.

  14. Nitric oxide: Mediator of nonadrenergic noncholinergic nerve-induced responses of opossum esophageal muscle

    SciTech Connect

    Murray, J.; Du, C.; Conklin, J.L.; Ledlow, A.; Bates, J.N. )

    1991-03-15

    Nonadrenergic noncholinergic (NANC) nerves of the opossum esophagus mediate relaxation of circular muscle from the lower esophageal sphincter (LES) and the off contraction of circular esophageal muscle. The latencies between the end of the stimulus and the off contraction describe a gradient such that the latency is longest in muscle from the caudad esophagus. N{sup G}-nitro-L-arginine (L-NNA), an inhibitor of nitric oxide synthase, and nitric oxide were used to test the hypothesis that NO is a mediator of these nerve-induced responses. Both electrical field stimulation (EFS) of intrinsic esophageal nerves and exogenous NO relaxed LES muscle. Only EFS-induced relaxation was inhibited by L-NNA. L-arginine, the substrate for NO synthase, antagonized the inhibitory effect of L-NNA. Exogenous NO neither relaxed nor contracted circular esophageal muscle. Both the amplitude and the latency of the off contraction were diminished by L-NNA. L-arginine antagonized the action of L-NNA. N{sup G}-nitro-L-arginine also attenuated the gradient in the latency of the off response by shortening latencies in muscle form the caudad esophagus. It had no effect on cholinergic nerve-induced contraction of longitudinal esophageal muscle. These data support the hypothesis that NO or an NO-containing compound mediates NANC nerve-induced responses of the esophagus and LES.

  15. Nitric oxide is overproduced by peritoneal macrophages in rat taurocholate pancreatitis: the mechanism of inducible nitric oxide synthase expression.

    PubMed

    Satoh, A; Shimosegawa, T; Kimura, K; Moriizumi, S; Masamune, A; Koizumi, M; Toyota, T

    1998-11-01

    To investigate the pathobiology of severe acute pancreatitis, we studied the expression of inducible nitric oxide synthase (iNOS) in peritoneal macrophages of experimental pancreatitis. Taurocholate (TCA) pancreatitis and cerulein (CE) pancreatitis were used as models of lethal and self-limited pancreatitis, respectively, and the mechanism of iNOS expression in peritoneal macrophages was studied. Serum nitrate and nitrite (NOx) concentrations increased during the course of TCA pancreatitis, and iNOS-immunoreactivity was detected in the peritoneal macrophages 12 h after the induction of TCA pancreatitis, but these phenomena were not observed in CE pancreatitis. Despite the difference in the iNOS expression, the iNOS messenger RNA (mRNA) and the activation of nuclear factor-kappa B (NF-kappa B) were detected in the peritoneal macrophages of both pancreatitis models. The supernatant of TCA pancreatitis ascites could induce iNOS in the peritoneal macrophages of normal rats in vitro, but the peritoneal lavage fluid of CE pancreatitis rats could not. The results indicated that there may be qualitative or quantitative differences in the macrophage activation between the two types of experimental pancreatitis and suggested that the ascites of rats with lethal acute pancreatitis contains some soluble factors that activate the macrophage/monocyte system and cause an overproduction of NO by the iNOS expression.

  16. Retinoic acid inhibits inducible nitric oxide synthase expression in 3T3-L1 adipocytes.

    PubMed

    Yang, Jeong-Yeh; Koo, Bon-Sun; Kang, Mi-Kyung; Rho, Hye-Won; Sohn, Hee-Sook; Jhee, Eun-Chung; Park, Jin-Woo

    2002-11-30

    The present study was undertaken to explore whether retinoids, which are known to have immunomodulatory actions, could attenuate tumor necrosis factor-alpha (TNF)-stimulated inducible nitric oxide synthase (iNOS) expression in 3T3-L1 adipocytes. Adipocytes incubated with TNF induced dose- and time-dependent accumulation of nitrite in the culture medium through the iNOS induction as confirmed by Western blotting. Treatment of cells with TNF in the presence of all-trans-retinoic acid (RA) significantly decreased their ability to produce nitrite and iNOS induction. Both 13-cis- and all- trans-RA-induced suppression was dose-dependent, and all-trans-RA was somewhat potent than 13-cis-RA. The inhibitory effect of RA on TNF-induced iNOS induction was reversible, completely recovered after 2 days, and was exerted through the inhibition of NF-kappaB activation. TNF also suppressed the lipoprotein lipase (LPL) activity of 3T3-L1 adipocytes. RA could not reverse the TNF- induced LPL suppression at RA levels causing near complete inhibition of the TNF-induced NO production. These results indicate that RAs attenuate iNOS expression reversibly in TNF-stimulated 3T3-L1 adipocytes, and that the TNF-induced LPL suppression is not the result of NO overproduction.

  17. Chitosan and blueberry treatment induces arginase activity and inhibits nitric oxide production during acetaminophen-induced hepatotoxicity

    PubMed Central

    Ozcelik, Eda; Uslu, Sema; Burukoglu, Dilek; Musmul, Ahmet

    2014-01-01

    Background: Liver diseases have become a major problem of the worldwide. More than 50% of all cases of liver failure can be attributed to drugs. Among these, acetaminophen is the most common cause. Objective: The aim of this study was to investigate the the hepatoprotective effects of blueberry and chitosan on tissue arginase activity, ornithine and nitric oxide levels during the acetaminophen-induced hepatotoxicity. Materials and Methods: Acetaminophen (250 mg/kg body weight per day), blueberry (60 mg/kg body weight per day) and, chitosan (200 mg/kg body weight per day) were administered to the rats by oral gavage during the experimental period. Results: Blueberry and chitosan significantly decreased liver arginase activity and ornithine levelsand and increased nitric oxide levels. Glutathione levels were remarkably increased by chitosan and blueberry treatments. Conclusion: The results of the present study indicate that blueberry and chitosan effectively protected against the acetaminophen-induced hepatotoxicity. The hepatoprotective effect afforded by blueberry and chitosan can be attributed to its antioxidant and anti-inflammatory activities. PMID:24991095

  18. Nitric oxide mediates Fos expression in the spinal cord induced by mechanical noxious stimulation.

    PubMed

    Lee, J H; Wilcox, G L; Beitz, A J

    1992-10-01

    Immunocytochemical localization of Fos protein was used to analyze the involvement of nitric oxide (NO) in the expression of Fos in the spinal cord, induced by mechanical noxious stimulation (NS). Mechanical NS was applied to the left hindpaw 30 minutes after intrathecal administration of the NO synthase inhibitor, N omega-nitro-L-arginine methyl ester (L-NAME) and the resulting Fos expression in the spinal cord dorsal horn was compared with that obtained in rats exposed only to the mechanical NS. Pretreatment with L-NAME but not its stereoisomer N omega-nitro-D-arginine methyl ester (D-NAME), produced a dose-dependent suppression of Fos expression induced by mechanical noxious stimulation. These results indicate that NO modulates the expression of Fos in the dorsal horn induced by mechanical noxious stimulation and further support the hypothesis that NO is involved in nociceptive events occurring in the spinal cord in response to a peripheral noxious stimulus.

  19. Nitric oxide-induced calcium release via ryanodine receptors regulates neuronal function.

    PubMed

    Kakizawa, Sho; Yamazawa, Toshiko; Chen, Yili; Ito, Akihiro; Murayama, Takashi; Oyamada, Hideto; Kurebayashi, Nagomi; Sato, Osamu; Watanabe, Masahiko; Mori, Nozomu; Oguchi, Katsuji; Sakurai, Takashi; Takeshima, Hiroshi; Saito, Nobuhito; Iino, Masamitsu

    2012-01-18

    Mobilization of intracellular Ca(2+) stores regulates a multitude of cellular functions, but the role of intracellular Ca(2+) release via the ryanodine receptor (RyR) in the brain remains incompletely understood. We found that nitric oxide (NO) directly activates RyRs, which induce Ca(2+) release from intracellular stores of central neurons, and thereby promote prolonged Ca(2+) signalling in the brain. Reversible S-nitrosylation of type 1 RyR (RyR1) triggers this Ca(2+) release. NO-induced Ca(2+) release (NICR) is evoked by type 1 NO synthase-dependent NO production during neural firing, and is essential for cerebellar synaptic plasticity. NO production has also been implicated in pathological conditions including ischaemic brain injury, and our results suggest that NICR is involved in NO-induced neuronal cell death. These findings suggest that NICR via RyR1 plays a regulatory role in the physiological and pathophysiological functions of the brain.

  20. Inducible nitric oxide synthase in rat hepatic lipocytes and the effect of nitric oxide on lipocyte contractility.

    PubMed Central

    Rockey, D C; Chung, J J

    1995-01-01

    In liver injury, perisinusoidal cells known as lipocytes (Ito cells) undergo "activation," acquiring smooth muscle-like features and a contractile phenotype. To assess whether contraction of these cells is regulated by nitric oxide (NO), we examined the production of NO by lipocytes and the effect of NO on lipocyte contractility. Cultured lipocytes were exposed to cytokines and/or LPS. Single agents had little or no effect on the level of inducible NO synthase (iNOS) mRNA. However, interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), or LPS in combination with interferon-gamma (IFN-gamma) stimulated iNOS mRNA, which was present within 4 h after exposure. iNOS mRNA levels were paralleled by changes in nitrite (a metabolic product of NO). Intraperitoneal administration of IFN-gamma, TNF-alpha, and LPS led to rapid induction of iNOS mRNA in lipocytes, confirming in vivo the culture findings. Ligation of the common hepatic bile duct, which induces periportal-based liver injury, stimulated iNOS mRNA in lipocytes. Transforming growth factor-beta 1 decreased IFN-gamma/TNF-alpha--stimulated iNOS mRNA and nitrite. Finally, the effect of NO on lipocyte contractility was examined. In cells incubated with IFN-gamma and TNF-alpha, the contractile response to either serum or endothelin-1 was blocked. Contraction was restored entirely by an inhibitor of NO synthase, NG-monomethylarginine. Furthermore, 8-bromoguanosine 3':5'-cyclic monophosphate and sodium nitroprusside inhibited lipocyte contractility, consistent with the effect of NO induced by cytokines. We conclude that NO is a potent modulator of lipocyte contractility and may regulate this function by autocrine (or intracrine) mechanisms. Moreover, NO may play an important role in liver injury, countering the effect of contractile agonists on lipocytes. Images PMID:7533786

  1. Calcium is involved in nitric oxide- and auxin-induced lateral root formation in rice.

    PubMed

    Chen, Yi Hsuan; Kao, Ching Huei

    2012-01-01

    In the present study, the role of nitric oxide (NO) in the regulation of lateral root (LR) formation in rice was examined. Application of sodium nitroprusside (SNP; a NO donor) and indole-3-butyric acid (IBA; a naturally occurring auxin) to rice seedlings induced LR formation. The effect is specific for NO because the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3- oxide (cPTIO) blocked the action of SNP and IBA. Endogenous NO was detected by the specific fluorescence probe 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate. SNP- and IBA-induced NO fluorescence was specifically suppressed by cPTIO. Nitrate reductase (NR) inhibitor sodium tungstate completely inhibited IBA-induced LR formation and NO fluorescence. However, nitric oxide synthase inhibitor N (G)-nitro-L: -arginine methyl ester hydrochloride slightly reduced IBA-induced LR formation and NO generation. It appears that NO generation that occurs in response to IBA might primarily involve NR activity. Moreover, NO production caused by SNP and IBA was localized in root area corresponding to LR emergence. The effects of Ca(2+) chelators, Ca(2+)-channel inhibitors, and calmodulin antagonists on LR formation induced by SNP and IBA were also examined. All these inhibitors were effective in reducing the action of SNP and IBA. However, Ca(2+) chelators and Ca(2+)-channel inhibitors had no effect on SNP- and IBA-induced NO generation. It is concluded that cytosolic levels of Ca(2+) may regulate SNP and IBA action through calmodulin-dependent mechanism.

  2. Nitric Oxide Mediates Bleomycin-Induced Angiogenesis and Pulmonary Fibrosis via Regulation of VEGF

    PubMed Central

    Iyer, Anand Krishnan V.; Ramesh, Vani; Castro, Carlos A.; Kaushik, Vivek; Kulkarni, Yogesh M.; Wright, Clayton A.; Venkatadri, Rajkumar; Rojanasakul, Yon; Azad, Neelam

    2015-01-01

    Pulmonary fibrosis is a progressive lung disease hallmarked by increased fibroblast proliferation, amplified levels of extracellular matrix deposition and increased angiogenesis. Although dysregulation of angiogenic mediators has been implicated in pulmonary fibrosis, the specific rate-limiting angiogenic markers involved and their role in the progression of pulmonary fibrosis remains unclear. We demonstrate that bleomycin treatment induces angiogenesis, and inhibition of the central angiogenic mediator VEGF using anti-VEGF antibody CBO-P11 significantly attenuates bleomycin-induced pulmonary fibrosis in vivo. Bleomycin-induced nitric oxide (NO) was observed to be the key upstream regulator of VEGF via the PI3k/Akt pathway. VEGF regulated other important angiogenic proteins including PAI-1 and IL-8 in response to bleomycin exposure. Inhibition of NO and VEGF activity significantly mitigated bleomycin-induced angiogenic and fibrogenic responses. NO and VEGF are key mediators of bleomycin-induced pulmonary fibrosis, and could serve as important targets against this debilitating disease. Overall, our data suggests an important role for angiogenic mediators in the pathogenesis of bleomycin-induced pulmonary fibrosis. PMID:25919965

  3. Reversal of hemoglobin-induced vasoconstriction with sustained release of nitric oxide

    PubMed Central

    Han, George; Nacharaju, Parimala; Friedman, Adam J.; Friedman, Joel M.

    2011-01-01

    Erythrocyte free hemoglobin (Hb) induces vasoconstriction due to nitric oxide (NO) scavenging, limiting the NO available for vascular smooth muscle. The central objective of this study was to restore NO bioavailability using long-lived circulating NO-releasing nanoparticles (NO-np) to reverse the vasoconstriction and hypertension induced by polymerized bovine Hb (PBH) NO scavenging. PBH (13 g/dl) was infused in a volume equal to 10% of the animal blood volume. Intravascular NO supplementation was provided with an infusion of NO-np (10 and 20 mg/kg body wt). This study was performed using the hamster window chamber model to concurrently access systemic and microvascular hemodynamics. Infusion of PBH increased blood pressure and induced vasoconstriction. Treatment with 10 and 20 mg/kg NO-np reduced the blood pressure and vasoconstriction induced by PBH. Moreover, the higher dose of NO-np decreased blood pressure and induced vasodilation compared with baseline, respectively. Treatment with NO-np to decrease PBH-induced vasoconstriction increased methemoglobin levels and plasma nitrite and nitrate. In conclusion, NO-np counteracted both systemic hypertension and decreased the vasoconstrictor effects of PBH infusion, improving systemic and microvascular function. Based on the observed physiological properties, NO-np has clear potential as a therapeutic agent to replenish NO in situations where NO production is impaired, insufficient, or consumed, thereby preventing vascular complications. PMID:21057038

  4. Effects of the inducible nitric oxide synthase inhibitor aminoguanidine in two different rat models of schizophrenia.

    PubMed

    Lafioniatis, Anastasios; Orfanidou, Martha A; Papadopoulou, Evangelia S; Pitsikas, Nikolaos

    2016-08-01

    Several lines evidence indicate that the non-competitive N-methyl-d-aspartate (NMDA) receptor antagonist ketamine and the mixed dopamine (DA) D1/D2 receptor agonist apomorphine induce schizophrenia-like symptoms in rodents, including memory impairments and social withdrawal. Nitric oxide (NO) has been proposed to act as an intracellular messenger in the brain and its overproduction is associated with schizophrenia. The current study was designed to investigate the ability of the inducible NO synthase (iNOS) inhibitor aminoguanidine (AG) to counteract schizophrenia-like behavioural deficits produced by ketamine and apomorphine in rats. The efficacy of AG to antagonize extinction of recognition memory, ketamine and apomorphine-induced recognition memory impairments was tested utilizing the novel object recognition task (NORT). Further, the efficacy of AG to attenuate ketamine-induced social withdrawal was examined in the social interaction test. AG (25 and 50mg/kg) antagonized extinction of recognition memory and reversed ketamine (3mg/kg) and apomorphine (1mg/kg)-induced recognition memory deficits. In contrast, AG (50 and 100mg/kg) did not counteract the ketamine (8mg/kg)-induced social isolation. The present data show that the iNOS inhibitor AG counteracted extinction of recognition memory and reversed recognition memory deficits produced by dysfunction of the glutamatergic and the dopaminergic (DAergic) system in rats. Therefore, AG may be efficacious in attenuating memory impairments often observed in schizophrenia patients.

  5. Nitric oxide mediates bleomycin-induced angiogenesis and pulmonary fibrosis via regulation of VEGF.

    PubMed

    Iyer, Anand Krishnan V; Ramesh, Vani; Castro, Carlos A; Kaushik, Vivek; Kulkarni, Yogesh M; Wright, Clayton A; Venkatadri, Rajkumar; Rojanasakul, Yon; Azad, Neelam

    2015-11-01

    Pulmonary fibrosis is a progressive lung disease hallmarked by increased fibroblast proliferation, amplified levels of extracellular matrix deposition and increased angiogenesis. Although dysregulation of angiogenic mediators has been implicated in pulmonary fibrosis, the specific rate-limiting angiogenic markers involved and their role in the progression of pulmonary fibrosis remains unclear. We demonstrate that bleomycin treatment induces angiogenesis, and inhibition of the central angiogenic mediator VEGF using anti-VEGF antibody CBO-P11 significantly attenuates bleomycin-induced pulmonary fibrosis in vivo. Bleomycin-induced nitric oxide (NO) was observed to be the key upstream regulator of VEGF via the PI3k/Akt pathway. VEGF regulated other important angiogenic proteins including PAI-1 and IL-8 in response to bleomycin exposure. Inhibition of NO and VEGF activity significantly mitigated bleomycin-induced angiogenic and fibrogenic responses. NO and VEGF are key mediators of bleomycin-induced pulmonary fibrosis, and could serve as important targets against this debilitating disease. Overall, our data suggests an important role for angiogenic mediators in the pathogenesis of bleomycin-induced pulmonary fibrosis.

  6. Photodynamic therapy-induced nitric oxide production in neuronal and glial cells

    NASA Astrophysics Data System (ADS)

    Kovaleva, Vera D.; Uzdensky, Anatoly B.

    2016-10-01

    Nitric oxide (NO) has been recently demonstrated to enhance apoptosis of glial cells induced by photodynamic therapy (PDT), but to protect glial cells from PDT-induced necrosis in the crayfish stretch receptor, a simple neuroglial preparation that consists of a single mechanosensory neuron enveloped by satellite glial cells. We used the NO-sensitive fluorescent probe 4,5-diaminofluorescein diacetate to study the distribution and dynamics of PDT-induced NO production in the mechanosensory neuron and surrounding glial cells. The NO production in the glial envelope was higher than in the neuronal soma axon and dendrites both in control and in experimental conditions. In dark NO generator, DEA NONOate or NO synthase substrate L-arginine hydrochloride significantly increased the NO level in glial cells, whereas NO scavenger 2-Phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO) or inhibitors of NO synthase L-NG-nitro arginine methyl ester and Nω-nitro-L-arginine decreased it. PDT induced the transient increase in NO production with a maximum at 4 to 7 min after the irradiation start followed by its inhibition at 10 to 40 min. We suggested that PDT stimulated neuronal rather than inducible NO synthase isoform in glial cells, and the produced NO could mediate PDT-induced apoptosis.

  7. TRPV1 and TRPA1 mediate peripheral nitric oxide-induced nociception in mice.

    PubMed

    Miyamoto, Takashi; Dubin, Adrienne E; Petrus, Matt J; Patapoutian, Ardem

    2009-10-29

    Nitric oxide (NO) can induce acute pain in humans and plays an important role in pain sensitization caused by inflammation and injury in animal models. There is evidence that NO acts both in the central nervous system via a cyclic GMP pathway and in the periphery on sensory neurons through unknown mechanisms. It has recently been suggested that TRPV1 and TRPA1, two polymodal ion channels that sense noxious stimuli impinging on peripheral nociceptors, are activated by NO in heterologous systems. Here, we investigate the relevance of this activation. We demonstrate that NO donors directly activate TRPV1 and TRPA1 in isolated inside-out patch recordings. Cultured primary sensory neurons display both TRPV1- and TRPA1-dependent responses to NO donors. BH4, an essential co-factor for NO production, causes activation of a subset of DRG neurons as assayed by calcium imaging, and this activation is at least partly dependent on nitric oxide synthase activity. We show that BH4-induced calcium influx is ablated in DRG neurons from TRPA1/TRPV1 double knockout mice, suggesting that production of endogenous levels of NO can activate these ion channels. In behavioral assays, peripheral NO-induced nociception is compromised when TRPV1 and TRPA1 are both ablated. These results provide genetic evidence that the peripheral nociceptive action of NO is mediated by both TRPV1 and TRPA1.

  8. The contradictory effects of nitric oxide in caerulein-induced acute pancreatitis in rats.

    PubMed

    Ozturk, Feral; Gul, Mehmet; Esrefoglu, Mukaddes; Ates, Burhan

    2008-04-01

    This study was planned to observe the effects of nitric oxide synthesis on the antioxidative defense enzymes and pancreatic tissue histology in caerulein-induced acute pancreatitis. Acute pancreatitis was induced by intraperitoneal injections of 50 microg/kg caerulein, L-arginine used for NO induction and N(omega)-nitro-L-arginine methyl ester (L-NAME) used for NO inhibition. In the caerulein group acinar cell degeneration, interstitial inflammation, oedema and haemorrhage were detected. Pancreatic damage scores were decreased with both NO induction and inhibition (p<0.05). MDA, GSH-Px, CAT, GSH and SOD activities were significantly changed in the caerulein group and indicated increased oxidative stress. Both NO induction and inhibition decreased this oxidative stress. It is concluded that both nitric oxide induction and inhibition ameliorated caerulein-induced acute pancreatitis. The findings indicate that a certain amount of NO production has beneficial effects in experimental acute pancreatitis, but uncontrolled over-production of NO may be detrimental.

  9. TRPV1 and TRPA1 Mediate Peripheral Nitric Oxide-Induced Nociception in Mice

    PubMed Central

    Miyamoto, Takashi; Dubin, Adrienne E.; Petrus, Matt J.; Patapoutian, Ardem

    2009-01-01

    Nitric oxide (NO) can induce acute pain in humans and plays an important role in pain sensitization caused by inflammation and injury in animal models. There is evidence that NO acts both in the central nervous system via a cyclic GMP pathway and in the periphery on sensory neurons through unknown mechanisms. It has recently been suggested that TRPV1 and TRPA1, two polymodal ion channels that sense noxious stimuli impinging on peripheral nociceptors, are activated by NO in heterologous systems. Here, we investigate the relevance of this activation. We demonstrate that NO donors directly activate TRPV1 and TRPA1 in isolated inside-out patch recordings. Cultured primary sensory neurons display both TRPV1- and TRPA1-dependent responses to NO donors. BH4, an essential co-factor for NO production, causes activation of a subset of DRG neurons as assayed by calcium imaging, and this activation is at least partly dependent on nitric oxide synthase activity. We show that BH4-induced calcium influx is ablated in DRG neurons from TRPA1/TRPV1 double knockout mice, suggesting that production of endogenous levels of NO can activate these ion channels. In behavioral assays, peripheral NO-induced nociception is compromised when TRPV1 and TRPA1 are both ablated. These results provide genetic evidence that the peripheral nociceptive action of NO is mediated by both TRPV1 and TRPA1. PMID:19893614

  10. Neuronal nitric oxide synthase contributes to pentylenetetrazole-kindling-induced hippocampal neurogenesis.

    PubMed

    Zhu, Xinjian; Dong, Jingde; Shen, Kai; Bai, Ying; Chao, Jie; Yao, Honghong

    2016-03-01

    Neuronal nitric oxide synthase (nNOS), the major nitric oxide synthase isoform in the mammalian brain, is implicated in the pathophysiology of several neurological conditions, including epilepsy. Neurogenesis in hippocampal dentate gyrus (DG) persists throughout life in the adult brain. Alterations in this process occur in many neurological diseases, including epilepsy. Few studies, however, have addressed the role of nNOS in hippocampal DG neurogenesis in epileptic brain. The present study, therefore, investigated the role of nNOS in pentylenetetrazole (PTZ)-kindling-induced neurogenesis in hippocampal DG. Our results showed that nNOS expression and enzymatic activity were significantly increased in the hippocampus of PTZ-kindled mice. Meanwhile, these PTZ-kindled mice were characterized by significant enhancement of new born cells proliferation and survival in hippocampal DG, and these survived cells are co-labeled with NeuN and GFAP. Selective inhibition of nNOS by 7-NI, however, suppressed PTZ-kindling-induced hippocampal DG new born cells proliferation and survival, suggesting that nNOS contributes to PTZ-kindling-induced hippocampal neurogenesis.

  11. Enhancement of fracture healing in the rat, modulated by compounds that stimulate inducible nitric oxide synthase

    PubMed Central

    Rajfer, R. A.; Kilic, A.; Neviaser, A. S.; Schulte, L. M.; Hlaing, S. M.; Landeros, J.; Ferrini, M. G.; Ebramzadeh, E.

    2017-01-01

    Objectives We investigated the effects on fracture healing of two up-regulators of inducible nitric oxide synthase (iNOS) in a rat model of an open femoral osteotomy: tadalafil, a phosphodiesterase inhibitor, and the recently reported nutraceutical, COMB-4 (consisting of L-citrulline, Paullinia cupana, ginger and muira puama), given orally for either 14 or 42 days. Materials and Methods Unilateral femoral osteotomies were created in 58 male rats and fixed with an intramedullary compression nail. Rats were treated daily either with vehicle, tadalafil or COMB-4. Biomechanical testing of the healed fracture was performed on day 42. The volume, mineral content and bone density of the callus were measured by quantitative CT on days 14 and 42. Expression of iNOS was measured by immunohistochemistry. Results When compared with the control group, the COMB-4 group exhibited 46% higher maximum strength (t-test, p = 0.029) and 92% higher stiffness (t-test, p = 0.023), but no significant changes were observed in the tadalafil group. At days 14 and 42, there was no significant difference between the three groups with respect to callus volume, mineral content and bone density. Expression of iNOS at day 14 was significantly higher in the COMB-4 group which, as expected, had returned to baseline levels at day 42. Conclusion This study demonstrates an enhancement in fracture healing by an oral natural product known to augment iNOS expression. Cite this article: R. A. Rajfer, A. Kilic, A. S. Neviaser, L. M. Schulte, S. M. Hlaing, J. Landeros, M. G. Ferrini, E. Ebramzadeh, S-H. Park. Enhancement of fracture healing in the rat, modulated by compounds that stimulate inducible nitric oxide synthase: Acceleration of fracture healing via inducible nitric oxide synthase. Bone Joint Res 2017:6:–97. DOI: 10.1302/2046-3758.62.BJR-2016-0164.R2. PMID:28188129

  12. 6-Hydroxydopamine and lipopolysaccharides induced DNA damage in astrocytes: involvement of nitric oxide and mitochondria.

    PubMed

    Gupta, Sonam; Goswami, Poonam; Biswas, Joyshree; Joshi, Neeraj; Sharma, Sharad; Nath, C; Singh, Sarika

    2015-01-15

    The present study was conducted to investigate the effect of the neurotoxins 6-hydroxydopamine and lipopolysaccharide on astrocytes. Rat astrocyte C6 cells were treated with different concentration of 6-hydroxydopamine (6-OHDA)/lipopolysaccharides (LPS) for 24 h. Both neurotoxins significantly decreased the viability of astrocytes, augmented the expression of inducible nitric oxide synthase (iNOS) and the astrocyte marker--glial fibrillar acidic protein. A significantly decreased mitochondrial dehydrogenase activity, mitochondrial membrane potential, augmented reactive oxygen species (ROS) level, caspase-3 mRNA level, chromatin condensation and DNA damage was observed in 6-OHDA/LPS treated astroglial cells. 6-OHDA/LPS treatment also caused the significantly increased expression of iNOS and nitrite level. Findings showed that 6-OHDA/LPS treatment caused mitochondrial dysfunction mediated death of astrocytes, which significantly involve the nitric oxide. Since we have observed significantly increased level of iNOS along with mitochondrial impairment and apoptotic cell death in astrocytes, therefore to validate the role of iNOS, the cells were co-treated with iNOS inhibitor aminoguanidine (AG, 100 μM). Co-treatment of AG significantly attenuated the 6-OHDA/LPS induced cell death, mitochondrial activity, augmented ROS level, chromatin condensation and DNA damage. GFAP and caspase-3 expression were also inhibited with co-treatment of AG, although the extent of inhibition was different in both experimental sets. In conclusion, the findings showed that iNOS mediated increased level of nitric oxide acts as a key regulatory molecule in 6-OHDA/LPS induced mitochondrial dysfunction, DNA damage and apoptotic death of astrocytes.

  13. Differential roles of nitric oxide synthases in regulation of ultraviolet B light-induced apoptosis.

    PubMed

    Liu, Wei; Wu, Shiyong

    2010-11-01

    Ultraviolet B light (UVB) activates nitric oxide synthase(s) (NOSs) and nitric oxide (NO()) production, which plays a role in regulation of apoptosis. However, the role of NO() in UVB-induced apoptosis remains controversial. In this study, we analyzed expression and activation of constitutive NOSs (cNOSs) and their roles in UV-induced apoptosis of HaCaT keratinocytes. Our data showed that the expression of neuronal NOS (nNOS) was increased while endothelial NOS (eNOS) was uncoupled in the early phase (0-6 h) post-UVB. The expression of both cNOSs peaked at 12h post-UVB and NO() was transiently elevated with 30 min and then steadily rose from 6 to 18 h post-UVB. The expression of iNOS was detected at 6h post-UVB and then sturdily increased. Inhibition of cNOSs with L-NAME reduced the inducibility of NO(*) in the early and late phases of irradiation. Along with the eNOS uncoupling, an increased level of peroxynitrite (ONOO(-)) was detected in the early phase, but not in the late phase post-UVB. Inhibition of cNOSs reduced the production of ONOO(-) in the early time, but led to an increase of ONOO(-) in the late time after UVB-irradiation. The results indicate that cNOSs regulate NO()/ONOO(-) imbalance after UVB-irradiation. Our data suggested that the activation of cNOSs in the early phase post-UVB leads to NO()/ONOO(-) imbalance and promotes apoptosis via a caspase 3-independent pathway. The elevation of NO() in the late phase of UVB-irradiation is mainly produced by inducible NOS (iNOS). However, cNOSs also contribute to the NO() production and to maintain a higher NO()/ONOO(-) ratio, which reduces caspase 3 activity and protects cells from UVB-induced apoptosis.

  14. Radiation-induced nitric oxide mitigates tumor hypoxia and radioresistance in a murine SCCVII tumor model

    SciTech Connect

    Nagane, Masaki; Yasui, Hironobu; Yamamori, Tohru; Zhao, Songji; Kuge, Yuji; Tamaki, Nagara; Kameya, Hiromi; Nakamura, Hideo; Fujii, Hirotada; Inanami, Osamu

    2013-08-02

    Highlights: •IR-induced NO increased tissue perfusion and pO{sub 2}. •IR increased NO production in tumors without changes in the mRNA and protein levels of NOS isoforms. •NOS activity assay showed that IR upregulated eNOS activity in tumors. •IR-induced NO decreased tumor hypoxia and altered tumor radiosensitivity. -- Abstract: Tumor hypoxia, which occurs mainly as a result of inadequate tissue perfusion in solid tumors, is a well-known challenge for successful radiotherapy. Recent evidence suggests that ionizing radiation (IR) upregulates nitric oxide (NO) production and that IR-induced NO has the potential to increase intratumoral circulation. However, the kinetics of NO production and the responsible isoforms for NO synthase in tumors exposed to IR remain unclear. In this study, we aimed to elucidate the mechanism by which IR stimulates NO production in tumors and the effect of IR-induced NO on tumor radiosensitivity. Hoechst33342 perfusion assay and electron spin resonance oxymetry showed that IR increased tissue perfusion and pO{sub 2} in tumor tissue. Immunohistochemical analysis using two different hypoxic probes showed that IR decreased hypoxic regions in tumors; treatment with a nitric oxide synthase (NOS) inhibitor, L-NAME, abrogated the effects of IR. Moreover, IR increased endothelial NOS (eNOS) activity without affecting its mRNA or protein expression levels in SCCVII-transplanted tumors. Tumor growth delay assay showed that L-NAME decreased the anti-tumor effect of fractionated radiation (10 Gy × 2). These results suggested that IR increased eNOS activity and subsequent tissue perfusion in tumors. Increases in intratumoral circulation simultaneously decreased tumor hypoxia. As a result, IR-induced NO increased tumor radiosensitivity. Our study provides a new insight into the NO-dependent mechanism for efficient fractionated radiotherapy.

  15. Lysophosphatidic acid induces vasodilation mediated by LPA1 receptors, phospholipase C, and endothelial nitric oxide synthase

    PubMed Central

    Ruisanchez, Éva; Dancs, Péter; Kerék, Margit; Németh, Tamás; Faragó, Bernadett; Balogh, Andrea; Patil, Renukadevi; Jennings, Brett L.; Liliom, Károly; Malik, Kafait U.; Smrcka, Alan V.; Tigyi, Gabor; Benyó, Zoltán

    2014-01-01

    Lysophosphatidic acid (LPA) has been implicated as a mediator of several cardiovascular functions, but its potential involvement in the control of vascular tone is obscure. Here, we show that both LPA (18:1) and VPC31143 (a synthetic agonist of LPA1–3 receptors) relax intact mouse thoracic aorta with similar Emax values (53.9 and 51.9% of phenylephrine-induced precontraction), although the EC50 of LPA- and VPC31143-induced vasorelaxations were different (400 vs. 15 nM, respectively). Mechanical removal of the endothelium or genetic deletion of endothelial nitric oxide synthase (eNOS) not only diminished vasorelaxation by LPA or VPC31143 but converted it to vasoconstriction. Freshly isolated mouse aortic endothelial cells expressed LPA1, LPA2, LPA4 and LPA5 transcripts. The LPA1,3 antagonist Ki16425, the LPA1 antagonist AM095, and the genetic deletion of LPA1, but not that of LPA2, abolished LPA-induced vasorelaxation. Inhibition of the phosphoinositide 3 kinase–protein kinase B/Akt pathway by wortmannin or MK-2206 failed to influence the effect of LPA. However, pharmacological inhibition of phospholipase C (PLC) by U73122 or edelfosine, but not genetic deletion of PLCε, abolished LPA-induced vasorelaxation and indicated that a PLC enzyme, other than PLCε, mediates the response. In summary, the present study identifies LPA as an endothelium-dependent vasodilator substance acting via LPA1, PLC, and eNOS.—Ruisanchez, É., Dancs, P., Kerék, M., Németh, T., Faragó, B., Balogh, A., Patil, R., Jennings, B. L., Liliom, K., Malik, K. U., Smrcka, A. V., Tigyi, G., Benyó, Z. Lysophosphatidic acid induces vasodilation mediated by LPA1 receptors, phospholipase C, and endothelial nitric oxide synthase. PMID:24249637

  16. Morphine-induced changes in cerebral and cerebellar nitric oxide synthase activity.

    PubMed

    Leza, J C; Lizasoain, I; San-Martín-Clark, O; Lorenzo, P

    1995-10-04

    The effect of acute and chronic morphine treatment on nitric oxide (NO) synthase activity (determined by the rate of conversion of [14C]arginine into [14C]citrulline) on mouse brain was studied. Acute morphine treatment induced an increased in Ca2+ -dependent NO synthase in cerebellum. This effect was blocked by coadministration with naloxone. Chronic morphine treatment (by s.c. pellet) also produced an increase in cerebellar NO synthase, with a maximum on the second day of implantation. No significant changes were found in frontal cortex and forebrain during acute or chronic morphine treatment. The relationship between opiate effects and the L-arginine: NO pathway is discussed.

  17. Metal release in metallothioneins induced by nitric oxide: X-ray absorption spectroscopy study.

    PubMed

    Casero, Elena; Martín-Gago, José A; Pariente, Félix; Lorenzo, Encarnación

    2004-12-01

    Metallothioneins (MTs) are low molecular weight proteins that include metal ions in thiolate clusters. The capability of metallothioneins to bind different metals has suggested their use as biosensors for different elements. We study here the interaction of nitric oxide with rat liver MTs by using in situ X-ray absorption spectroscopy techniques. We univocally show that the presence of NO induces the release of Zn atoms from the MT structure to the solution. Zn ions transform in the presence of NO from a tetrahedral four-fold coordinated environment in the MT into a regular octahedral six-fold coordinated state, with interatomic distances compatible with those of Zn solvated in water.

  18. Malarial anaemia and nitric oxide induced megaloblastic anaemia: a review on the causes of malarial anaemia.

    PubMed

    Pradhan, Prasanna

    2009-06-01

    Direct destruction and ineffective erythropoesis does not adequately explain the cause of anaemia in malaria. It is possible that there are more other mechanisms involved besides the causes described till date in malarial anaemia. The effect of NO on erythropoesis and a major haematological abnormality (microcytic/normocytic/megaloblastic picture) can significantly be observed on repeated exposure. In addition, NO can inhibit the enzyme methionine synthase so functional vit B12 deficiency state may occur which can lead to megaloblastic anaemia. This review will focus on causation of malarial anaemia and nitric oxide induced megaloblastic anaemia.

  19. Proliferation of macrophages due to the inhibition of inducible nitric oxide synthesis by oxidized low-density lipoproteins

    PubMed Central

    Brunner, Monika; Gruber, Miriam; Schmid, Diethart; Baran, Halina; Moeslinger, Thomas

    2015-01-01

    Oxidized low-density lipoprotein (ox-LDL) is assumed to be a major causal agent in hypercholesteraemia-induced atherosclerosis. Because the proliferation of lipid-loaden macrophages within atherosclerotic lesions has been described, we investigated the dependence of macrophage proliferation on the inhibition of inducible nitric oxide synthase (iNOS) by hypochlorite oxidized LDL. Ox-LDL induces a dose dependent inhibition of inducible nitric oxide synthesis in lipopolysaccharide-interferon stimulated mouse macrophages (J774.A1) with concomitant macrophage proliferation as assayed by cell counting, tritiated-thymidine incorporation and measurement of cell protein. Native LDL did not influence macrophage proliferation and inducible nitric oxide synthesis. iNOS protein and mRNA was reduced by HOCl-oxidized LDL (0-40 µg/ml) as revealed by immunoblotting and competitive semiquantitative PCR. Macrophage proliferation was increased by the addition of the iNOS inhibitor L-NAME. The addition of ox-LDL to L-NAME containing incubations induced no further statistically significant increase in cell number. Nitric oxide donors decreased ox-LDL induced macrophage proliferation and nitric oxide scavengers restored macrophage proliferation to the initial values achieved by ox-LDL. The decrease of cytosolic DNA fragments in stimulated macrophages incubated with ox-LDL demonstrates that the proliferative actions of ox-LDL are associated with a decrease of NO-induced apoptosis. Our data show that inhibition of iNOS dependent nitric oxide production caused by hypochlorite oxidized LDL enhances macrophage proliferation. This might be a key event in the pathogenesis of atherosclerotic lesions. PMID:26600745

  20. Nitric oxide production occurs after cytosolic alkalinization during stomatal closure induced by abscisic acid.

    PubMed

    Gonugunta, Vijay K; Srivastava, Nupur; Puli, Mallikarjuna R; Raghavendra, Agepati S

    2008-11-01

    Abscisic acid (ABA) raised the cytosolic pH and nitric oxide (NO) levels in guard cells while inducing stomatal closure in epidermis of Pisum sativum. Butyrate (a weak acid) reduced the cytosolic pH/NO production and prevented stomatal closure by ABA. Methylamine (a weak base) enhanced the cytosolic alkalinization and aggravated stomatal closure by ABA. The rise in guard cell pH because of ABA became noticeable after 6 min and peaked at 12 min, while NO production started at 9 min and peaked at 18 min. These results suggested that NO production was downstream of the rise in cytosolic pH. The ABA-induced increase in NO of guard cells and stomatal closure was prevented by 2-phenyl-4,4,5,5-tetramethyl imidazoline-1-oxyl 3-oxide (cPTIO, a NO scavenger) and partially by N-nitro-L-Arg-methyl ester (L-NAME, an inhibitor of NO synthase). In contrast, cPTIO or L-NAME had only a marginal effect on the pH rise induced by ABA. Ethylene glycol tetraacetic acid (EGTA, a calcium chelator) prevented ABA-induced stomatal closure while restricting cytosolic pH rise and NO production. We suggest that during ABA-induced stomatal closure, a rise in cytosolic pH is necessary for NO production. Calcium may act upstream of cytosolic alkalinization and NO production, besides its known function as a downstream component.

  1. Guard cell hydrogen peroxide and nitric oxide mediate elevated CO2 -induced stomatal movement in tomato.

    PubMed

    Shi, Kai; Li, Xin; Zhang, Huan; Zhang, Guanqun; Liu, Yaru; Zhou, Yanhong; Xia, Xiaojian; Chen, Zhixiang; Yu, Jingquan

    2015-10-01

    Climate change as a consequence of increasing atmospheric CO2 influences plant photosynthesis and transpiration. Although the involvement of stomata in plant responses to elevated CO2 has been well established, the underlying mechanism of elevated CO2 -induced stomatal movement remains largely unknown. We used diverse techniques, including laser scanning confocal microscopy, transmission electron microscopy, biochemical methodologies and gene silencing to investigate the signaling pathway for elevated CO2 -induced stomatal movement in tomato (Solanum lycopersicum). Elevated CO2 -induced stomatal closure was dependent on the production of RESPIRATORY BURST OXIDASE 1 (RBOH1)-mediated hydrogen peroxide (H2 O2 ) and NITRATE REDUCTASE (NR)-mediated nitric oxide (NO) in guard cells in an abscisic acid (ABA)-independent manner. Silencing of OPEN STOMATA 1 (OST1) compromised the elevated CO2 -induced accumulation of H2 O2 and NO, upregulation of SLOW ANION CHANNEL ASSOCIATED 1 (SLAC1) gene expression and reduction of stomatal aperture, whereas silencing of RBOH1 or NR had no effects on the expression of OST1. Our results demonstrate that as critical signaling molecules, RBOH1-dependent H2 O2 and NR-dependent NO act downstream of OST1 that regulate SLAC1 expression and elevated CO2 -induced stomatal movement. This information is crucial to deepen the understanding of CO2 signaling pathway in guard cells.

  2. Nitric oxide reverses prednisolone-induced inactivation of muscle satellite cells.

    PubMed

    Betters, Jenna L; Long, Jodi H D; Howe, Kathleen S; Braith, Randy W; Soltow, Quinlyn A; Lira, Vitor A; Criswell, David S

    2008-02-01

    Long-term corticosteroid therapy causes myopathy and can inhibit regeneration of skeletal muscle. Therefore, we hypothesized that corticosteroid exposure reduces satellite cell activity in skeletal myofibers. Male Swiss-Webster mice were injected daily for 8 weeks with prednisolone (GC) or vehicle (control). Single myofibers were isolated from the gastrocnemius, centrifuged to mechanically activate satellite cells, and maintained in culture for 48 h. Both constitutive nitric oxide synthase (NOS) isoforms were reduced in muscle by GC treatment (nNOS: -30%, eNOS: -34%). Fewer myogenic (myoD+) cells emanated from GC myofibers compared to control (-61%, P < 0.05). Supplementation of culture media with the nitric oxide donor, diethylenetriamine NONOate (DETA-NO; 5-50 microM), caused a dose-dependent increase in the number of myoD+ cells arising from both control and GC myofibers (P < 0.05), and 10 and 50 microM DETA-NO eliminated the GC-induced deficit in myogenic cells (P > 0.05). Therefore, supplementation of GC myofibers with DETA-NO restores satellite cell activity to control levels. Nitric oxide production could be an important therapeutic target for the prevention of corticosteroid myopathy.

  3. Histamine receptor type coupled to nitric oxide-induced relaxation of guinea-pig nasal mucosa.

    PubMed

    Bockman, C S; Zeng, W

    2002-01-01

    1 The aim of this study was to characterize the histamine receptor type mediating relaxation of the vascular bed of the nasal mucosa from the guinea-pig, and to determine the role of cyclo-oxygenase products and nitric oxide in this relaxant response to histamine. These studies were performed in isolated nasal mucosae examined in vitro to obtain potencies of histamine receptor-type selective agonists in causing vasorelaxation and to determine affinities of histamine receptor antagonists for inhibiting histamine-induced relaxation. 2 After contraction of nasal mucosae with noradrenaline, histamine caused a maximal relaxation response that was 75 +/- 6% of the contraction caused by noradrenaline with a mean EC50 value of 4.3 +/- 0.5 microM. Neither dimaprit (H2-receptor selective) nor R-alpha-methylhistamine (H3-receptor selective) caused significant relaxation of nasal mucosae. In contrast, betahistine (H1-receptor selective) caused an 81 +/- 7% relaxation of noradrenaline-induced tone with an EC50 value of 15 +/- 1 microM. 3 pA2 experiments were performed to obtain KB values of chlorpheniramine (H1-receptor selective) and diphenhydramine (H1-receptor selective) for blocking histamine-stimulated relaxation of nasal mucosae. KB values for chlorpheniramine (0.87 nM) and diphenhydramine (7.4 nM) were consistent with their interaction at the H1-receptor type. Additionally, neither 10 microM cimetidine (H2-receptor selective) nor 1 microM thioperamide (H3-receptor selective) had any effect on the relaxation curve for histamine. 4 In the presence of 10 microM indomethacin (cyclo-oxygenase inhibitor), histamine caused a maximal relaxation response of 73 +/- 5% of the noradrenaline-induced tone with an EC50 value of 2.9 +/- 0.2 microM, which was not different from control values (EC50 = 5.0 +/- 0.4 microM; maximal relaxation = 71 +/- 6%). In contrast, 200 microM NG-nitro-L-arginine (nitric oxide synthase inhibitor) completely inhibited histamine-induced relaxation of nasal

  4. The attenuation of hemorrhage-induced liver injury by exogenous nitric oxide, L-arginine, and inhibition of inducible nitric oxide synthase.

    PubMed

    Anaya-Prado, Roberto; Toledo-Pereyra, Luis H; Guo, R F; Reuben, J; Ward, Peter A; Walsh, J

    2003-01-01

    We investigated the role of nitric oxide (NO) in its ability to reduce liver injury in an animal model of hemorrhagic shock (HS). Ninety-six Sprague-Dawley rats weighing 250 to 300 g were divided in 6 groups (n = 16 per group) that included treatment at the beginning of resuscitation with normal saline (groups 1, 3) sodium nitroprusside (NP) (0.5 mg/kg) (groups 2, 4) L-arginine (300 mg/kg) (group 5), and L-N6-(1-iminoethyl) lysine (L-NIL, 40 mg/kg) (group 6). The experimental model of HS consisted of the withdrawal of 3 mL blood per 100 g in a 15-min period, tail amputation (75%), and drug administration at 30 min. This was followed by fluid resuscitation (FR) with lactated Ringer's (LR) solution to reach a mean arterial pressure (MAP) of 40 mm Hg, then a hospital phase of 60 min with hemostasis and FR with LR solution to reach a MAP of 70 mm Hg with a 3-day observation phase. NP, L-Arginine, and L-NIL significantly reduced fluid requirements for resuscitation (p =.0001) as well as significantly increased MAP after resuscitation from hemorrhage. We also observed an improved statistically significant difference (p =.001) in tests demonstrating less hepatic injury and histology damage. The mRNA expression of cytokines in the liver (interleukin [IL]-1alpha, IL-beta1, tumor necrosis factor [TNF]beta, IL-3, IL-4, IL-5, IL-6, IL-10, TNFalpha, IL-2, interferon [IFN]gamma) was reduced by NP treatment, L-arginine, and L-NIL. These data suggest that excess NO mediates hemorrhage-induced liver injury and that the suppression of inducible nitric oxide synthase (iNOS)-generated NO bioavailability with the NO donor sodium nitroprusside may reduce the pathophysiologic consequences of severe hemorrhage. This effect could be possibly related to the scavenging of to superoxide radicals (O2-) or the blockade of the deleterious effects of TNF and other inflammatory cytokines. The protective action noted with L-arginine cannot be fully explained within the context of this article

  5. Expression of inducible nitric oxide synthase (iNOS) in microglia of the developing quail retina.

    PubMed

    Sierra, Ana; Navascués, Julio; Cuadros, Miguel A; Calvente, Ruth; Martín-Oliva, David; Ferrer-Martín, Rosa M; Martín-Estebané, María; Carrasco, María-Carmen; Marín-Teva, José L

    2014-01-01

    Inducible nitric oxide synthase (iNOS), which produce large amounts of nitric oxide (NO), is induced in macrophages and microglia in response to inflammatory mediators such as LPS and cytokines. Although iNOS is mainly expressed by microglia that become activated in different pathological and experimental situations, it was recently reported that undifferentiated amoeboid microglia can also express iNOS during normal development. The aim of this study was to investigate the pattern of iNOS expression in microglial cells during normal development and after their activation with LPS by using the quail retina as model. iNOS expression was analyzed by iNOS immunolabeling, western-blot, and RT-PCR. NO production was determined by using DAR-4M AM, a reliable fluorescent indicator of subcellular NO production by iNOS. Embryonic, postnatal, and adult in situ quail retinas were used to analyze the pattern of iNOS expression in microglial cells during normal development. iNOS expression and NO production in LPS-treated microglial cells were investigated by an in vitro approach based on organotypic cultures of E8 retinas, in which microglial cell behavior is similar to that of the in situ retina, as previously demonstrated in our laboratory. We show here that amoeboid microglia in the quail retina express iNOS during normal development. This expression is stronger in microglial cells migrating tangentially in the vitreal part of the retina and is downregulated, albeit maintained, when microglia differentiate and become ramified. LPS treatment of retina explants also induces changes in the morphology of amoeboid microglia compatible with their activation, increasing their lysosomal compartment and upregulating iNOS expression with a concomitant production of NO. Taken together, our findings demonstrate that immature microglial cells express iNOS during normal development, suggesting a certain degree of activation. Furthermore, LPS treatment induces overactivation of amoeboid

  6. Interleukin-13 inhibits inducible nitric oxide synthase expression in human mesangial cells.

    PubMed Central

    Saura, M; Martínez-Dalmau, R; Minty, A; Pérez-Sala, D; Lamas, S

    1996-01-01

    The synthesis of nitric oxide in inflammatory situations requires the expression of an inducible isoform of nitric oxide synthase (iNOS). Human mesangial cells (HMC) express an iNOS enzyme after exposure to multiple co-stimuli. In this study we have observed that while tumour necrosis factor-alpha, interleukin (IL)-1 beta, interferon-gamma and bacterial lipopolysaccharide (LPS) were unable to significantly induce NO synthesis when used alone, they induced an evident stimulation of NO synthesis when used in various combinations. A mixture of the three cytokines (CM) and LPS resulted in a 10-15-fold stimulation of NO synthesis over control values which started to be significant after 16 h. The addition of IL-13, a cytokine with anti-inflammatory properties, inhibited CM/LPS-induced NO synthesis in a concentration-dependent manner. A marked inhibitory effect (60-65%) could be observed when HMC were treated with IL-13 (10 ng/ml) 24 h before, at the same time as, or even 4 h after the addition of CM/LPS. This inhibitory effect was still significant (25%) when IL-13 was added 16 h after CM/LPS. Northern analysis showed that IL-13-mediated iNOS inhibition was closely correlated with the suppression of iNOS mRNA expression. These results identify IL-13 as a powerful regulatory tool for the inhibition of NO synthesis in human cells, a property which may be pathophysiologically relevant in NO-related inflammatory processes. PMID:8573104

  7. Pharmacological characterization of KLYP961, a dual inhibitor of inducible and neuronal nitric-oxide synthases.

    PubMed

    Symons, Kent T; Nguyen, Phan M; Massari, Mark E; Anzola, John V; Staszewski, Lena M; Wang, Li; Yazdani, Nahid; Dorow, Steven; Muhammad, Jerry; Sablad, Marciano; Rozenkrants, Natasha; Bonefous, Celine; Payne, Joseph E; Rix, Peter J; Shiau, Andrew K; Noble, Stewart A; Smith, Nicholas D; Hassig, Christian A; Zhang, Yan; Rao, Tadimeti S

    2011-02-01

    Nitric oxide (NO) derived from neuronal nitric-oxide synthase (nNOS) and inducible nitric-oxide synthase (iNOS) plays a key role in various pain and inflammatory states. KLYP961 (4-((2-cyclobutyl-1H-imidazo[4,5-b]pyrazin-1-yl)methyl)-7,8-difluoroquinolin-2(1H)-one) inhibits the dimerization, and hence the enzymatic activity of human, primate, and murine iNOS and nNOS (IC(50) values 50-400 nM), with marked selectivity against endothelial nitric-oxide synthase (IC(50) >15,000 nM). It has ideal drug like-properties, including excellent rodent and primate pharmacokinetics coupled with a minimal off-target activity profile. In mice, KLYP961 attenuated endotoxin-evoked increases in plasma nitrates, a surrogate marker of iNOS activity in vivo, in a sustained manner (ED(50) 1 mg/kg p.o.). KLYP961 attenuated pain behaviors in a mouse formalin model (ED(50) 13 mg/kg p.o.), cold allodynia in the chronic constriction injury model (ED(50) 25 mg/kg p.o.), or tactile allodynia in the spinal nerve ligation model (ED(50) 30 mg/kg p.o.) with similar efficacy, but superior potency relative to gabapentin, pregabalin, or duloxetine. Unlike morphine, the antiallodynic activity of KLYP961 did not diminish upon repeated dosing. KLYP961 also attenuated carrageenin-induced edema and inflammatory hyperalgesia and writhing response elicited by phenylbenzoquinone with efficacy and potency similar to those of celecoxib. In contrast to gabapentin, KLYP961 did not impair motor coordination at doses as high as 1000 mg/kg p.o. KLYP961 also attenuated capsaicin-induced thermal allodynia in rhesus primates in a dose-related manner with a minimal effective dose (≤ 10 mg/kg p.o.) and a greater potency than gabapentin. In summary, KLYP961 represents an ideal tool with which to probe the physiological role of NO derived from iNOS and nNOS in human pain and inflammatory states.

  8. Inducible nitric oxide synthase (NOS-2) in subarachnoid hemorrhage: Regulatory mechanisms and therapeutic implications.

    PubMed

    Iqbal, Sana; Hayman, Erik G; Hong, Caron; Stokum, Jesse A; Kurland, David B; Gerzanich, Volodymyr; Simard, J Marc

    2016-01-01

    Aneurysmal subarachnoid hemorrhage (SAH) typically carries a poor prognosis. Growing evidence indicates that overabundant production of nitric oxide (NO) may be responsible for a large part of the secondary injury that follows SAH. Although SAH modulates the activity of all three isoforms of nitric oxide synthase (NOS), the inducible isoform, NOS-2, accounts for a majority of NO-mediated secondary injuries after SAH. Here, we review the indispensable physiological roles of NO that must be preserved, even while attempting to downmodulate the pathophysiologic effects of NO that are induced by SAH. We examine the effects of SAH on the function of the various NOS isoforms, with a particular focus on the pathological effects of NOS-2 and on the mechanisms responsible for its transcriptional upregulation. Finally, we review interventions to block NOS-2 upregulation or to counteract its effects, with an emphasis on the potential therapeutic strategies to improve outcomes in patients afflicted with SAH. There is still much to be learned regarding the apparently maladaptive response of NOS-2 and its harmful product NO in SAH. However, the available evidence points to crucial effects that, on balance, are adverse, making the NOS-2/NO/peroxynitrite axis an attractive therapeutic target in SAH.

  9. Inhibition of inducible nitric oxide synthase and osteoclastic differentiation by Atractylodis Rhizoma Alba extract

    PubMed Central

    Choi, Sung-Ho; Kim, Sung-Jin

    2014-01-01

    Background: Atractylodis Rhizoma Alba (ARA) has been used in Korean folk medicine for constipation, dizziness, and anticancer agent. In the present study, we performed to test whether the methanolic extract of ARA has antioxidant and antiosteoclastogenesis activity in RAW 264.7 macrophage cells. Materials and Methods: Antioxidant capacities were tested by measuring free radical scavenging activity, nitric oxide (NO) levels, reducing power, and inducible nitric oxide synthase (iNOS) expression in response to lipopolysaccharides (LPS). Antiosteoclastogenesis activity was evaluated by performing tartrate-resistant acid phosphatase assay in RAW 264.7 macrophage cells. Results: The extract exerted significant 1,1-diphenyl-2-picrylhydrazyl and NO radical scavenging activity, and it exerted dramatic reducing power. Induction of iNOS and NO by LPS in RAW 264.7 cells was significantly inhibited by the extract, suggesting that the ARA extract inhibits NO production by suppressing iNOS expression. Strikingly, the ARA extracts substantially inhibited the receptor activator of NF-κB ligand-induced osteclastic differentiation of LPS-activated RAW 264.7 cells. The ARA extract contains a significant amount of antioxidant components, including phenolics, flavonoids and anthocyanins. Conclusion: These results suggest that the methanolic extract of ARA exerts significant antioxidant activities potentially via inhibiting free radicals and iNOS induction, thereby leading to the inhibition of osteoclastogenesis. PMID:25298665

  10. Molecular cloning, structure, and chromosomal localization of the human inducible nitric oxide synthase gene.

    PubMed

    Chartrain, N A; Geller, D A; Koty, P P; Sitrin, N F; Nussler, A K; Hoffman, E P; Billiar, T R; Hutchinson, N I; Mudgett, J S

    1994-03-04

    Nitric oxide, a multifunctional effector molecule synthesized by nitric oxide synthase (NOS) from L-arginine, conveys signals for vasorelaxation, neurotransmission, and cytotoxicity. Three different NOS isoforms have been identified which fall into two distinct types, constitutive and inducible. The inducible NOS (iNOS) isoform is expressed in a variety of cell types and tissues in response to inflammatory agents and cytokines. The human iNOS (NOS2) gene was isolated on overlapping cosmid clones from a human genomic library using both the murine macrophage and the human hepatocyte iNOS cDNAs as probes. All isolated cosmids were part of a single genomic locus and no other genomic loci were identified or isolated. Analysis of this locus indicated that the human iNOS gene is approximately 37 kilobases in length and consists of 26 exons and 25 introns. Primer extension analysis of lipopolysaccharide and cytokine-stimulated human hepatocyte RNA mapped the transcriptional initiation site 30 base pairs downstream of a TATA sequence, and a 400-base pair 5'-flanking region was found to be structurally similar to the recently described murine iNOS promoter. Polymerase chain reaction analysis of a human/rodent genomic DNA somatic cell hybrid panel and fluorescent in situ hybridization indicated that the human iNOS gene is located on chromosome 17 at position 17cen-q11.2.

  11. Does restraining nitric oxide biosynthesis rescue from toxins-induced parkinsonism and sporadic Parkinson's disease?

    PubMed

    Gupta, Satya Prakash; Yadav, Sharawan; Singhal, Naveen Kumar; Tiwari, Manindra Nath; Mishra, Sarad Kumar; Singh, Mahendra Pratap

    2014-02-01

    Nitric oxide (NO) is an important inorganic molecule of the biological system owing to diverse physiological implications. NO is synthesised from a semi-essential amino acid L-arginine. NO biosynthesis is catalysed by a family of enzymes referred to as nitric oxide synthases (NOSs). NO is accused in many acute and chronic illnesses, which include central nervous system disorders, inflammatory diseases, reproductive impairments, cancer and cardiovascular anomalies. Owing to very unstable nature, NO gets converted into nitrite, peroxynitrite and other reactive nitrogen species that could lead to nitrosative stress in the nigrostriatal system. Nitrosative stress is widely implicated in Parkinson's disease (PD), and its beneficial and harmful effects are demonstrated in in vitro, rodent and primate models of toxins-induced parkinsonism and in the blood, cerebrospinal fluid and nigrostriatal tissues of sporadic PD patients. The current article updates the roles of NO and NOSs in sporadic PD and toxins-induced parkinsonism in rodents along with the scrutiny of how inhibitors of NOSs could open a new line of approach to moderately rescue from PD pathogenesis based on the existing literature. The article also provides a perspective concerning the lack of ample admiration to such an approach and how to minimise the underlying lacunae.

  12. Endurance training enhances vasodilation induced by nitric oxide in human skin.

    PubMed

    Boegli, Yann; Gremion, Gerald; Golay, Sandrine; Kubli, Sandrine; Liaudet, Lucas; Leyvraz, Pierre-François; Waeber, Bernard; Feihl, François

    2003-11-01

    Endurance training modifies the thermoregulatory control of skin blood flow, as manifested by a greater augmentation of skin perfusion for the same increase in core temperature in athletes, in comparison with sedentary subjects. In this study, we tested the hypothesis that a component of this adaptation might reside in a higher ability of cutaneous blood vessels to respond to vasodilatory stimuli. We recruited healthy nonsmoking males, either endurance trained or sedentary, in two different age ranges (18-35 y and >50 y). Skin blood flow was measured in the forearm skin, using a laser Doppler imager, allowing to record the vasodilatory responses to the following stimuli: iontophoresis of acetylcholine (an endothelium-dependent vasodilator), iontophoresis of sodium nitroprusside (a nitric oxide donor), and release of a temporary interruption of arterial inflow (reactive hyperemia). There was no effect of training on reactive hyperemia or the response to acetylcholine. In contrast, the increase in perfusion following the iontophoresis of sodium nitroprusside, expressed in perfusion units, was larger in trained than in sedentary subjects (younger: 398 +/- 54 vs 350 +/- 87, p < 0.05; older 339 +/- 72 vs 307 +/- 66, p < 0.05). In conclusion, endurance training enhances the vasodilatory effects of nitric oxide in the human dermal microcirculation, at least in forearm skin. These observations have considerable physiologic interest in view of recent data indicating that nitric oxide mediates in part the cutaneous vasodilation induced by heat stress in humans. Therefore, the augmentation of nitric oxide bioactivity in the dermal microcirculation might be one mechanism whereby endurance training modifies the thermoregulatory control of skin blood flow.

  13. Role of nitric oxide in adenosine-induced vasodilation in humans

    NASA Technical Reports Server (NTRS)

    Costa, F.; Biaggioni, I.; Robertson, D. (Principal Investigator)

    1998-01-01

    Vasodilation is one of the most prominent effects of adenosine and one of the first to be recognized, but its mechanism of action is not completely understood. In particular, there is conflicting information about the potential contribution of endothelial factors. The purpose of this study was to explore the role of nitric oxide in the vasodilatory effect of adenosine. Forearm blood flow responses to intrabrachial adenosine infusion (125 microg/min) were assessed with venous occlusion plethysmography during intrabrachial infusion of saline or the nitric oxide synthase inhibitor NG-monomethyl-L-arginine (L-NMMA) (12.5 mg/min). Intrabrachial infusions of acetylcholine (50 microg/min) and nitroprusside (3 microg/min) were used as a positive and negative control, respectively. These doses were chosen to produce comparable levels of vasodilation. In a separate study, a second saline infusion was administered instead of L-NMMA to rule out time-related effects. As expected, pretreatment with L-NMMA reduced acetylcholine-induced vasodilation; 50 microg/min acetylcholine increased forearm blood flow by 150+/-43% and 51+/-12% during saline and L-NMMA infusion, respectively (P<.01, n=6). In contrast, L-NMMA did not affect the increase in forearm blood flow produced by 3 microg/min nitroprusside (165+/-30% and 248+/-41% during saline and L-NMMA, respectively) or adenosine (173+/-48% and 270+/-75% during saline and L-NMMA, respectively). On the basis of our observations, we conclude that adenosine-induced vasodilation is not mediated by nitric oxide in the human forearm.

  14. Nitric oxide acts as a positive regulator to induce metamorphosis of the ascidian Herdmania momus.

    PubMed

    Ueda, Nobuo; Degnan, Sandie M

    2013-01-01

    Marine invertebrates commonly have a biphasic life cycle in which the metamorphic transition from a pelagic larva to a benthic post-larva is mediated by the nitric oxide signalling pathway. Nitric oxide (NO) is synthesised by nitric oxide synthase (NOS), which is a client protein of the molecular chaperon heat shock protein 90 (HSP90). It is notable, then, that both NO and HSP90 have been implicated in regulating metamorphosis in marine invertebrates as diverse as urochordates, echinoderms, molluscs, annelids, and crustaceans. Specifically, the suppression of NOS activity by the application of either NOS- or HSP90-inhibiting pharmacological agents has been shown consistently to induce the initiation of metamorphosis, leading to the hypothesis that a negative regulatory role of NO is widely conserved in biphasic life cycles. Further, the induction of metamorphosis by heat-shock has been demonstrated for multiple species. Here, we investigate the regulatory role of NO in induction of metamorphosis of the solitary tropical ascidian, Herdmania momus. By coupling pharmacological treatments with analysis of HmNOS and HmHSP90 gene expression, we present compelling evidence of a positive regulatory role for NO in metamorphosis of this species, in contrast to all existing ascidian data that supports the hypothesis of NO as a conserved negative regulator of metamorphosis. The exposure of competent H. momus larvae to a NOS inhibitor or an NO donor results in an up-regulation of NOS and HSP90 genes. Heat shock of competent larvae induces metamorphosis in a temperature dependent manner, up to a thermal tolerance that approaches 35°C. Both larval/post-larval survival and the appearance of abnormal morphologies in H. momus post-larvae reflect the magnitude of up-regulation of the HSP90 gene in response to heat-shock. The demonstrated role of NO as a positive metamorphic regulator in H. momus suggests the existence of inter-specific adaptations of NO regulation in ascidian

  15. Nitric Oxide Acts as a Positive Regulator to Induce Metamorphosis of the Ascidian Herdmania momus

    PubMed Central

    Ueda, Nobuo; Degnan, Sandie M.

    2013-01-01

    Marine invertebrates commonly have a biphasic life cycle in which the metamorphic transition from a pelagic larva to a benthic post-larva is mediated by the nitric oxide signalling pathway. Nitric oxide (NO) is synthesised by nitric oxide synthase (NOS), which is a client protein of the molecular chaperon heat shock protein 90 (HSP90). It is notable, then, that both NO and HSP90 have been implicated in regulating metamorphosis in marine invertebrates as diverse as urochordates, echinoderms, molluscs, annelids, and crustaceans. Specifically, the suppression of NOS activity by the application of either NOS- or HSP90-inhibiting pharmacological agents has been shown consistently to induce the initiation of metamorphosis, leading to the hypothesis that a negative regulatory role of NO is widely conserved in biphasic life cycles. Further, the induction of metamorphosis by heat-shock has been demonstrated for multiple species. Here, we investigate the regulatory role of NO in induction of metamorphosis of the solitary tropical ascidian, Herdmania momus. By coupling pharmacological treatments with analysis of HmNOS and HmHSP90 gene expression, we present compelling evidence of a positive regulatory role for NO in metamorphosis of this species, in contrast to all existing ascidian data that supports the hypothesis of NO as a conserved negative regulator of metamorphosis. The exposure of competent H. momus larvae to a NOS inhibitor or an NO donor results in an up-regulation of NOS and HSP90 genes. Heat shock of competent larvae induces metamorphosis in a temperature dependent manner, up to a thermal tolerance that approaches 35°C. Both larval/post-larval survival and the appearance of abnormal morphologies in H. momus post-larvae reflect the magnitude of up-regulation of the HSP90 gene in response to heat-shock. The demonstrated role of NO as a positive metamorphic regulator in H. momus suggests the existence of inter-specific adaptations of NO regulation in ascidian

  16. Nitric oxide mediates the anticonvulsant effects of thalidomide on pentylenetetrazole-induced clonic seizures in mice.

    PubMed

    Payandemehr, Borna; Rahimian, Reza; Gooshe, Maziar; Bahremand, Arash; Gholizadeh, Ramtin; Berijani, Sina; Ahmadi-Dastgerdi, Mohammad; Aminizade, Mehdi; Sarreshte-Dari, Ali; Dianati, Vahid; Amanlou, Massoud; Dehpour, Ahmad Reza

    2014-05-01

    Thalidomide is an old glutamic acid derivative which was initially used as a sedative medication but withdrawn from the market due to the high incidence of teratogenicity. Recently, it has reemerged because of its potential for counteracting number of diseases, including neurodegenerative disorders. Other than the antiemetic and hypnotic aspects, thalidomide exerts some anticonvulsant properties in experimental settings. However, the underlying mechanisms of thalidomide actions are not fully realized yet. Some investigations revealed that thalidomide could elicit immunomodulatory or neuromodulatory properties by affecting different targets, including cytokines (such as TNF α), neurotransmitters, and nitric oxide (NO). In this regard, we used a model of clonic seizure induced by pentylenetetrazole (PTZ) in male NMRI mice to investigate whether the anticonvulsant effect of thalidomide is affected through modulation of the l-arginine-nitric oxide pathway or not. Injection of a single effective dose of thalidomide (10 mg/kg, i.p. or higher) significantly increased the seizure threshold (P<0.05). On the one hand, pretreatment with low and per se noneffective dose of l-arginine [NO precursor] (10, 30 and 60 mg/kg) prevented the anticonvulsant effect of thalidomide. On the other hand, NOS inhibitors [l-NAME and 7-NI] augmented the anticonvulsant effect of a subeffective dose of thalidomide (1 and 5 mg/kg, i.p.) at relatively low doses. Meanwhile, several doses of aminoguanidine [an inducible NOS inhibitor] (20, 50 and 100 mg/kg) failed to alter the anticonvulsant effect of thalidomide significantly. In summary, our findings demonstrated that the l-arginine-nitric oxide pathway can be involved in the anticonvulsant properties of thalidomide, and the role of constitutive nNOS is prominent in the reported neuroprotective feature.

  17. Beneficial Effects of Concomitant Neuronal and Inducible Nitric Oxide Synthase Inhibition in Ovine Burn and Inhalation Injury

    PubMed Central

    Lange, Matthias; Hamahata, Atsumori; Enkhbaatar, Perenlei; Cox, Robert A.; Nakano, Yoshimitsu; Westphal, Martin; Traber, Lillian D.; Herndon, David N.; Traber, Daniel L.

    2013-01-01

    Different isoforms of nitric oxide synthase are critically involved in the development of pulmonary failure secondary to acute lung injury. Here we tested the hypothesis that simultaneous blockade of inducible and neuronal nitric oxide synthase effectively prevents the pulmonary lesions in an ovine model of acute respiratory distress syndrome (ARDS) induced by combined burn and smoke inhalation injury. Chronically instrumented sheep were allocated to a sham-injured group (n = 6), an injured and untreated group (n = 6), or an injured group treated with simultaneous infusion of selective inducible and neuronal nitric oxide synthase inhibitors (n = 5). The injury was induced by 48 breath of cotton smoke and a 3rd degree burn of 40% total body surface area. All sheep were mechanically ventilated and fluid resuscitated. The injury induced severe pulmonary dysfunction as indicated by decreases in PaO2/FiO2 ratio and increases in pulmonary shunt fraction, ventilatory pressures, lung lymph flow, and lung wet/dry weight ratio. The treatment fully prevented the elevations in lymph and plasma nitrate/nitrite levels, pulmonary shunting, ventilatory pressures, lung lymph flow, and wet/dry weight ratio and significantly attenuated the decline in PaO2/FiO2 ratio. In conclusion, simultaneous blockade of inducible and neuronal nitric oxide synthase exerts beneficial pulmonary effects in an ovine model of ARDS secondary to combined burn and smoke inhalation injury. This novel treatment strategy may represent a useful therapeutic adjunct for patients with these injuries. PMID:21263377

  18. Inhibition of nitric oxide production rescues LPS-induced fetal abortion in mice.

    PubMed

    Athanassakis, I; Aifantis, I; Ranella, A; Giouremou, K; Vassiliadis, S

    1999-06-01

    In this report, we examined the involvement of the cytokines tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, interleukin (IL)-4, and IL-10 as well as nitric oxide (NO) in the lipopolysaccharide (LPS)-induced experimental abortion model in BALB/c mice. Although in vivo administration of LPS in pregnant mice showed a 72% decrease of serum IL-10, no significant difference in serum TNF-alpha, IFN-gamma, and IL-4 levels, compared to controls, could be detected. At the same time, a correlation of fetal abortion and maternal splenomegaly with an important increase of NO synthesis in the serum was obtained. Simultaneous administration of LPS and aminoguanidine (AG; an inhibitor to NO synthase) rescued the LPS-induced fetal abortion, reduced maternal spleen weight to physiological levels, and decreased serum NO concentration to control levels. In vitro experiments showed that LPS directly induced NO production in primary placental cells and the TPOPHO-1 trophoblast cell line by stimulating the inducible isoform of NO synthase, which ultimately could be blocked by the NO synthase inhibitors AG and L-NAME. The results indicate that LPS, despite its beneficial involvement in intracellular infections, participates in inflammatory/autoimmune damage during pregnancy, leading to embryotoxicity, which is closely linked to the NO pathway.

  19. Nitric oxide is required for hydrogen gas-induced adventitious root formation in cucumber.

    PubMed

    Zhu, Yongchao; Liao, Weibiao; Wang, Meng; Niu, Lijuan; Xu, Qingqing; Jin, Xin

    2016-05-20

    Hydrogen gas (H2) is involved in plant development and stress responses. Cucumber explants were used to study whether nitric oxide (NO) is involved in H2-induced adventitious root development. The results revealed that 50% and 100% hydrogen-rich water (HRW) apparently promoted the development of adventitious root in cucumber. While, the responses of HRW-induced adventitious rooting were blocked by a specific NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (cPTIO), NO synthase (NOS) enzyme inhibitor N(G)-nitro-l-arginine methylester hydrochloride (l-NAME) and nitrate reductase (NR) inhibitor NaN3. HRW also increased NO content and NOS and NR activity both in a dose- and time-dependent fashion. Moreover, molecular evidence showed that HRW up-regulated NR genes expression in explants. The results indicate the importance of NOS and NR enzymes, which might be responsible for NO production in explants during H2-induced root organogenesis. Additionally, peroxidase (POD) and indoleacetic acid oxidase (IAAO) activity was significantly decreased in the explants treated with HRW, while HRW treatment significantly increased polyphenol oxidase (PPO) activity. In addition, cPTIO, l-NAME and NaN3 inhibited the actions of HRW on the activity of these enzymes. Together, NO may be involved in H2-induced adventitious rooting, and NO may be acting downstream in plant H2 signaling cascade.

  20. Nitric oxide synthases and tubal ectopic pregnancies induced by Chlamydia infection: basic and clinical insights.

    PubMed

    Shao, Ruijin; Zhang, Sean X; Weijdegård, Birgitta; Zou, Shien; Egecioglu, Emil; Norström, Anders; Brännström, Mats; Billig, Håkan

    2010-12-01

    Human ectopic pregnancy (EP) remains a common cause of pregnancy-related first trimester death. Nitric oxide (NO) is synthesized from L-arginine by three NO synthases (NOS) in different tissues, including the Fallopian tube. Studies of knockout mouse models have improved our understanding of the function of NOS isoforms in reproduction, but their roles and specific mechanisms in infection-induced tubal dysfunction have not been fully elucidated. Here, we provide an overview of the expression, regulation and possible function of NOS isoforms in the Fallopian tube, highlighting the effects of infection-induced changes in the tubal cellular microenvironment (imbalance of NO production) on tubal dysfunction and the potential involvement of NOS isoforms in tubal EP after Chlamydia trachomatis genital infection. The non-equivalent regulation of tubal NOS isoforms during the menstrual cycle suggests that endogenous ovarian steroid hormones regulate NOS in an isoform-specific manner. The current literature suggests that infection with C. trachomatis induces an inflammatory response that eventually leads to tubal epithelial destruction and functional impairment, caused by a high NO output mediated by inducible NOS (iNOS). Therefore, tissue-specific therapeutic approaches to suppress iNOS expression may help to prevent ectopic implantation in patients with prior C. trachomatis infection of the Fallopian tube.

  1. Phospholipase Dδ is involved in nitric oxide-induced stomatal closure.

    PubMed

    Distéfano, Ayelen M; Scuffi, Denise; García-Mata, Carlos; Lamattina, Lorenzo; Laxalt, Ana M

    2012-12-01

    Nitric oxide (NO) has recently emerged as a second messenger involved in the complex network of signaling events that regulate stomatal closure. Little is known about the signaling events occurring downstream of NO. Previously, we demonstrated the involvement of phospholipase D (PLD) in NO signaling during stomatal closure. PLDδ, one of the 12 Arabidopsis PLDs, is involved in dehydration stress responses. To investigate the role of PLDδ in NO signaling in guard cells, we analyzed guard cells responses using Arabidopsis wild type and two independent pldδ single mutants. In this work, we show that pldδ mutants failed to close the stomata in response to NO. Treatments with phosphatidic acid, the product of PLD activity, induced stomatal closure in pldδ mutants. Abscisic acid (ABA) signaling in guard cells involved H(2)O(2) and NO production, both required for ABA-induced stomatal closure. pldδ guard cells produced similar NO and H(2)O(2) levels as the wild type in response to ABA. However, ABA- or H(2)O(2)-induced stomatal closure was impaired in pldδ plants. These data indicate that PLDδ is downstream of NO and H(2)O(2) in ABA-induced stomatal closure.

  2. Inhibition of inducible Nitric Oxide Synthase by a mustard gas analog in murine macrophages

    PubMed Central

    Qui, Min; Paromov, Victor M; Yang, Hongsong; Smith, Milton; Stone, William L

    2006-01-01

    Background 2-Chloroethyl ethyl sulphide (CEES) is a sulphur vesicating agent and an analogue of the chemical warfare agent 2,2'-dichlorodiethyl sulphide, or sulphur mustard gas (HD). Both CEES and HD are alkylating agents that influence cellular thiols and are highly toxic. In a previous publication, we reported that lipopolysaccharide (LPS) enhances the cytotoxicity of CEES in murine RAW264.7 macrophages. In the present investigation, we studied the influence of CEES on nitric oxide (NO) production in LPS stimulated RAW264.7 cells since NO signalling affects inflammation, cell death, and wound healing. Murine macrophages stimulated with LPS produce NO almost exclusively via inducible nitric oxide synthase (iNOS) activity. We suggest that the influence of CEES or HD on the cellular production of NO could play an important role in the pathophysiological responses of tissues to these toxicants. In particular, it is known that macrophage generated NO synthesised by iNOS plays a critical role in wound healing. Results We initially confirmed that in LPS stimulated RAW264.7 macrophages NO is exclusively generated by the iNOS form of nitric oxide synthase. CEES treatment inhibited the synthesis of NO (after 24 hours) in viable LPS-stimulated RAW264.7 macrophages as measured by either nitrite secretion into the culture medium or the intracellular conversion of 4,5-diaminofluorescein diacetate (DAF-2DA) or dichlorofluorescin diacetate (DCFH-DA). Western blots showed that CEES transiently decreased the expression of iNOS protein; however, treatment of active iNOS with CEES in vitro did not inhibit its enzymatic activity Conclusion CEES inhibits NO production in LPS stimulated macrophages by decreasing iNOS protein expression. Decreased iNOS expression is likely the result of CEES induced alteration in the nuclear factor kappa B (NF-κB) signalling pathway. Since NO can act as an antioxidant, the CEES induced down-regulation of iNOS in LPS-stimulated macrophages could elevate

  3. Active site cysteine-null glyceraldehyde-3-phosphate dehydrogenase (GAPDH) rescues nitric oxide-induced cell death.

    PubMed

    Kubo, Takeya; Nakajima, Hidemitsu; Nakatsuji, Masatoshi; Itakura, Masanori; Kaneshige, Akihiro; Azuma, Yasu-Taka; Inui, Takashi; Takeuchi, Tadayoshi

    2016-02-29

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a homotetrameric enzyme involved in a key step of glycolysis, also has a role in mediating cell death under nitrosative stress. Our previous reports suggest that nitric oxide-induced intramolecular disulfide-bonding GAPDH aggregation, which occurs through oxidation of the active site cysteine (Cys-152), participates in a mechanism to account for nitric oxide-induced death signaling in some neurodegenerative/neuropsychiatric disorders. Here, we demonstrate a rescue strategy for nitric oxide-induced cell death accompanied by GAPDH aggregation in a mutant with a substitution of Cys-152 to alanine (C152A-GAPDH). Pre-incubation of purified wild-type GAPDH with C152A-GAPDH under exposure to nitric oxide inhibited wild-type GAPDH aggregation in a concentration-dependent manner in vitro. Several lines of structural analysis revealed that C152A-GAPDH extensively interfered with nitric oxide-induced GAPDH-amyloidogenesis. Overexpression of doxycycline-inducible C152A-GAPDH in SH-SY5Y neuroblastoma significantly rescued nitric oxide-induced death, concomitant with the decreased formation of GAPDH aggregates. Further, both co-immunoprecipitation assays and simulation models revealed a heterotetramer composed of one dimer each of wild-type GAPDH and C152A-GAPDH. These results suggest that the C152A-GAPDH mutant acts as a dominant-negative molecule against GAPDH aggregation via the formation of this GAPDH heterotetramer. This study may contribute to a new therapeutic approach utilizing C152A-GAPDH against brain damage in nitrosative stress-related disorders.

  4. Nitric oxide and NK(1)-tachykinin receptors in cyclophosphamide-induced cystitis, in rats.

    PubMed

    Alfieri, A B; Cubeddu, L X

    2000-11-01

    The present study was conducted to investigate the role of NK(1) receptors and of nitric oxide (NO) on the pathogenesis of cyclophosphamide-induced cystitis, in rats. This bladder toxicity was characterized by marked increases in protein plasma extravasation, urothelial damage, edema, white blood cell infiltrates, and vascular congestion. These changes were associated with appearance of Ca(2+)-independent NO-synthase (NOS) activity [characteristic of inducible NOS (iNOS)] in the bladder and with increases in urinary NO metabolites. GR205171, a selective NK(1) antagonist (10-20 mg/kg, i.p.) reduced cyclophosphamide-induced increases in protein plasma extravasation and in the urinary excretion of NO metabolites. N(G)-Nitro-L-arginine (L-NNA) (10 mg/kg, i.p.), a NOS inhibitor, reduced basal and cyclophosphamide-induced increases in NO metabolites and protected against cyclophosphamide-induced protein plasma extravasation. GR205171 had no effect, whereas L-NNA reduced basal NO metabolite excretion. Combined treatment with the NK(1) antagonist and the NO-synthesis inhibitor produced comparable reduction in protein plasma extravasation than that achieved with each drug given separately. Combined drug treatment ameliorated cyclophosphamideinduced urothelial damage, and the extent of edema, vascular congestion, and white blood cell infiltrates in the bladder. In summary, NK(1) receptors and iNOS play a role in NO formation and on cyclophosphamide-induced cystitis. Activation of NK(1) receptors mainly acts through the formation of NO. It is proposed that cyclophosphamide and/or its metabolites would stimulate primary afferent capsaicin-sensitive fibers in the bladder, releasing neuropeptides, which would activate NK(1) receptors. However, additional mechanisms are involved, because neither the NK(1) receptor antagonist nor the NO synthesis inhibitor, either alone or in combination, were able to completely prevent the toxicity.

  5. Inhibition of nitric oxide-induced apoptosis by nicotine in oral epithelial cells.

    PubMed

    Banerjee, Abhijit G; Gopalakrishnan, Velliyur K; Vishwanatha, Jamboor K

    2007-11-01

    Development of oral cancer is clearly linked to the usage of smokeless tobacco. The molecular mechanisms involved in this process are however not well understood. Toward this goal, we investigated the effect of smokeless tobacco exposure on apoptosis of oral epithelial cells. Exposure of oral epithelial cells to smokeless tobacco extract (STE) induces apoptosis in a dose-dependent manner, until a threshold level of nicotine is achieved upon which apoptosis is inhibited. 1 mM of nicotine is able to inhibit apoptosis significantly induced by STE in these oral cells. Exposure of cells to nicotine alone has no effect on apoptosis, but nicotine inhibits apoptosis induced by other agents present in STE. In this study we show that, the anti-apoptotic action of nicotine is specifically associated with down-regulation of nitric oxide (NO) production. Using specific inducers of NO, we have demonstrated that inhibition of apoptosis by nicotine is through down-regulation of NO production. Further, we observed that nicotine clearly acts as a sink of NO radicals, shown using peroxynitrite generator (SIN-1) in conjunction or absence of radical scavengers. Nicotine thus causes most damage in transformed epithelial cells as depicted by accumulation of nitrotyrosine in a 3-NT ELISA assay. Inhibition of apoptosis is a hallmark in tumor progression and propels development of cancer. It may further result in functional loss of apoptotic effector mechanisms in the transformed cells. Thus, our data clearly indicates that inhibition of NO-induced apoptosis by nicotine may lead to tobacco-induced oral carcinogenesis, and implies careful development of modalities in tobacco cessation programs.

  6. Endothelial Nitric Oxide Mediates Caffeine Antagonism of Alcohol-Induced Cerebral Artery Constriction

    PubMed Central

    Chang, Jennifer; Fedinec, Alexander L.; Kuntamallappanavar, Guruprasad; Leffler, Charles W.; Bukiya, Anna N.

    2016-01-01

    Despite preventive education, the combined consumption of alcohol and caffeine (particularly from “energy drinks”) continues to rise. Physiologic perturbations by separate intake of ethanol and caffeine have been widely documented. However, the biologic actions of the alcohol-caffeine combination and their underlying subcellular mechanisms have been scarcely studied. Using intravital microscopy on a closed-cranial window and isolated, pressurized vessels, we investigated the in vivo and in vitro action of ethanol-caffeine mixtures on cerebral arteries from rats and mice, widely recognized models to address cerebrovascular pathophysiology and pharmacology. Caffeine at concentrations found in human circulation after ingestion of one to two cups of coffee (10 µM) antagonized the endothelium-independent constriction of cerebral arteries evoked by ethanol concentrations found in blood during moderate-heavy alcohol intoxication (40–70 mM). Caffeine antagonism against alcohol was similar whether evaluated in vivo or in vitro, suggesting independence of systemic factors and drug metabolism, but required a functional endothelium. Moreover, caffeine protection against alcohol increased nitric oxide (NO•) levels over those found in the presence of ethanol alone, disappeared upon blocking NO• synthase, and could not be detected in pressurized cerebral arteries from endothelial nitric-oxide synthase knockout (eNOS−/−) mice. Finally, incubation of de-endothelialized cerebral arteries with the NO• donor sodium nitroprusside (10 µM) fully restored the protective effect of caffeine. This study demonstrates for the first time that caffeine antagonizes ethanol-induced cerebral artery constriction and identifies endothelial NO• as the critical caffeine effector on smooth muscle targets. Conceivably, situations that perturb endothelial function and/or NO• availability will critically alter caffeine antagonism of alcohol-induced cerebrovascular constriction without

  7. Endothelial Nitric Oxide Mediates Caffeine Antagonism of Alcohol-Induced Cerebral Artery Constriction.

    PubMed

    Chang, Jennifer; Fedinec, Alexander L; Kuntamallappanavar, Guruprasad; Leffler, Charles W; Bukiya, Anna N; Dopico, Alex M

    2016-01-01

    Despite preventive education, the combined consumption of alcohol and caffeine (particularly from "energy drinks") continues to rise. Physiologic perturbations by separate intake of ethanol and caffeine have been widely documented. However, the biologic actions of the alcohol-caffeine combination and their underlying subcellular mechanisms have been scarcely studied. Using intravital microscopy on a closed-cranial window and isolated, pressurized vessels, we investigated the in vivo and in vitro action of ethanol-caffeine mixtures on cerebral arteries from rats and mice, widely recognized models to address cerebrovascular pathophysiology and pharmacology. Caffeine at concentrations found in human circulation after ingestion of one to two cups of coffee (10 µM) antagonized the endothelium-independent constriction of cerebral arteries evoked by ethanol concentrations found in blood during moderate-heavy alcohol intoxication (40-70 mM). Caffeine antagonism against alcohol was similar whether evaluated in vivo or in vitro, suggesting independence of systemic factors and drug metabolism, but required a functional endothelium. Moreover, caffeine protection against alcohol increased nitric oxide (NO•) levels over those found in the presence of ethanol alone, disappeared upon blocking NO• synthase, and could not be detected in pressurized cerebral arteries from endothelial nitric-oxide synthase knockout (eNOS(-/-)) mice. Finally, incubation of de-endothelialized cerebral arteries with the NO• donor sodium nitroprusside (10 µM) fully restored the protective effect of caffeine. This study demonstrates for the first time that caffeine antagonizes ethanol-induced cerebral artery constriction and identifies endothelial NO• as the critical caffeine effector on smooth muscle targets. Conceivably, situations that perturb endothelial function and/or NO• availability will critically alter caffeine antagonism of alcohol-induced cerebrovascular constriction without

  8. Selective Inducible Nitric Oxide Synthase Inhibitor Reversed Zinc Chloride-Induced Spatial Memory Impairment via Increasing Cholinergic Marker Expression.

    PubMed

    Tabrizian, Kaveh; Azami, Kian; Belaran, Maryam; Soodi, Maliheh; Abdi, Khosrou; Fanoudi, Sahar; Sanati, Mehdi; Mottaghi Dastjerdi, Negar; Soltany Rezaee-Rad, Mohammad; Sharifzadeh, Mohammad

    2016-10-01

    Zinc, an essential micronutrient and biochemical element of the human body, plays structural, catalytic, and regulatory roles in numerous physiological functions. In the current study, the effects of a pretraining oral administration of zinc chloride (10, 25, and 50 mg/kg) for 14 consecutive days and post-training bilateral intra-hippocampal infusion of 1400W as a selective inducible nitric oxide synthase (iNOS) inhibitor (10, 50, and 100 μM/side), alone and in combination, on the spatial memory retention in Morris water maze (MWM) were investigated. Animals were trained for 4 days and tested 48 h after completion of training. Also, the molecular effects of these compounds on the expression of choline acetyltransferase (ChAT), as a cholinergic marker in the CA1 region of the hippocampus and medial septal area (MSA), were evaluated. Behavioral and molecular findings of this study showed that a 2-week oral administration of zinc chloride (50 mg/kg) impaired spatial memory retention in MWM and decreased ChAT expression. Immunohistochemical analysis of post-training bilateral intra-hippocampal infusion of 1400W revealed a significant increase in ChAT immunoreactivity. Furthermore, post-training bilateral intra-hippocampal infusion of 1400W into the CA1 region of the hippocampus reversed zinc chloride-induced spatial memory impairment in MWM and significantly increased ChAT expression in comparison with zinc chloride-treated animals. Taken together, these results emphasize the role of selective iNOS inhibitors in reversing zinc chloride-induced spatial memory deficits via modulation of cholinergic marker expression.

  9. Nitric oxide-mediated bystander signal transduction induced by heavy-ion microbeam irradiation.

    PubMed

    Tomita, Masanori; Matsumoto, Hideki; Funayama, Tomoo; Yokota, Yuichiro; Otsuka, Kensuke; Maeda, Munetoshi; Kobayashi, Yasuhiko

    2015-07-01

    In general, a radiation-induced bystander response is known to be a cellular response induced in non-irradiated cells after receiving bystander signaling factors released from directly irradiated cells within a cell population. Bystander responses induced by high-linear energy transfer (LET) heavy ions at low fluence are an important health problem for astronauts in space. Bystander responses are mediated via physical cell-cell contact, such as gap-junction intercellular communication (GJIC) and/or diffusive factors released into the medium in cell culture conditions. Nitric oxide (NO) is a well-known major initiator/mediator of intercellular signaling within culture medium during bystander responses. In this study, we investigated the NO-mediated bystander signal transduction induced by high-LET argon (Ar)-ion microbeam irradiation of normal human fibroblasts. Foci formation by DNA double-strand break repair proteins was induced in non-irradiated cells, which were co-cultured with those irradiated by high-LET Ar-ion microbeams in the same culture plate. Foci formation was suppressed significantly by pretreatment with an NO scavenger. Furthermore, NO-mediated reproductive cell death was also induced in bystander cells. Phosphorylation of NF-κB and Akt were induced during NO-mediated bystander signaling in the irradiated and bystander cells. However, the activation of these proteins depended on the incubation time after irradiation. The accumulation of cyclooxygenase-2 (COX-2), a downstream target of NO and NF-κB, was observed in the bystander cells 6 h after irradiation but not in the directly irradiated cells. Our findings suggest that Akt- and NF-κB-dependent signaling pathways involving COX-2 play important roles in NO-mediated high-LET heavy-ion-induced bystander responses. In addition, COX-2 may be used as a molecular marker of high-LET heavy-ion-induced bystander cells to distinguish them from directly irradiated cells, although this may depend on the time

  10. Nitric oxide-mediated bystander signal transduction induced by heavy-ion microbeam irradiation

    NASA Astrophysics Data System (ADS)

    Tomita, Masanori; Matsumoto, Hideki; Funayama, Tomoo; Yokota, Yuichiro; Otsuka, Kensuke; Maeda, Munetoshi; Kobayashi, Yasuhiko

    2015-07-01

    In general, a radiation-induced bystander response is known to be a cellular response induced in non-irradiated cells after receiving bystander signaling factors released from directly irradiated cells within a cell population. Bystander responses induced by high-linear energy transfer (LET) heavy ions at low fluence are an important health problem for astronauts in space. Bystander responses are mediated via physical cell-cell contact, such as gap-junction intercellular communication (GJIC) and/or diffusive factors released into the medium in cell culture conditions. Nitric oxide (NO) is a well-known major initiator/mediator of intercellular signaling within culture medium during bystander responses. In this study, we investigated the NO-mediated bystander signal transduction induced by high-LET argon (Ar)-ion microbeam irradiation of normal human fibroblasts. Foci formation by DNA double-strand break repair proteins was induced in non-irradiated cells, which were co-cultured with those irradiated by high-LET Ar-ion microbeams in the same culture plate. Foci formation was suppressed significantly by pretreatment with an NO scavenger. Furthermore, NO-mediated reproductive cell death was also induced in bystander cells. Phosphorylation of NF-κB and Akt were induced during NO-mediated bystander signaling in the irradiated and bystander cells. However, the activation of these proteins depended on the incubation time after irradiation. The accumulation of cyclooxygenase-2 (COX-2), a downstream target of NO and NF-κB, was observed in the bystander cells 6 h after irradiation but not in the directly irradiated cells. Our findings suggest that Akt- and NF-κB-dependent signaling pathways involving COX-2 play important roles in NO-mediated high-LET heavy-ion-induced bystander responses. In addition, COX-2 may be used as a molecular marker of high-LET heavy-ion-induced bystander cells to distinguish them from directly irradiated cells, although this may depend on the time

  11. Nitric Oxide Synthase Inhibitor Improves De Novo and Long-Term l-DOPA-Induced Dyskinesia in Hemiparkinsonian Rats

    PubMed Central

    Padovan-Neto, Fernando Eduardo; Echeverry, Marcela Bermúdez; Chiavegatto, Silvana; Del-Bel, Elaine

    2011-01-01

    Inhibitors of neuronal and endothelial nitric oxide synthase decrease l-3,4-dihidroxifenilalanine (l-DOPA)-induced dyskinesias in rodents. The mechanism of nitric oxide inhibitor action is unknown. The aims of the present study were to investigate the decrease of l-DOPA-induced abnormal involuntary movements (AIMs) in 6-hydroxydopamine (6-OHDA)-lesioned rats by nitric oxide inhibitors following either acute or chronic treatment. The primary findings of this study were that NG-nitro-l-Arginine, an inhibitor of endothelial and neuronal nitric oxide synthase, attenuated AIMs induced by chronic and acute l-DOPA. In contrast, rotational behavior was attenuated only after chronic l-DOPA. The 6-OHDA lesion and the l-DOPA treatment induced a bilateral increase (1.5 times) in the neuronal nitric oxide synthase (nNOS) protein and nNOS mRNA in the striatum and in the frontal cortex. There was a parallel increase, bilaterally, of the FosB/ΔFosB, primarily in the ipsilateral striatum. The exception was in the contralateral striatum and the ipsilateral frontal cortex, where chronic l-DOPA treatment induced an increase of approximately 10 times the nNOS mRNA. Our results provided further evidence of an anti-dyskinetic effect of NOS inhibitor. The effect appeared under l-DOPA acute and chronic treatment. The l-DOPA treatment also revealed an over-expression of the neuronal NOS in the frontal cortex and striatum. Our results corroborated findings that l-DOPA-induced rotation differs between acute and chronic treatment. The effect of the NOS inhibitor conceivably relied on the l-DOPA structural modifications in the Parkinsonian brain. Taken together, these data provided a rationale for further evaluation of NOS inhibitors in the treatment of l-DOPA-induced dyskinesia. PMID:21713068

  12. Estriol-induced fibrinolysis due to the activation of plasminogen to plasmin by nitric oxide synthesis in platelets.

    PubMed

    Jana, Pradipta; Maiti, Smarajit; Kahn, Nighat N; Sinha, Asru K

    2015-04-01

    Estriol, an oestrogen, at 0.6 nmol/l was reported to inhibit ADP-induced platelet aggregation through nitric oxide synthesis. As nitric oxide has been reported to cause fibrinolysis due to the activation of plasminogen to plasmin, the role of estriol as a fibrinolytic agent was investigated. Also, the mechanism of estriol-induced nitric oxide synthesis in anucleated platelets was investigated. The estriol-induced lysis of platelet-rich plasma (PRP) clot was determined by photography of the clot lysis and by the assay of fibrin degradation products in the lysate and was obtained by SDS-PAGE. Nitric oxide was determined by methemoglobin method. The platelet membrane protein was isolated from the platelets by using Triton X-100 (0.05% v/v). The binding of estriol to the protein was determined by Scatchard plot by using an ELISA for estriol. Estriol at 0.6 nmol/l was found to lyse the clotted PRP due to fibrinolysis that produced fibrin degradation products in the lysate. The amino acid analysis of the platelet membrane protein, which resembles with nitric oxide synthase (NOS) activity, was activated nearly 10-fold over the control in the presence of estriol and was identified to be a human serum albumin precursor (Mr. 69 kDa) that binds to estriol with Kd1 of 6.0 × 10 mol/l and 39 ± 2 molecules of estriol bound the NOS molecule. The estriol-induced nitric oxide is capable of inducing fibrinolysis of the clotted PRP. The binding of estriol to platelet membrane NOS activated the enzyme in the absence of DNA in the platelet.

  13. Prolactin, TNF alpha and nitric oxide expression in nitroso-N-methylurea-induced-mammary tumours

    PubMed Central

    Vegh, Irene; de Salamanca, Rafael Enríquez

    2007-01-01

    Background The N-Nitrosomethylurea breast cancer model induced in rats is used for the study of carcinogenesis in mammary cancer, prostate, pancreas, etc. This model is very similar to human neoplastic disease. Methods The present experimental study was designed to assess whether metoclopramide administration has any effect on development of MNU-induced tumours, and evaluate the treatment of goserelin acetate on PRL, TNF alpha and NO expression. NMU was administered to female Wistar rats on 2 occasions (5 mg/100 g body w/rat). PRL and TNF alpha were performed by immune-assay. Nitric Oxide by semi automated-assay and ploidy analyses by flow cytometry. Results The administration of metoclopramide made the induction time shorter and increased the incidence and average of tumours per rat. Tumours development was inhibited by a goserelin chronic administration. The ploidy of adenocarcinoma was polyploid-aneuploid type (average S = 60%). It was higher basal PRL plasma levels in rats with NMU induced tumours than in basal controls without tumour (p < 0.001). The goserelin "in bolus" administration showed maximal inhibition of plasma PRL at 90 min. Plasmatic TNF alpha expression was inhibited at 60 min and also remained inhibited in tissue homogenate post chronic treatment (P < 0.0125). Plasmatic NO expression is higher in rats with induced tumours than healthy controls (P < 0.001). In tissue homogenate NO values were inhibited at 90 min (P < 0.01), as well during chronically goserelin treatment (P < 0.005). Conclusion The increase of blood PRL levels in NMU-induced rats may be an indicator of a poor prognosis of mammary cancer evolution. The metoclopramide administration accelerates tumour growth. However goserelin administration achieves regression in tumour development associated to inhibition PRL, TNF alpha and NO expression. PMID:18045456

  14. Nitric oxide activates superoxide dismutase and ascorbate peroxidase to repress the cell death induced by wounding.

    PubMed

    Lin, Chih-Ching; Jih, Pei-Ju; Lin, Hsin-Hung; Lin, Jeng-Shane; Chang, Ling-Lan; Shen, Yu-Hsing; Jeng, Shih-Tong

    2011-10-01

    Wounding caused by rain, wind, and pathogen may lead plants to onset defense response. Previous studies indicated that mechanical wounding stimulates plants to generate nitric oxide (NO) and hydrogen peroxide (H(2)O(2)). In this study, the functions of NO and H(2)O(2) after wounding in sweet potato (Ipomoea batatas cv. Tainung 57) was further analyzed. Mechanical wounding damaged cells and resulted in necrosis, but the presence of NO donors or NO scavenger might reduce or enhance the cell death caused by wounding, respectively. The amount of H(2)O(2) induced by wounding was also decreased or increased when plants were incubated with NO donors or NO scavenger, individually. These results indicate that NO may regulate H(2)O(2) generation to affect cell death. NO-induced proteins isolated from two-dimensional electrophoresis were identified to be Copper/Zinc superoxide dismutases (CuZnSODs). The activities of CuZnSODs and ascorbate peroxidase (APX) could be enhanced by NO. In addition, the expression of CuZnSOD and APX was induced by wounding via NO, and their expression was further stimulated by NO through the generation of cGMP. The influx of calcium ions and the activity of NADPH oxidase were also involved in the NO signal transduction pathway inducing APX expression. Collectively, the generation of H(2)O(2) in wounded plants might trigger cell death. Meanwhile, the production of NO induced by wounding stimulated signal transducers including cGMP, calcium ions, and H(2)O(2) to activate CuZnSOD and APX, which further decreased H(2)O(2) level and reduced the cell death caused by wounding.

  15. Therapeutic insight into molsidomine, a nitric oxide donor in streptozotocin-induced diabetic nephropathy in rats

    PubMed Central

    Minaz, Nathani; Razdan, Rema

    2016-01-01

    Background: Diabetes-induced oxidative stress and hypertension play a major role in the development of nephropathy. Hence, the present study was undertaken to evaluate the protective effects of molsidomine, a nitric oxide donor in streptozotocin (STZ)-induced diabetic nephropathy (DN) in rats. Materials and Methods: Type 1 diabetes was induced through a single dose of STZ (52 mg/kg, i.p.) in male Wistar rats and then treated with molsidomine (5 and 10 mg/kg; p.o.) for 8 weeks. Physical parameters, vital and renal function test including blood glucose, albuminuria, blood urine nitrogen, serum creatinine, and kidney index were determined. Oxidative stress and lipid peroxidation were assessed in the kidney homogenate by means of antioxidant enzymes and malondialdehyde levels. Results: DN rats exhibited a significant renal dysfunction with a reduction in body weight, excessive oxidative stress, and pathological changes. Molsidomine treatment significantly improved vital sign, renal functions, and oxidative stress in DN rats in a dose-dependent manner. The protective effect of molsidomine was also substantiated by pathological changes in the architect of the kidney. Conclusion: Molsidomine shows a significant beneficial effect in Type 1 DN in rats. PMID:27721541

  16. Effects of phenylethanoid glycosides from Digitalis purpurea L. on the expression of inducible nitric oxide synthase.

    PubMed

    Oh, Jae Wook; Lee, Jeong Yong; Han, Song Hee; Moon, Young Hee; Kim, Yoon Gyoon; Woo, Eun-Rhan; Kang, Keon Wook

    2005-07-01

    We have isolated four different phenylethanoid glycosides (purpureaside A, desrhamnosyl acteoside, calceolarioside B and plantainoside D) from the leaves of Digitalis purpurea (foxglove). The effects of these glycosides on activator protein-1 (AP-1)-mediated inducible nitric oxide synthase (iNOS) gene expression in the Raw264.7 macrophage cell line have been studied. Of these four glycosides, purpureaside A potently inhibited iNOS induction by lipopolysaccharide (LPS). Increase in iNOS mRNA by LPS was completely suppressed by purpureaside A. Purpureaside A did not significantly affect LPS-inducible nuclear factor-kappaB (NF-kappaB) activation or the nuclear translocation of p65. Moreover, a reporter gene assay using AP-1 specific luciferase reporter revealed that the enhanced activity of AP-1 by LPS was completely abolished in cells treated with purpureaside A. These results demonstrated that purpureaside A inhibited LPS-inducible iNOS expression in macrophages through the suppression of AP-1, but not of NF-kappaB.

  17. Nitric oxide synthesis contributes to IL-2-induced antitumor responses against intraperitoneal Meth A tumor.

    PubMed

    Yim, C Y; McGregor, J R; Kwon, O D; Bastian, N R; Rees, M; Mori, M; Hibbs, J B; Samlowski, W E

    1995-11-01

    IL-2 therapy is a potent inductive stimulus for nitric oxide (NO.) synthesis in mice and humans. It is not yet clear whether NO. can contribute to IL-2-induced therapeutic responses. The murine skin cancer Meth A is relatively resistant to lymphokine-activated killer (LAK) cell killing, allowing evaluation of the role of IL-2-induced NO. synthesis in vivo, without contribution by LAK cells. Subcutaneous IL-2 treatment of mice bearing i.p. Meth A tumor increased nitrite production by cells derived from ascites (63 +/- 14 microM vs 3.2 +/- 1.5 microM in untreated controls). N omega-monomethyl-L-arginine (MLA), NO. synthase inhibitor, prevented this increase. NO. production correlated in an inverse fashion with tumor cell proliferation in vitro. Evidence for IL-2-induced heme nitrosylation was demonstrated in tumor cells by electron paramagnetic resonance spectroscopy. By immunomagnetic depletion experiments, macrophages were implicated as a major source of NO. synthesis. Cytologic and flow-cytometric evaluation revealed that IL-2 treatment resulted in enhanced lymphocyte and macrophage recruitment into malignant ascites, and decreases in tumor cell recovery. MLA administration further increased host cell recovery. Subcutaneous IL-2 therapy increased urinary nitrate excretion up to eightfold in mice, and appeared to produce a significant survival advantage that was prevented by MLA administration.

  18. Nitric oxide protects carbon assimilation process of watermelon from boron-induced oxidative injury.

    PubMed

    Farag, Mohamed; Najeeb, Ullah; Yang, Jinghua; Hu, Zhongyuan; Fang, Zhang Ming

    2017-02-01

    Nitric oxide (NO) mediates plant response to a variety of abiotic stresses; however, limited information is available on its effect on boron (B)-stressed watermelon plants. The present study investigates the mechanism through which NO protects watermelon seedlings from B deficiency and toxicity stresses. Five days old watermelon seedlings were exposed to B (0, 0.5 and 10 mg L(-1)) alone or with 75 μmole of NO donor sodium nitroprusside (SNP) for 30 days. Both low and high B concentrations in the media altered nutrient accumulation and impaired various physiological processes of watermelon seedlings, leading to a significant reduction in biomass production. The plants exposed to B deficient or toxic concentrations had 66 and 69% lower shoot dry weight, respectively compared with optimum B levels. B toxicity-induced growth inhibition of watermelon seedlings was associated with high B translocation to shoot tissues, which caused lipid membrane peroxidation (12% increase) and chlorophyll destruction (25% reduction). In contrast, B deficiency accelerated generation of reactive oxygen species (ROS), specifically OH(-1) and induced cellular oxidative injury. Exogenously applied SNP promoted leaf chlorophyll, photosynthesis and consequently biomass production in B-stressed watermelon seedlings by reducing B accumulation, lipid membrane peroxidation and ROS generation. It also activated antioxidant enzymes such as SOD, POD and APX, and protected the seedlings from ROS-induced cellular burst.

  19. Hypoxia inducible factor-1: regulation by nitric oxide in posthypoxic microvascular endothelium.

    PubMed

    Natarajan, Ramesh; Jones, Drew G; Fisher, Bernard J; Wallace, Timothy J; Ghosh, Shobha; Fowler, Alpha A

    2005-10-01

    Microvascular endothelial cells provide a critical regulatory interface between blood constituents and tissue. Hypoxia inducible factor-1 (HIF-1) is a key transcription factor required for expression of hypoxia-dependent genes. We employed a model of hypoxia and reoxygenation (H/R) using the dermal microvascular endothelial cell line HMEC-1 to examine the effects of altered oxygen concentrations on microvascular HIF-1 expression and nitric oxide (NO) formation. Hypoxia increased inducible NO synthase (iNOS) mRNA in a time-dependent manner in HMEC-1. However, endothelial NO synthase mRNA progressively declined during hypoxia. H/R promoted significant increases in cellular nitrite levels that were significantly abrogated by the specific iNOS inhibitor N6-(1-iminoethyl)-L-lysine, di hy drochloride. Exogenous NO promoted stabilization of the alpha subunit of HIF-1 and produced functional DNA binding. Exposure of HMEC-1 to H/R resulted in previously unrecognized biphasic HIF-1alpha stabilization during reoxygenation. When the iNOS gene was silenced through the use of iNOS-specific small interfering RNA, HIF-1alpha stabilization and HIF-1 activation were dramatically diminished, suggesting that inducible NOS-derived NO is a key factor sustaining HIF-1 activation during both hypoxia and reoxygenation.

  20. Nitric oxide is a mediator of tachykinin NK3 receptor-induced relaxation in rat mesenteric artery.

    PubMed Central

    Mizuta, A.; Takano, Y.; Honda, K.; Saito, R.; Matsumoto, T.; Kamiya, H.

    1995-01-01

    1. The mechanism of vasodilatation induced by tachykinin peptides was studied in isolated mesenteric arteries of rats. 2. Senktide, a selective NK3 agonist, elicited potent endothelium-dependent relaxation of arteries precontracted with phenylephrine (10(-5) M), but an NK1 agonist did not. 3. A non-peptide NK3 antagonist, SR 142801, inhibited senktide-induced relaxation. However, a non-peptide NK1 antagonist, CP-96,345, and a peptide-based NK2 antagonist, L-659,877, had no effect on senktide-induced relaxation. 4. N omega-nitro-L-arginine (L-NOARG), a nitric oxide synthesis inhibitor, markedly attenuated the relaxant response to senktide. 5. These results suggest that the endothelium of rat mesenteric arteries possesses tachykinin NK3 receptors, and that NK3 agonist-induced vasodilatation is mediated by release of nitric oxide (NO) from the endothelium. PMID:8680725

  1. Chitosan oligosaccharides suppress production of nitric oxide in lipopolysaccharide-induced N9 murine microglial cells in vitro.

    PubMed

    Wei, Peng; Ma, Pan; Xu, Qing-Song; Bai, Qun-Hua; Gu, Jian-Guo; Xi, Hao; Du, Yu-Guang; Yu, Chao

    2012-08-01

    Chitosan oligosaccharides (COS) have been reported to exert many biological activities, such as antioxidant, antitumor and anti-inflammatory effects. In the present study, we examined the effect of COS on nitric oxide (NO) production in LPS induced N9 microglial cells. Pretreatment with COS (50~200 μg/ml) could markedly inhibit NO production by suppressing inducible nitric oxide synthase (iNOS) expression in activated microglial cells. Signal transduction studies showed that COS remarkably inhibited LPS-induced phosphorylation of p38 MAPK and ERK1/2. COS pretreatment could also inhibit the activation of both nuclear factor-κB (NF-κB) and activator protein-1 (AP-1). In conclusion, our results suggest that COS could suppress the production of NO in LPS-induced N9 microglial cells, mediated by p38 MAPK and ERK1/2 pathways.

  2. Spironolactone Prevents Endothelial Nitric Oxide Synthase Uncoupling and Vascular Dysfunction Induced by β-Adrenergic Overstimulation

    PubMed Central

    Victorio, Jamaira A.; Clerici, Stefano P.; Palacios, Roberto; Alonso, María J.; Vassallo, Dalton V.; Jaffe, Iris Z.; Rossoni, Luciana V.

    2016-01-01

    Sustained stimulation of β-adrenoceptors (β-ARs) and activation of renin–angiotensin–aldosterone system are common features of cardiovascular diseases with rising sympathetic activation, including essential hypertension, myocardial infarction, and heart failure. In this study, we investigated the role of AT1 receptor and mineralocorticoid receptor (MR) in the vascular alterations caused by β-AR overstimulation. β-AR overstimulation with associated cardiac hypertrophy and increased vasoconstrictor response to phenylephrine in aorta were modeled in rats by 7-day isoproterenol treatment. The increased vasoconstrictor response to phenylephrine in this model was blunted by the MR antagonist spironolactone, but not by the AT1 receptor antagonist losartan, despite the blunting of cardiac hypertrophy with both drugs. Spironolactone, but not losartan, restored NO bioavailability in association with lower endothelial nitric oxide synthase–derived superoxide production, increased endothelial nitric oxide synthase dimerization, and aortic HSP90 upregulation. MR genomic and nongenomic functions were activated in aortas from isoproterenol-treated rats. Isoproterenol did not modify plasma levels of MR ligands aldosterone and corticosterone but rather increased perivascular adipose tissue–derived corticosterone in association with increased expression of 11β-hydroxysteroid dehydrogenase type 1. The anticontractile effect of aortic perivascular adipose tissue was impaired by β-AR overstimulation and restored by MR blockade. These results suggest that activation of vascular MR signaling contributes to the vascular dysfunction induced by β-AR overstimulation associated with endothelial nitric oxide synthase uncoupling. These findings reveal an additional explanation for the protective effects of MR antagonists in cardiovascular disorders with sympathetic activation. PMID:27432866

  3. CCTTT-repeat polymorphism of the inducible nitric oxide synthase is not associated with HIV pathogenesis

    PubMed Central

    HERSBERGER, M; BONHOEFFER, S; RAMPINI, S K; OPRAVIL, M; MARTI-JAUN, J; TELENTI, A; HÄNSELER, E; LEDERGERBER, B; Speck, R F

    2004-01-01

    Nitric oxide (NO) produced by the inducible form of nitric oxide synthase (iNOS) has bactericidal and virocidal effects. Although NO synthesis and iNOS expression in macrophages affect several aspects of human immunodeficiency virus (HIV) type-1 pathogenesis, their role in HIV disease remains largely unknown. In humans, the expression of iNOS is influenced by a functional CCTTT-repeat polymorphism in the promoter region of the gene. We investigated the association of this polymorphism with HIV pathogenesis in naive HIV-infected patients before the initiation of antiretroviral therapy. The allele frequencies of the iNOS CCTTT-repeat polymorphism were assessed by PCR in 857 patients from the Swiss HIV Cohort Study, including rapid progressors and long-term nonprogressors, and in 240 healthy volunteers. In HIV-infected patients, the initial viral load and the decline in total CD4 cells was calculated to estimate disease progression. Allele frequencies of the iNOS CCTTT-repeat polymorphism were similar between the HIV-infected and noninfected blood donors. In treatment-naive HIV-positive patients, there was no association of the iNOS polymorphism with viral load or with the course of CD4 cells. Regulation of iNOS expression by the functional CCTTT-polymorphism does not modify HIV pathogenesis. PMID:15320907

  4. Inducible and endothelial nitric oxide synthase expression during development of transplant arteriosclerosis in rat aortic grafts.

    PubMed Central

    Akyürek, L. M.; Fellström, B. C.; Yan, Z. Q.; Hansson, G. K.; Funa, K.; Larsson, E.

    1996-01-01

    In the vascular system, distinct isoforms of nitric oxide synthase (NOS) generate nitric oxide (NO), which acts as a biological messenger. Its role in the development of transplant arteriosclerosis (TA) is still unclear. To investigate whether NO is involved in TA, we studied the expression of NOS isoforms, inducible NOS (iNOS) and endothelial NOS (eNOS), by immunohistochemistry and in situ hybridization during the first two post-transplantation months and their relation with cold ischemia (1 to 24 hours) and reperfusion injury using an aortic transplantation model in the rat. We found an increased iNOS expression in the intima and adventitia and a decreased expression in the media, whereas eNOS expression was not significantly altered during the development of TA. Co-localization studies suggested that iNOS-positive cells were vascular smooth muscle cells, monocyte-derived macrophages, and endothelial cells. Prolonged ischemic storage time resulted in an increase in eNOS expression in the neointima. In situ hybridization showed iNOS mRNA expression by vascular cells in the neointima and media. NO produced by iNOS and eNOS may be involved, at least in part, in the pathogenesis of TA in aortic grafts. Additional studies are needed to confirm the modulatory mechanism of NO during the development of TA. Images Figure 3 Figure 4 Figure 6 PMID:8952533

  5. Bile acid induced colonic irritation stimulates intracolonic nitric oxide release in humans.

    PubMed Central

    Casellas, F; Mourelle, M; Papo, M; Guarner, F; Antolin, M; Armengol, J R; Malagelada, J R

    1996-01-01

    AIM--To measure the intracolonic release of nitric oxide end products (nitrates plus nitrites) and eicosanoids in response to intraluminal irritation with deoxycholic acid (DCA). PATIENTS--Seven patients with irritable bowel syndrome. METHODS--The left colon was perfused with a solution with or without 3 mM deoxycholic acid. Aspirates were assayed for eicosanoids by specific radioimmuno-assay, and for nitrates plus nitrites by the Griess reaction. To confirm that stimulated colonic mucosa can produce nitric oxide (NO), ancillary studies were performed in vitro using samples of normal mucosa obtained from five surgically resected colons. Samples were incubated for 30 minutes in Kreb's solution, 3 mM DCA or DCA with 1 mM L-nitro-arginine-methyl-ester (L-NAME) to inhibit the NO synthase. Finally, NO synthase activity was measured in five samples of human colonic mucosa. RESULTS--Intracolonic release of nitrates plus nitrites was basally undetectable in six of seven patients. Bile acid considerably increased the release of prostaglandin E2 and nitrates plus nitrites (p < 0.01). By contrast, no increase in thromboxane and leukotriene was seen. In vitro mucosal incubation with DCA increased the production of NO synthase products, which was blocked by L-NAME. Activity of Ca+2 independent NO synthase was detectable in four of five samples of human colonic mucosa. CONCLUSION--The human colonic mucosa responds to bile acid induced irritation by a surge in NO generation via NO synthase. PMID:8707118

  6. Inducible nitric oxide synthase in pulmonary alveolar macrophages from patients with tuberculosis

    PubMed Central

    1996-01-01

    The high-output pathway of nitric oxide production helps protect mice from infection by several pathogens, including Mycobacterium tuberculosis. However, based on studies of cells cultured from blood, it is controversial whether human mononuclear phagocytes can express the corresponding inducible nitric oxide synthase (iNOS;NOS2). The present study examined alveolar macrophages fixed directly after bronchopulmonary lavage. An average of 65% of the macrophages from 11 of 11 patients with untreated, culture-positive pulmonary tuberculosis reacted with an antibody documented herein to be monospecific for human NOS2. In contrast, a mean of 10% of bronchoalveolar lavage cells were positive from each of five clinically normal subjects. Tuberculosis patients' macrophages displayed diaphorase activity in the same proportion that they stained for NOS2, under assay conditions wherein the diaphorase reaction was strictly dependent on NOS2 expression. Bronchoalveolar lavage specimens also contained NOS2 mRNA. Thus, macrophages in the lungs of people with clinically active Mycobacterium tuberculosis infection often express catalytically competent NOS2. PMID:8642338

  7. Manganese-induced oxidative stress in two ontogenetic stages of chamomile and amelioration by nitric oxide.

    PubMed

    Kováčik, Jozef; Babula, Petr; Hedbavny, Josef; Švec, Pavel

    2014-02-01

    Impact of manganese (Mn(2+)) excess (100, 500 and 1000 μM over 7 days) on two ontogenetic stages (7-week-old plants and 7-day-old seedlings) of Matricaria chamomilla was compared. Mn excess depressed growth of seedlings (but not germination) and stimulated oxidative stress (ROS and lipid peroxidation) in both plants and seedlings. Growth inhibition could be evoked by higher Mn uptake and higher translocation factor in seedlings than in plants. Total thiols staining revealed elevation in almost all treatments. In 7-week-old plants, activity of peroxidases increased slightly and rather decreased under high Mn doses. Superoxide rather than hydrogen peroxide contributed to visualized ROS presence. Fluorescence of nitric oxide (NO) showed stimulation in plants but decrease in seedlings. Impact of exogenous nitric oxide donor (sodium nitroprusside/SNP) was therefore tested and results showed amelioration of 1000 μM Mn-induced oxidative stress in seedlings (decrease in H2O2 and increase in NO content while antioxidative enzyme activities were variably affected) concomitantly with depleted Mn accumulation. It is concluded that NO participates in tolerance to Mn excess but negative effects of the highest SNP dose were also observed. Extensive fluorescence microscopy is also explanatively discussed.

  8. Cloning and Characterization of Inducible Nitric Oxide Synthase from Mouse Macrophages

    NASA Astrophysics Data System (ADS)

    Xie, Qiao-Wen; Cho, Hearn J.; Calaycay, Jimmy; Mumford, Richard A.; Swiderek, Kristine M.; Lee, Terry D.; Ding, Aihao; Troso, Tiffany; Nathan, Carl

    1992-04-01

    Nitric oxide (NO) conveys a variety of messages between cells, including signals for vasorelaxation, neurotransmission, and cytotoxicity. In some endothelial cells and neurons, a constitutive NO synthase is activated transiently by agonists that elevate intracellular calcium concentrations and promote the binding of calmodulin. In contrast, in macrophages, NO synthase activity appears slowly after exposure of the cells to cytokines and bacterial products, is sustained, and functions independently of calcium and calmodulin. A monospecific antibody was used to clone complementary DNA that encoded two isoforms of NO synthase from immunologically activated mouse macrophages. Liquid chromatography-mass spectrometry was used to confirm most of the amino acid sequence. Macrophage NO synthase differs extensively from cerebellar NO synthase. The macrophage enzyme is immunologically induced at the transcriptional level and closely resembles the enzyme in cytokine-treated tumor cells and inflammatory neutrophils.

  9. Nitric oxide, a new player in L-DOPA-induced dyskinesia?

    PubMed

    Del-Bel, Elaine; Padovan-Neto, Fernando E; Bortolanza, Mariza; Tumas, Vitor; Aguiar, Aderbal S; Raisman-Vozari, Rita; Prediger, Rui D

    2015-01-01

    L-3,4-Dihydroxyphenylalanine (L-DOPA) remains the most effective symptomatic treatment of Parkinson's disease (PD). However, the long-term use of L-DOPA causes, in combination with disease progression, the development of motor complications termed L-DOPA-induced dyskinesia (LID). LID is the result of profound modifications in the functional organization of the basal ganglia circuitry. There is increasing evidence of the involvement of non-dopaminergic systems on the pathophysiology of LID. This raises the possibility of novel promising therapeutic approaches in the future, including agents that interfere with glutamatergic, serotonergic, adenosine, adrenergic, and cholinergic neurotransmission that are currently in preclinical testing or clinical development. Herein, we summarize the current knowledge of the pharmacology of LID in PD. More importantly, this review attempts to highlight the role of nitric oxide (NO) in PD and provide a comprehensive picture of recent preclinical findings from our group and others showing its potential involvement in dyskinesia.

  10. Lignans from Arctium lappa and their inhibition of LPS-induced nitric oxide production.

    PubMed

    Park, So Young; Hong, Seong Su; Han, Xiang Hua; Hwang, Ji Sang; Lee, Dongho; Ro, Jai Seup; Hwang, Bang Yeon

    2007-01-01

    A new butyrolactone sesquilignan, isolappaol C (1), together with four known lignans, lappaol C (2), lappaol D (3), lappaol F (4), and diarctigenin (5), were isolated from the methanolic extract of the seeds from the Arctium lappa plant. The structure of isolappaol C (1) was determined by spectral analysis including 1D- and 2D-NMR. All the isolates were evaluated for their inhibitory effects on the LPS-induced nitric oxide production using murine macrophage RAW264.7 cells. Lappaol F (4) and diarctigenin (5) strongly inhibited NO production in the LPS-stimulated RAW264.7 cells with IC(50) values of 9.5 and 9.6 microM, respectively.

  11. Immunohistochemical localization of endothelial and inducible nitric oxide synthase within neurons of cattle with rabies.

    PubMed

    Shin, Taekyun; Weinstock, Daniel; Castro, Marlene D; Hamir, Amir N; Wampler, Thomas; Walter, Mark; Kim, Hyun Young; Acland, Helen

    2004-05-01

    The expression of constitutive endothelial nitric oxide synthase (eNOS) and inducible NOS (iNOS) in the brains of cattle with natural rabies was studied. Increased expression of eNOS was detected in neurons of the brain stem and Purkinje cells of cerebellum. By contrast, iNOS was diffusely localized in the cytoplasm of affected neurons, and some inflammatory cells were positive. eNOS and rabies antigen were co-localized in inclusion bodies (Negri bodies) in neurons. The specific localization of eNOS, but not iNOS, in the Negri bodies suggests that eNOS is involved in the formation of rabies virus inclusion bodies.

  12. Polarized distribution of inducible nitric oxide synthase regulates activity in intestinal epithelial cells

    PubMed Central

    Rumbo, Martin; Courjault-Gautier, Françoise; Sierro, Frédéric; Sirard, Jean-Claude; Felley-Bosco, Emanuela

    2005-01-01

    Summary Inducible nitric oxide synthase (iNOS) functions as a homodimer. In cell extracts, iNOS molecules partition both in cytosolic and particulate fractions, indicating that iNOS exist as soluble and membrane associated forms. In this study, iNOS features were investigated in human intestinal epithelial cells stimulated with cytokines and in duodenum from mice exposed to flagellin. Our experiments indicate that iNOS is mainly associated to the particulate fraction of cell extracts. Confocal microscopy showed a preferential localization of iNOS at the apical pole of intestinal epithelial cells. In particulate fractions, iNOS dimers were more abundant than in the cytosolic fraction. Similar observations were done in mouse duodenum samples. These results suggest that, in epithelial cells, iNOS activity is regulated by localization-dependent processes. PMID:15654882

  13. The monocyte locomotion inhibitory factor produced by Entamoeba histolytica inhibits induced nitric oxide production in human leukocytes.

    PubMed

    Rico, G; Leandro, E; Rojas, S; Giménez, J A; Kretschmer, R R

    2003-07-01

    The monocyte locomotion inhibitory factor, an anti-inflammatory pentapeptide produced by Entamoeba histolytica, inhibits the in vitro production of nitric oxide induced by cytokines (INF-gamma, TNF-alpha) or PMA in human leukocytes. This can be added to the other previously reported functional effects of this factor, such as the inhibition of monocyte locomotion and the synthesis of reactive oxygen intermediates in both monocytes and neutrophils. The decreased nitric oxide production may interfere with the killing of amebas by neutrophils in the early invasive stages of amebiasis, when oxidative mechanisms are used [reactive oxygen and nitrogen intermediates either individually or synergistically via peroxynitrite (ONOO(-))], and in the advanced stages, when both non-oxidative and oxidative (including nitric oxide) mechanisms are employed by macrophages. Diminished nitric oxide production by leukocytes may also contribute to the paucity of late inflammatory components in amebic abscess of the liver and other amebic lesions.

  14. Citrus nobiletin suppresses inducible nitric oxide synthase gene expression in interleukin-1β-treated hepatocytes

    SciTech Connect

    Yoshigai, Emi; Machida, Toru; Okuyama, Tetsuya; Mori, Masatoshi; Murase, Hiromitsu; Yamanishi, Ryota; Okumura, Tadayoshi; Ikeya, Yukinobu; Nishino, Hoyoku; Nishizawa, Mikio

    2013-09-13

    Highlights: •Nobiletin is a polymethoxylated flavone that is abundant in citrus peels. •Nobiletin is a major constituent of the Citrus unshiu peel extract. •Nobiletin suppresses induction of NO and reduces iNOS expression in hepatocytes. •Nobiletin reduces the iNOS promoter activity and the DNA-binding activity of NF-κB. -- Abstract: Background: Nobiletin is a polymethoxylated flavone that is abundant in the peels of citrus fruits, such as Citrus unshiu (Satsuma mandarin) and Citrus sinensis. The dried peels of C. unshiu (chinpi) have been included in several formulae of Japanese Kampo medicines. Nobiletin may suppress the induction of inducible nitric oxide synthase (iNOS), which synthesizes the inflammatory mediator nitric oxide (NO) in hepatocytes. Methods: A C. unshiu peel (CUP) extract was prepared. Primary cultured rat hepatocytes were treated with the CUP extract or nobiletin in the presence of interleukin 1β (IL-1β), which induces iNOS expression. NO production and iNOS gene expression were analyzed. Results: High-performance liquid chromatography analyses revealed that the nobiletin content in the CUP extract was 0.14%. Nobiletin dose-dependently reduced the NO levels and decreased iNOS expression at the protein, mRNA and antisense transcript levels. Flavone, which does not contain any methoxy groups, also suppressed iNOS induction. Nobiletin reduced the transcriptional activity of iNOS promoter-luciferase constructs and the DNA-binding activity of nuclear factor κB (NF-κB) in the nuclei. Conclusions: The suppression of iNOS induction by nobiletin suggests that nobiletin may be responsible for the anti-inflammatory effects of citrus peels and have a therapeutic potential for liver diseases.

  15. Hepatitis B virus X protein regulates hepatic glucose homeostasis via activation of inducible nitric oxide synthase.

    PubMed

    Shin, Hye-Jun; Park, Young-Ho; Kim, Sun-Uk; Moon, Hyung-Bae; Park, Do Sim; Han, Ying-Hao; Lee, Chul-Ho; Lee, Dong-Seok; Song, In-Sung; Lee, Dae Ho; Kim, Minhye; Kim, Nam-Soon; Kim, Dae-Ghon; Kim, Jin-Man; Kim, Sang-Keun; Kim, Yo Na; Kim, Su Sung; Choi, Cheol Soo; Kim, Young-Bum; Yu, Dae-Yeul

    2011-08-26

    Dysregulation of liver functions leads to insulin resistance causing type 2 diabetes mellitus and is often found in chronic liver diseases. However, the mechanisms of hepatic dysfunction leading to hepatic metabolic disorder are still poorly understood in chronic liver diseases. The current work investigated the role of hepatitis B virus X protein (HBx) in regulating glucose metabolism. We studied HBx-overexpressing (HBxTg) mice and HBxTg mice lacking inducible nitric oxide synthase (iNOS). Here we show that gene expressions of the key gluconeogenic enzymes were significantly increased in HepG2 cells expressing HBx (HepG2-HBx) and in non-tumor liver tissues of hepatitis B virus patients with high levels of HBx expression. In the liver of HBxTg mice, the expressions of gluconeogenic genes were also elevated, leading to hyperglycemia by increasing hepatic glucose production. However, this effect was insufficient to cause systemic insulin resistance. Importantly, the actions of HBx on hepatic glucose metabolism are thought to be mediated via iNOS signaling, as evidenced by the fact that deficiency of iNOS restored HBx-induced hyperglycemia by suppressing the gene expression of gluconeogenic enzymes. Treatment of HepG2-HBx cells with nitric oxide (NO) caused a significant increase in the expression of gluconeogenic genes, but JNK1 inhibition was completely normalized. Furthermore, hyperactivation of JNK1 in the liver of HBxTg mice was also suppressed in the absence of iNOS, indicating the critical role for JNK in the mutual regulation of HBx- and iNOS-mediated glucose metabolism. These findings establish a novel mechanism of HBx-driven hepatic metabolic disorder that is modulated by iNOS-mediated activation of JNK.

  16. Stimulation of Inducible Nitric Oxide by Hepatitis B Virus Transactivator Protein HBx Requires MTA1 Coregulator*

    PubMed Central

    Bui-Nguyen, Tri M.; Pakala, Suresh B.; Sirigiri, Divijendranatha Reddy; Martin, Emil; Murad, Ferid; Kumar, Rakesh

    2010-01-01

    Nitric oxide has been implicated in the pathogenesis of inflammatory disorders, including hepatitis B virus-associated hepatocellular carcinoma. Transactivator protein HBx, a major regulator of cellular responses of hepatitis B virus, is known to induce the expression of MTA1 (metastasis-associated protein 1) coregulator via NF-κB signaling in hepatic cells. However, the underlying mechanism of HBx regulation of the inducible nitric-oxide synthase (iNOS) pathway remains unknown. Here we provide evidence that MTA1 is a positive regulator of iNOS transcription and plays a mechanistic role in HBx stimulation of iNOS expression and activity. We found that the HBx-MTA1 complex is recruited onto the human iNOS promoter in an NF-κB-dependent manner. Pharmacological inhibition of the NF-κB signaling prevented the ability of HBx to stimulate the transcription, the expression, and the activity of iNOS; nevertheless, these effects could be substantially rescued by MTA1 dysregulation. We further discovered that HBx-mediated stimulation of MTA1 is paralleled by the suppression of miR-661, a member of the small noncoding RNAs, recently shown to target MTA1. We observed that miR-661 controls of MTA1 expression contributed to the expression and activity of iNOS in HBx-expressing HepG2 cells. Accordingly, depletion of MTA1 by either miR-661 or siRNA in HBx-expressing cells severely impaired the ability of HBx to modulate the endogenous levels of iNOS and nitrite production. Together, these findings reveal an inherent role of MTA1 in HBx regulation of iNOS expression and consequently its function in the liver cancer cells. PMID:20022949

  17. Bilirubin prevents acute DSS-induced colitis by inhibiting leukocyte infiltration and suppressing upregulation of inducible nitric oxide synthase.

    PubMed

    Zucker, Stephen D; Vogel, Megan E; Kindel, Tammy L; Smith, Darcey L H; Idelman, Gila; Avissar, Uri; Kakarlapudi, Ganesh; Masnovi, Michelle E

    2015-11-15

    Bilirubin is thought to exert anti-inflammatory effects by inhibiting vascular cell adhesion molecule-1 (VCAM-1)-dependent leukocyte migration and by suppressing the expression of inducible nitric oxide synthase (iNOS). As VCAM-1 and iNOS are important mediators of tissue injury in the dextran sodium sulfate (DSS) murine model of inflammatory colitis, we examined whether bilirubin prevents colonic injury in DSS-treated mice. Male C57BL/6 mice were administered 2.5% DSS in the drinking water for 7 days, while simultaneously receiving intraperitoneal injections of bilirubin (30 mg/kg) or potassium phosphate vehicle. Disease activity was monitored, peripheral blood counts and serum nitrate levels were determined, and intestinal specimens were analyzed for histological injury, leukocyte infiltration, and iNOS expression. The effect of bilirubin on IL-5 production by HSB-2 cells and on Jurkat cell transendothelial migration also was determined. DSS-treated mice that simultaneously received bilirubin lost less body weight, had lower serum nitrate levels, and exhibited reduced disease severity than vehicle-treated animals. Concordantly, histopathological analyses revealed that bilirubin-treated mice manifested significantly less colonic injury, including reduced infiltration of eosinophils, lymphocytes, and monocytes, and diminished iNOS expression. Bilirubin administration also was associated with decreased eosinophil and monocyte infiltration into the small intestine, with a corresponding increase in peripheral blood eosinophilia. Bilirubin prevented Jurkat migration but did not alter IL-5 production. In conclusion, bilirubin prevents DSS-induced colitis by inhibiting the migration of leukocytes across the vascular endothelium and by suppressing iNOS expression.

  18. YC-1 potentiates cAMP-induced CREB activation and nitric oxide production in alveolar macrophages

    SciTech Connect

    Hwang, Tsong-Long; Tang, Ming-Chi; Kuo, Liang-Mou; Chang, Wen-De; Chung, Pei-Jen; Chang, Ya-Wen; Fang, Yao-Ching

    2012-04-15

    Alveolar macrophages play significant roles in the pathogenesis of several inflammatory lung diseases. Increases in exhaled nitric oxide (NO) are well documented to reflect disease severity in the airway. In this study, we investigated the effect of 3-(5′-hydroxymethyl-2′-furyl)-1-benzyl indazole (YC-1), a known activator of soluble guanylyl cyclase, on prostaglandin (PG)E{sub 1} (a stable PGE{sub 2} analogue) and forskolin (a adenylate cyclase activator) induced NO production and inducible NO synthase (iNOS) expression in rat alveolar macrophages (NR8383). YC-1 did not directly cause NO production or iNOS expression, but drastically potentiated PGE{sub 1}- or forskolin-induced NO production and iNOS expression in NR8383 alveolar macrophages. Combination treatment with YC-1 and PGE{sub 1} significantly increased phosphorylation of the cAMP response element-binding protein (CREB), but not nuclear factor (NF)-κB activation. The combined effect on NO production, iNOS expression, and CREB phosphorylation was reversed by a protein kinase (PK)A inhibitor (H89), suggesting that the potentiating functions were mediated through a cAMP/PKA signaling pathway. Consistent with this, cAMP analogues, but not the cGMP analogue, caused NO release, iNOS expression, and CREB activation. YC-1 treatment induced an increase in PGE{sub 1}-induced cAMP formation, which occurred through the inhibition of cAMP-specific phosphodiesterase (PDE) activity. Furthermore, the combination of rolipram (an inhibitor of PDE4), but not milronone (an inhibitor of PDE3), and PGE{sub 1} also triggered NO production and iNOS expression. In summary, YC-1 potentiates PGE{sub 1}-induced NO production and iNOS expression in alveolar macrophages through inhibition of cAMP PDE activity and activation of the cAMP/PKA/CREB signaling pathway. Highlights: ► YC-1 potentiated PGE1-induced iNOS expression in alveolar macrophages. ► The combination of YC-1 and PGE1 increased CREB but not NFκB activation.

  19. Nitric oxide is involved in phosphorus deficiency-induced cluster root development and citrate exudation in white lupin

    Technology Transfer Automated Retrieval System (TEKTRAN)

    White lupin (Lupinus albus) forms specialized cluster roots characterized by exudation of organic anions under phosphorus (P) deficiency. Here, we evaluated the role of nitric oxide (NO) in P deficiency-induced cluster-root formation and citrate exudation in white lupin. Plants were treated with NO ...

  20. Hemorrhage-induced Vascular Hyporeactivity to Norepinephrine in Select Vasculatures of Rats and the Roles of Nitric Oxide and Endothelin

    DTIC Science & Technology

    2003-03-01

    Hepatology 27:755– 764, 1998. 20. Iglarz M, Levy BI, Henrion D: Chronic endothelin-1 induced changes in vascu- lar reactivity in rat resistance...Mitchell JA, Vane JR: Vascular hyporeactivity to vasoconstrictor agents and hemodynamic decompensation in hemorrhagic shock is mediated by nitric oxide

  1. Inducible nitric oxide synthase expression in chronic viral hepatitis. Evidence for a virus-induced gene upregulation.

    PubMed Central

    Majano, P L; García-Monzón, C; López-Cabrera, M; Lara-Pezzi, E; Fernández-Ruiz, E; García-Iglesias, C; Borque, M J; Moreno-Otero, R

    1998-01-01

    Increased nitric oxide (NO) production may contribute to the pathological changes featuring in some inflammatory diseases, but the role of NO in chronic viral hepatitis is still unknown. We compared the inducible NO synthase (NOS2) expression in the liver of patients with chronic viral hepatitis with that of both nonviral liver disease and histologically normal liver. NOS2 expression was assessed by immunohistochemical and in situ hybridization studies of liver biopsy sections. An intense hepatocellular NOS2 reactivity was detected in chronic viral hepatitis, whereas it was weakly or not observed in nonviral liver disease or normal liver, respectively. In addition, we determined whether the hepatitis B virus (HBV) might regulate the synthesis of this enzyme. NOS2 mRNA and protein levels as well as enzyme activity were assessed in cytokine-stimulated HBV-transfected and untransfected hepatoma cells. Transfection with either HBV genome or HBV X gene resulted in induction of NOS2 mRNA expression, and the maximal induction of this transcript and NO production was observed in cytokine-stimulated HBV-transfected cells. These results indicate that hepatotropic viral infections are able to upregulate the NOS2 gene expression in human hepatocytes, suggesting that NO may mediate important pathogenic events in the course of chronic viral hepatitis. PMID:9525976

  2. Trimethyltin-induced apoptosis is associated with upregulation of inducible nitric oxide synthase and Bax in a hippocampal cell line

    SciTech Connect

    Zhang, L.; Li, L.; Prabhakaran, K.; Borowitz, J.L.; Isom, G.E. . E-mail: geisom@purdue.edu

    2006-10-01

    Trimethyltin (TMT) produces selective neuronal degeneration in the central nervous system (CNS), in which the hippocampus is the most sensitive area. Since previous studies have been conducted in either non-neural cells or mixed primary cultures, an immortalized hippocampal neuronal cell line (HT-22 cell) was used to assess the mechanism and mode of death produced by TMT. The compound produced a time- and concentration-dependent apoptotic death that was caspase-mediated. Excessive generation of reactive oxygen species (ROS) and subsequent reduction of mitochondrial membrane potential ({delta}{psi}{sub m}) were involved in the cytotoxicity{sub .} Scavenging of ROS by a free radical trapping agent or inhibition of the mitochondrial permeability transition (MPT) pore significantly reduced cell death. Additionally, TMT increased expression of inducible nitric oxide synthase (iNOS) by activation of the redox-sensitive transcription factor NF{kappa}B. Pharmacologic inhibition studies showed that the iNOS-mediated NO generation increased expression of Bax and then mitochondrial-mediated apoptosis. It was concluded that excessive ROS generation initiated the apoptotic cell death by upregulating iNOS followed by increased Bax expression which then led to loss of {delta}{psi}{sub m} and caspase-executed cell death. This study is the first to report in a neuronal cell model that TMT stimulates induction of iNOS, which then increases cellular levels of reactive nitrogen species (RNS) to initiate apoptotic death.

  3. Integrins mediate mechanical compression-induced endothelium-dependent vasodilation through endothelial nitric oxide pathway.

    PubMed

    Lu, Xiao; Kassab, Ghassan S

    2015-09-01

    Cardiac and skeletal muscle contraction lead to compression of intramuscular arterioles, which, in turn, leads to their vasodilation (a process that may enhance blood flow during muscle activity). Although endothelium-derived nitric oxide (NO) has been implicated in compression-induced vasodilation, the mechanism whereby arterial compression elicits NO production is unclear. We cannulated isolated swine (n = 39) myocardial (n = 69) and skeletal muscle (n = 60) arteriole segments and exposed them to cyclic transmural pressure generated by either intraluminal or extraluminal pressure pulses to simulate compression in contracting muscle. We found that the vasodilation elicited by internal or external pressure pulses was equivalent; moreover, vasodilation in response to pressure depended on changes in arteriole diameter. Agonist-induced endothelium-dependent and -independent vasodilation was used to verify endothelial and vascular smooth muscle cell viability. Vasodilation in response to cyclic changes in transmural pressure was smaller than that elicited by pharmacological activation of the NO signaling pathway. It was attenuated by inhibition of NO synthase and by mechanical removal of the endothelium. Stemming from previous observations that endothelial integrin is implicated in vasodilation in response to shear stress, we found that function-blocking integrin α5β1 or αvβ3 antibodies attenuated cyclic compression-induced vasodilation and NOx (NO(-)2 and NO(-)3) production, as did an RGD peptide that competitively inhibits ligand binding to some integrins. We therefore conclude that integrin plays a role in cyclic compression-induced endothelial NO production and thereby in the vasodilation of small arteries during cyclic transmural pressure loading.

  4. Hybrid inhibitor of peripheral cannabinoid-1 receptors and inducible nitric oxide synthase mitigates liver fibrosis

    PubMed Central

    Iyer, Malliga R.; Liu, Ziyi; Cao, Zongxian; Jourdan, Tony; Erdelyi, Katalin; Godlewski, Grzegorz; Szanda, Gergő; Liu, Jie; Park, Joshua K.; Mukhopadhyay, Bani; Rosenberg, Avi Z.; Liow, Jeih-San; Lorenz, Robin G.; Pacher, Pal; Innis, Robert B.

    2016-01-01

    Liver fibrosis, a consequence of chronic liver injury and a way station to cirrhosis and hepatocellular carcinoma, lacks effective treatment. Endocannabinoids acting via cannabinoid-1 receptors (CB1R) induce profibrotic gene expression and promote pathologies that predispose to liver fibrosis. CB1R antagonists produce opposite effects, but their therapeutic development was halted due to neuropsychiatric side effects. Inducible nitric oxide synthase (iNOS) also promotes liver fibrosis and its underlying pathologies, but iNOS inhibitors tested to date showed limited therapeutic efficacy in inflammatory diseases. Here, we introduce a peripherally restricted, orally bioavailable CB1R antagonist, which accumulates in liver to release an iNOS inhibitory leaving group. In mouse models of fibrosis induced by CCl4 or bile duct ligation, the hybrid CB1R/iNOS antagonist surpassed the antifibrotic efficacy of the CB1R antagonist rimonabant or the iNOS inhibitor 1400W, without inducing anxiety-like behaviors or CB1R occupancy in the CNS. The hybrid inhibitor also targeted CB1R-independent, iNOS-mediated profibrotic pathways, including increased PDGF, Nlrp3/Asc3, and integrin αvβ6 signaling, as judged by its ability to inhibit these pathways in cnr1–/– but not in nos2–/– mice. Additionally, it was able to slow fibrosis progression and to attenuate established fibrosis. Thus, dual-target peripheral CB1R/iNOS antagonists have therapeutic potential in liver fibrosis. PMID:27525312

  5. Mechanisms Of Hypoxia-Induced Immune Escape In Cancer And Their Regulation By Nitric Oxide.

    PubMed

    Graham, Charles; Barsoum, Ivraym; Kim, Judy; Black, Madison; Siemens, Robert D

    2015-08-01

    The acquired ability of tumour cells to avoid destruction by immune effector mechanisms (immune escape) is important for malignant progression. Also associated with malignant progression is tumour hypoxia, which induces aggressive phenotypes such as invasion, metastasis and drug resistance in cancer cells. Our studies revealed that hypoxia contributes to escape from innate immunity by increasing tumour cell expression of the metalloproteinase ADAM10 in a manner dependent on accumulation of the alpha subunit of the transcription factor hypoxia-inducible factor-1 (HIF-1α). Increased ADAM10 expression leads to shedding of the NK cell-activating ligand, MICA, from the surface of tumour cells, thereby resulting in resistance to NK cell-mediated lysis. Our more recent studies demonstrated that hypoxia, also via HIF-1α accumulation, increases the expression of the inhibitory co-stimulatory ligand PD-L1 on tumour cells. Elevated PD-L1 expression leads to escape from adaptive immunity via increased apoptosis of CD8(+) cytotoxic T lymphocytes. Accumulating evidence indicates that hypoxia-induced acquisition of malignant phenotypes, including immune escape, is in part due to impaired nitric oxide (NO)-mediated activation of cGMP signalling and that restoration of cGMP signalling prevents such hypoxic responses. We have shown that NO/cGMP signalling inhibits hypoxia-induced malignant phenotypes likely in part by interfering with HIF-1α accumulation via a mechanism involving calpain. These findings indicate that activation of NO/cGMP signalling may have useful applications in cancer therapy.

  6. Protein kinase A mediates inhibition of the thrombin-induced platelet shape change by nitric oxide.

    PubMed

    Jensen, Baard Olav; Selheim, Frode; Døskeland, Stein Ove; Gear, Adrian R L; Holmsen, Holm

    2004-11-01

    The thrombin-induced platelet shape change was blocked by nitric oxide (NO), as revealed by scanning electron microscopy, light transmission, and resistive-particle volume determination. The inhibitory effect of NO was accompanied by an increase in levels of both cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) and phosphorylation of the vasodilator-stimulated phosphoprotein (VASP). However, the inhibition of the shape change was only mimicked by cAMP analogs (Sp-5,6-DClcBIMPS, 8-AHA-cAMP, and 8-CPT-cAMP) and not by cGMP analogs (8-Br-PET-cGMP, 8-Br-cGMP, and 8-pCPT-cGMP). The effect of NO on the thrombin-induced shape change was prevented by the protein kinase A (PKA) antagonists Rp-8-Br-cAMPS and Rp-cAMPS. The protein kinase G (PKG) antagonist Rp-8-CPT-cGMPS strongly inhibited PKG-mediated 46-kDa VASP Ser239 phosphorylation, but did not inhibit the thrombin-induced shape change or the PKA-mediated VASP Ser157 phosphorylation. Whereas an inhibitor of cyclic nucleotide phosphodiesterase (PDE) 3A (milrinone) mimicked the effect of NO, inhibitors of PDE2 (erythro-9-(2-hydroxy-3-nonyl)adenine) and PDE5 (dipyridamole) were poorly effective. We concluded that (1) NO was a potent and reversible inhibitor of the platelet shape change, (2) the shape change was reversible, (3) the inhibitory effect of NO was mediated through activation of PKA, (4) the onset of the NO effect coincided with VASP Ser157 phosphorylation, and (5) removal of NO and platelet shape change coincided with VASP Ser157 dephosphorylation. These findings are compatible with elevation of cGMP by NO in a compartment close to PDE3A, PKA, and VASP, leading to a local increase of cAMP able to block thrombin-induced shape change.

  7. Integrins mediate mechanical compression–induced endothelium-dependent vasodilation through endothelial nitric oxide pathway

    PubMed Central

    Lu, Xiao

    2015-01-01

    Cardiac and skeletal muscle contraction lead to compression of intramuscular arterioles, which, in turn, leads to their vasodilation (a process that may enhance blood flow during muscle activity). Although endothelium-derived nitric oxide (NO) has been implicated in compression-induced vasodilation, the mechanism whereby arterial compression elicits NO production is unclear. We cannulated isolated swine (n = 39) myocardial (n = 69) and skeletal muscle (n = 60) arteriole segments and exposed them to cyclic transmural pressure generated by either intraluminal or extraluminal pressure pulses to simulate compression in contracting muscle. We found that the vasodilation elicited by internal or external pressure pulses was equivalent; moreover, vasodilation in response to pressure depended on changes in arteriole diameter. Agonist-induced endothelium-dependent and -independent vasodilation was used to verify endothelial and vascular smooth muscle cell viability. Vasodilation in response to cyclic changes in transmural pressure was smaller than that elicited by pharmacological activation of the NO signaling pathway. It was attenuated by inhibition of NO synthase and by mechanical removal of the endothelium. Stemming from previous observations that endothelial integrin is implicated in vasodilation in response to shear stress, we found that function-blocking integrin α5β1 or αvβ3 antibodies attenuated cyclic compression–induced vasodilation and NOx (NO−2 and NO−3) production, as did an RGD peptide that competitively inhibits ligand binding to some integrins. We therefore conclude that integrin plays a role in cyclic compression–induced endothelial NO production and thereby in the vasodilation of small arteries during cyclic transmural pressure loading. PMID:26324675

  8. Differential cytokine expression in skin graft healing in inducible nitric oxide synthase knockout mice.

    PubMed

    Most, D; Efron, D T; Shi, H P; Tantry, U S; Barbul, A

    2001-10-01

    Inducible nitric oxide synthase (iNOS) and its product, nitric oxide, have been shown to play important roles in wound biology. The present study was performed to investigate the role of iNOS in modulating the cytokine cascade during the complex process of skin graft wound healing.Fifteen iNOS-knockout mice and 15 wild-type C57BL/6J mice were subjected to autogenous 1-cm2 intrascapular full-thickness skin grafts. Three animals in each group were killed on postoperative days 3, 5, 7, 10, and 14. Specimens were then analyzed using nonisotopic in situ hybridization versus mRNA of tumor growth factor-beta1, vascular endothelial growth factor, iNOS, endothelial nitric oxide synthase (eNOS), tumor necrosis factor-alpha, and basic fibroblast growth factor, as well as positive and negative control probes. Positive cells in both grafts and wound beds were counted using a Leica microgrid. Scar thickness was measured with a Leica micrometer. Data were analyzed using the unpaired Student's t test. Expression of iNOS was 2- to 4-fold higher in knockout mice than in wild-type mice on postoperative days 5, 7, and 14. Expression of eNOS was 2- to 2.5-fold higher in knockout mice than in wild-type mice on postoperative days 5 and 7. Tumor necrosis factor-alpha expression was 2- to 7-fold higher in knockout mice than in wild-type mice on all postoperative days. In contrast, expression levels of angiogenic/fibrogenic cytokines (vascular endothelial growth factor, basis fibroblast growth factor, and tumor growth factor-beta1) were 2.5- to 4-fold higher in wild-type mice than in knockout mice. Scars were 1.5- to 2.5-fold thicker in knockout mice than in wild-type mice at all time points. All of the above results represent statistically significant differences (p < 0.05). Significantly different patterns of cytokine expression were seen in knockout and wild-type mice. Although the scar layer was thicker in knockout mice, it showed much greater infiltration with inflammatory cells. These

  9. Role of Nitric Oxide in the Pathogenesis of Diabetic Nephropathy in Streptozotocin-Induced Diabetic Rats

    PubMed Central

    Choi, Ki Chul; Lee, Seong Cheol; Kim, Soo Wan; Kim, Nam Ho; Lee, Jong-Un; Kang, Young Joon

    1999-01-01

    Objectives Several reports suggest that enhanced generation or actions of nitric oxide (NO) have been implicated in the pathogenesis of glomerular hyperfiltration and hyperperfusion that occurs in early diabetes. However, the precise role of altered NO generation in the pathogenesis of diabetic nephropathy is unclear. The present study was aimed at investigating the role of nitric oxide in the pathogenesis of glomerular hyperfiltration and hyperperfusion in streptozotocin-induced diabetic rats. Methods To evaluate the role of NO in diabetic hyperfiltration, we measured plasma and urine concentrations of NO2−/NO3−, stable metabolic products of NO and protein expressions of three isoforms of nitric oxide synthase (NOS) in streptozotocin-induced diabetic rats. We also investigated renal hemodynamic changes, such as glomerular filtration rate (GFR) and renal plasma flow (RPF), in responses to acute and chronic administration of NO synthesis inhibitor, nitro-L-arginine methyl ester (L-NAME), in diabetic and control rats. Results Diabetic rats exhibited significantly elevated plasma and urinary NO2−/NO3− levels at 28 days after streptozotocin injection, and total excretion of NO2−/NO3− was approximately five-fold higher in diabetic rats than controls. Insulin and L-NAME treatment prevented the increases in plasma and urinary NO2−/NO3− concentrations in diabetic rats, respectively. The three isoforms of NOS (bNOS, iNOS, and ecNOS) were all increased in the renal cortex, whereas they remained unaltered in the renal medulla at day 28. GFR and RPF were significantly elevated in diabetic rats, and acute and chronic inhibition of NO synthesis by L-NAME attenuated the renal hemodynamic changes (increases in GFR and RPF) in diabetic rats, respectively. Conclusions NO synthesis was increased due to enhanced NOS expression in diabetic rats, and chronic NO blockade attenuated renal hyperfiltration and hyperperfusion in diabetic rats. In addition, diabetic rats

  10. Methamphetamine- and 1-methyl-4-phenyl- 1,2,3, 6-tetrahydropyridine-induced dopaminergic neurotoxicity in inducible nitric oxide synthase-deficient mice.

    PubMed

    Itzhak, Y; Martin, J L; Ali, S F

    1999-12-15

    Previous studies have suggested a role for the retrograde messenger, nitric oxide (NO), in methamphetamine (METH)- and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)- induced dopaminergic neurotoxicity. Since evidence supported the involvement of the neuronal nitric oxide synthase (nNOS) isoform in the dopaminergic neurotoxicity, the present study was undertaken to investigate whether the inducible nitric oxide synthase (iNOS) isoform is also associated with METH- and MPTP-induced neurotoxicity. The administration of METH (5mg/kg x 3) to iNOS deficient mice [homozygote iNOS(-/-)] and wild type mice (C57BL/6) resulted in significantly smaller depletion of striatal dopaminergic markers in the iNOS(-/-) mice compared with the wild-type mice. METH-induced hyperthermia was also significantly lower in the iNOS(-/-) mice than in wild-type mice. In contrast to the outcome of METH administration, MPTP injections (20 mg/kg x 3) resulted in a similar decrease in striatal dopaminergic markers in iNOS(-/-) and wild-type mice. In the set of behavioral experiments, METH-induced locomotor sensitization was investigated. The acute administration of METH (1.0 mg/kg) resulted in the same intensity of locomotor activity in iNOS(-/-) and wild-type mice. Moreover, 68 to 72 h after the exposure to the high-dose METH regimen (5 mg/kg x 3), a marked sensitized response to a challenge injection of METH (1.0 mg/kg) was observed in both the iNOS(-/-) and wild-type mice. The finding that iNOS(-/-) mice were unprotected from MPTP-induced neurotoxicity suggests that the partial protection against METH-induced neurotoxicity observed was primarily associated with the diminished hyperthermic effect of METH seen in the iNOS(-/-) mice. Moreover, in contrast to nNOS deficiency, iNOS deficiency did not affect METH-induced behavioral sensitization.

  11. Nitric oxide contributes to estrogen-induced vasodilation of the ovine uterine circulation.

    PubMed Central

    Rosenfeld, C R; Cox, B E; Roy, T; Magness, R R

    1996-01-01

    Estradiol-17beta (E2beta), a potent vasodilator, has its greatest effects on the uterine vasculature, blood flow (UBF) increasing > or = 10-fold. The mechanism(s) responsible for E2beta-induced vasodilation is unclear. We determined if nitric oxide (NO)-induced increases in cGMP modulate estrogen-induced increases in UBF, and if cyclooxygenase inhibition modifies E2beta responses. Nonpregnant (n = 15) and pregnant (n = 8) ewes had flow probes implanted on main uterine arteries and catheters in branches of the uterine vein and artery bilaterally for blood sampling and infusion of the NO synthase inhibitor L-nitro-arginine methyl ester (L-NAME), respectively. In nonpregnant ewes E2beta (1 microg/kg) caused parallel increases (P < 0.001) in UBF (15+/-3 to 130+/-16 ml/min) and uterine cGMP secretion (23+/-10 to 291+/-38 pmol/min); uterine venous cGMP also rose (4.98+/-1.4 to 9.43+/-3.2 pmol/ml; P < 0.001). Intra-arterial L-NAME partially inhibited increases in UBF dose-dependently (r = 0.66, n = 18, P < 0.003) while completely inhibiting cGMP secretion (P = 0.025). Indomethacin, 2 mg/kg intravenously, did not alter E2beta-induced responses. After E2beta-induced increases in UBF, intraarterial L-NAME partially decreased UBF dose dependently (r = 0.73, n = 46, P < 0.001) while inhibiting cGMP secretion (178+/-48 to 50+/-24 pmol/min; n = 5, P = 0.006); both were reversed by L-arginine. In pregnant ewes, E2beta increased UBF and venous cGMP (9.1+/-0.96 to 13.2+/-0.96 pmol/ml, P < 0.01); however, intraarterial L-NAME decreased basal cGMP secretion 66% (P = 0.02), but not UBF. Acute estrogen-induced increases in UBF are associated with NO-dependent increases in cGMP synthesis, but other mechanisms may also be involved. However, vasodilating prostanoids do not appear to be important. In ovine pregnancy NO is not essential for maintaining uteroplacental vasodilation. PMID:8903336

  12. Puerarin suppresses production of nitric oxide and inducible nitric oxide synthase in lipopolysaccharide-induced N9 microglial cells through regulating MAPK phosphorylation, O-GlcNAcylation and NF-κB translocation.

    PubMed

    Zheng, Gao-Ming; Yu, Chao; Yang, Zhu

    2012-05-01

    Microglial cells play a critical role in mediating central nervous system inflammatory processes. Activated microglial cells induced by proinflammatory factor, such as lipopolysaccharide (LPS), release many kinds of neurotoxic cytokines including reactive oxygen species (ROS) which contributes to the pathogenesis of neurodegenerative diseases. Puerarin, extracted from kudzu root, possesses the characteristic of neuroprotection, antioxidation and anticancer. In the present study, we observed that LPS induced over-production of nitric oxide (NO) and increased the level of intracellular ROS in N9 microglial cells, but it was inhibited by puerarin. Furthermore, treatment with puerarin on N9 cells suppressed the over-expression of inducible nitric oxide synthase (iNOS) induced by LPS which is implicated in intracellular O-linked β-N-acetylglucosamine (O-GlcNAc) level, phosphorylation of mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB) signaling pathway. We also observed that the enhanced phosphorylation of p38, JNK and ERK1/2 in N9 cells induced by LPS were inhibited by puerarin, otherwise the down-regulation of O-GlcNAcylation level of protein in N9 cell induced by LPS was up-regulated by pretreatment with puerarin. These results indicate that puerarin effectively inhibits microglia activation induced by LPS through inhibiting expression of iNOS, production of NO and ROS which was mediated via regulating O-GlcNAcylation, phosphorylation of MAPK and NF-κB translocation.

  13. Nitric oxide mitigates arsenic-induced oxidative stress and genotoxicity in Vicia faba L.

    PubMed

    Shukla, Pratiksha; Singh, A K

    2015-09-01

    The protective effects of nitric oxide (NO) against arsenic (As)-induced structural disturbances in Vicia faba have been investigated. As treatment (0.25, 0.50, and 1 mM) resulted in a declined growth of V. faba seedlings. Arsenic treatment stimulates the activity of SOD and CAT while the activities of APX and GST content were decreased. The oxidative stress markers such as superoxide radical, hydrogen peroxide and malondialdehyde (lipid peroxidation) contents were enhanced by As. Overall results revealed that significant accumulation of As suppressed growth, photosynthesis, antioxidant enzymes (SOD, CAT, APX, and GST activity), mitotic index, and induction of different chromosomal abnormalities, hence led to oxidative stress. The concentration of SNP (0.02 mM) was very effective in counteracting the adverse effect of As toxicity. These abnormalities use partially or fully reversed by a simultaneous application of As and NO donor and sodium nitroprusside and has an ameliorating effect against As-induced oxidative stress and genotoxicity in V. faba roots.

  14. Nitric oxide reduces Cu toxicity and Cu-induced NH4+ accumulation in rice leaves.

    PubMed

    Yu, Chia Chi; Hung, Kuo Tung; Kao, Ching Huei

    2005-12-01

    Nitric oxide (NO) is a highly reactive, membrane-permeable free radical, which has recently emerged as an important antioxidant. Here we investigated the protective effect of NO against the toxicity and NH4+ accumulation in rice leaves caused by excess CuSO4 (10mmol L(-1)). It was found that free radical scavengers (sodium benzoate, thiourea, and reduced glutathione) reduced the toxicity and NH4+ accumulation in rice leaves caused by excess CuSO4. NO donor sodium nitroprusside (SNP) was also effective in reducing CuSO4-induced toxicity and NH4+ accumulation in rice leaves. The protective effect of SNP on the toxicity and NH4+ accumulation can be reversed by 2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethyl- imidazoline-1-oxyl-3-oxide, a NO scavenger, suggesting that the protective effect of SNP is attributable to NO released. Results obtained in the present study suggest that reduction of CuSO4-induced toxicity and NH4+ accumulation by SNP is most likely mediated through its ability to scavenge active oxygen species.

  15. Carotid body chemosensory excitation induced by nitric oxide: involvement of oxidative metabolism.

    PubMed

    Mosqueira, Matias; Iturriaga, Rodrigo

    2002-08-01

    Nitric oxide (NO) produces a dual effect on carotid body (CB) oxygen chemoreception. At low concentration, NO inhibits chemosensory response to hypoxia, while in normoxia, medium and high [NO] increases the frequency of carotid chemosensory discharges (f(x)). Since NO and peroxynitrite inhibit mitochondrial respiration, it is plausible that the NO-induced excitation may depend on the mitochondrial oxidative metabolism. To test this hypothesis, we studied the effects of oligomycin, FCCP and antimycin A that produce selective blockade of hypoxic and NaCN-induced chemosensory responses, leaving nicotinic response less affected. CBs excised from pentobarbitone-anaesthetised cats were perfused in vitro with Tyrode (P(O(2)) approximately 125 Torr, pH 7.40 at 38 degrees C). Hypoxia (P(O(2)) approximately equal 30 Torr), NaCN and nicotine (1-100 microg) and S-nitroso-N-acetylpenicillamide (SNAP, 300-600 microg) increased f(x). Oligomycin (12.5-25 microg), antimycin A (10 microg) and FCCP (5 microM) transiently increased f(x). Subsequently, chemosensory responses to hypoxia, NaCN and SNAP were reduced or abolished, while the response to nicotine was less affected. The electron donor system tetramethyl-p-phenylene diamide and ascorbate that bypasses the electron chain blockade produced by antimycin A, restores the excitatory responses to NaCN and SNAP. Present results suggest that the chemoexcitatory effect of NO depends on the integrity of mitochondrial metabolism.

  16. The NADPH oxidase inhibitor apocynin induces nitric oxide synthesis via oxidative stress

    SciTech Connect

    Riganti, Chiara

    2008-05-01

    We have recently shown that apocynin elicits an oxidative stress in N11 mouse glial cells and other cell types. Here we report that apocynin increased the accumulation of nitrite, the stable derivative of nitric oxide (NO), in the extracellular medium of N11 cell cultures, and the NO synthase (NOS) activity in cell lysates. The increased synthesis of NO was associated with increased expression of inducible NOS (iNOS) mRNA, increased nuclear translocation of the redox-sensitive transcription factor NF-{kappa}B and decreased intracellular level of its inhibitor IkB{alpha}. These effects, accompanied by increased production of H{sub 2}O{sub 2}, were very similar to those observed after incubation with bacterial lipopolysaccharide (LPS) and were inhibited by catalase. These results suggest that apocynin, similarly to LPS, induces increased NO synthesis by eliciting a generation of reactive oxygen species (ROS), which in turn causes NF-{kappa}B activation and increased expression of iNOS. Therefore, the increased bioavailability of NO reported in the literature after in vivo or in vitro treatments with apocynin might depend, at least partly, on the drug-elicited induction of iNOS, and not only on the inhibition of NADPH oxidase and the subsequent decreased scavenging of NO by oxidase-derived ROS, as it is often supposed.

  17. Mechanisms of xylanase-induced nitric oxide and phosphatidic acid production in tomato cells.

    PubMed

    Lanteri, M Luciana; Lamattina, Lorenzo; Laxalt, Ana M

    2011-10-01

    The second messenger nitric oxide (NO), phosphatidic acid (PA) and reactive oxygen species (ROS) are involved in the plant defense response during plant-pathogen interactions. NO has been shown to participate in PA production in response to the pathogen-associated molecular pattern xylanase in tomato cell suspensions. Defense responses downstream of PA include ROS production. The goal of this work was to study the signaling mechanisms involved in PA production during the defense responses triggered by xylanase and mediated by NO in the suspension-cultured tomato cells. We analyzed the participation of protein kinases, guanylate cyclase and the NO-mediated posttranslational modification S-nitrosylation, by means of pharmacology and biochemistry. We showed that NO, PA and ROS levels are significantly diminished by treatment with the general protein kinase inhibitor staurosporine. This indicates that xylanase-induced protein phosphorylation events might be the important components leading to NO formation, and hence for the downstream regulation of PA and ROS levels. When assayed, a guanylate cyclase inhibitor or a cGMP analog did not alter the PA accumulation. These results suggest that a cGMP-mediated pathway is not involved in xylanase-induced PA formation. Finally, the inhibition of protein S-nitrosylation did not affect NO formation but compromised PA and ROS production. Data collectively indicate that upon xylanase perception, cells activate a protein kinase pathway required for NO formation and that, S-nitrosylation-dependent mechanisms are involved in downstream signaling leading to PA and ROS.

  18. Attenuation of malonate-induced degeneration of the nigrostriatal pathway by inhibitors of nitric oxide synthase.

    PubMed

    Connop, B P; Boegman, R J; Beninger, R J; Jhamandas, K

    1996-04-01

    Focal infusions of the succinate dehydrogenase inhibitor, malonate, into the substantia nigra pars compacta (SNc) of adult Sprague-Dawley rats resulted in a substantial depletion of ipsilateral striatal tyrosine hydroxylase (TH) activity. The percentage decrease in striatal TH activity following intranigral malonate (0.5 mumol/0.5 microliter) infusion was similar at 4 (58%) and 7 days (62%) post-infusion. To assess the role of N-methyl-D-aspartate (NMDA) receptor activation in malonate neurotoxicity, animals were pretreated with the NMDA receptor antagonist MK-801 (2 x 5 mg/kg, i.p.). Four days post-infusion of malonate (0.5 mumol/0.5 microliter) into the SNc, striatal TH activity was depleted by 58% in vehicle pretreated animals and 14% in the presence of MK-801 indicating a significant neuroprotective effect of MK-801 on malonate action. To determine the role of nitric oxide (NO) in malonate-induced nigral toxicity, the actions of malonate were evaluated in the presence of the nitric oxide synthase (NOS) inhibitors, 7-nitro indazole (7-NI) and N omega-nitro-L-arginine methyl ester (L- NAME). Systemic injections of 7-NI (20, 30, 40, 50 and 75 mg/kg, i.p.) produced a dose-related inhibition of nigral NOS activity which was maximal at a dose of 40 mg/kg. Intranigral infusion of malonate with 20 and 50 mg/kg 7-NI pretreatment produced a 46 and 31% decrease in striatal TH activity, respectively. Thus, a significant protective effect at the higher but not lower dose of 7-NI was observed. Pretreatment with a L- NAME regimen (2 x 250 mg/kg; i.p.), previously shown to inhibit brain NOS activity by greater than 86%, also produced a significant neuroprotective effect against malonate-induced neurotoxicity (30% decrease). The results of this study suggest that malonate-induced toxicity to the dopaminergic neurons of the nigrostriatal pathway is mediated, at least in part, by NMDA receptor activation and the formation of NO.

  19. Interactions between Nitric Oxide and Hypoxia-Inducible Factor Signaling Pathways in Inflammatory Disease

    PubMed Central

    Olson, Nels; van der Vliet, Albert

    2011-01-01

    Induction and activation of nitric oxide (NO) synthases (NOS) and excessive production of NO are common features of almost all diseases associated with infection and acute or chronic inflammation, although the contribution of NO to the pathophysiology of these diseases is highly multifactorial and often still a matter of controversy. Because of its direct impact on tissue oxygenation and cellular oxygen (O2) consumption and redistribution, the ability of NO to regulate various aspects of hypoxia-induced signaling has received widespread attention. Conditions of tissue hypoxia and the activation of hypoxia-inducible factors (HIF) have been implicated in hypoxia or in cancer biology, but are also being increasingly recognized as important features of acute and chronic inflammation. Thus, the activation of HIF transcription factors has been increasingly implicated in inflammatory diseases, and recent studies have indicated its critical importance in regulating phagocyte function, inflammatory mediator production, and regulation of epithelial integrity and repair processes. Finally, HIF also appears to contribute to important features of tissue fibrosis and epithelial-to-mesenchymal transition, processes that are associated with tissue remodeling in various non-malignant chronic inflammatory disorders. In this review, we briefly summarize the current state of knowledge with respect to the general mechanisms involved in HIF regulation and the impact of NO on HIF activation. Secondly, we will summarize the major recent findings demonstrating a role for HIF signaling in infection, inflammation, and tissue repair and remodeling, and will address the involvement of NO. The growing interest in hypoxia-induced signaling and its relation with NO biology is expected to lead to further insights into the complex roles of NO in acute or chronic inflammatory diseases and may point to the importance of HIF signaling as key feature of NO-mediated events during these disorders. PMID

  20. Roles of thioredoxin in nitric oxide-dependent preconditioning-induced tolerance against MPTP neurotoxin

    SciTech Connect

    Chiueh, C.C. . E-mail: chiueh@tmu.edu.tw; Andoh, Tsugunobu; Chock, P. Boon

    2005-09-01

    Hormesis, a stress tolerance, can be induced by ischemic preconditioning stress. In addition to preconditioning, it may be induced by other means, such as gas anesthetics. Preconditioning mechanisms, which may be mediated by reprogramming survival genes and proteins, are obscure. A known neurotoxicant, 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), causes less neurotoxicity in the mice that are preconditioned. Pharmacological evidences suggest that the signaling pathway of {center_dot}NO-cGMP-PKG (protein kinase G) may mediate preconditioning phenomenon. We developed a human SH-SY5Y cell model for investigating {sup {center_dot}}NO-mediated signaling pathway, gene regulation, and protein expression following a sublethal preconditioning stress caused by a brief 2-h serum deprivation. Preconditioned human SH-SY5Y cells are more resistant against severe oxidative stress and apoptosis caused by lethal serum deprivation and 1-mehtyl-4-phenylpyridinium (MPP{sup +}). Both sublethal and lethal oxidative stress caused by serum withdrawal increased neuronal nitric oxide synthase (nNOS/NOS1) expression and {sup {center_dot}}NO levels to a similar extent. In addition to free radical scavengers, inhibition of nNOS, guanylyl cyclase, and PKG blocks hormesis induced by preconditioning. S-nitrosothiols and 6-Br-cGMP produce a cytoprotection mimicking the action of preconditioning tolerance. There are two distinct cGMP-mediated survival pathways: (i) the up-regulation of a redox protein thioredoxin (Trx) for elevating mitochondrial levels of antioxidant protein Mn superoxide dismutase (MnSOD) and antiapoptotic protein Bcl-2, and (ii) the activation of mitochondrial ATP-sensitive potassium channels [K(ATP)]. Preconditioning induction of Trx increased tolerance against MPP{sup +}, which was blocked by Trx mRNA antisense oligonucleotide and Trx reductase inhibitor. It is concluded that Trx plays a pivotal role in {sup {center_dot}}NO-dependent preconditioning hormesis against

  1. Asbestos induces nitric oxide synthesis in mesothelioma cells via Rho signaling inhibition.

    PubMed

    Riganti, Chiara; Orecchia, Sara; Silvagno, Francesca; Pescarmona, Gianpiero; Betta, Pier Giacomo; Gazzano, Elena; Aldieri, Elisabetta; Ghigo, Dario; Bosia, Amalia

    2007-06-01

    We have observed that in three human malignant mesothelioma cell lines, crocidolite asbestos induced the activation of the transcription factor NF-kappaB and the synthesis of nitric oxide (NO) by inhibiting the RhoA signaling pathway. The incubation with crocidolite decreased the level of GTP-bound RhoA and the activity of Rho-dependent kinase, and induced the activation of Akt/PKB and IkBalpha kinase, leading to the nuclear translocation of NF-kappaB. The effects of crocidolite fibers on NF-kappaB activation and NO synthesis were mimicked by Y27632 (an inhibitor of the Rho-dependent kinases) and toxin B (an inhibitor of RhoA GTPase activity), while they were reverted by mevalonic acid, the product of 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase. Furthermore, crocidolite, similarly to mevastatin, inhibited the synthesis of cholesterol and ubiquinone and the prenylation of RhoA: these effects were prevented in the presence of mevalonic acid. This suggests that crocidolite fibers might inhibit the synthesis of isoprenoid molecules at the level of the HMGCoA reductase reaction or of an upstream step, thus impairing the prenylation and subsequent activation of RhoA. Akt can stimulate NO synthesis via a double mechanism: it can activate the inducible NO synthase via the NF-kappaB pathway and the endothelial NO synthase via a direct phosphorylation. Our results suggest that crocidolite increases the NO levels in mesothelioma cells by modulating both NO synthase isoforms.

  2. Evidence for the involvement of nitric oxide in cholestasis-induced itch associated response in mice.

    PubMed

    Ostadhadi, Sattar; Foroutan, Arash; Momeny, Majid; Norouzi-Javidan, Abbas; Azimi, Ehsan; Kordjazy, Nastaran; Dehpour, Ahmad Reza

    2016-12-01

    Cholestasis is a major systemic disorder associated with distressing pruritus (itch). Nitric oxide (NO) is a neurotransmitter, assumed to be involved in pruritus. Based on over-production of NO in cholestatic liver diseases, this project aimed to investigate involvement of NO in cholestasis-related itch in mice. To achieve this, cholestasis was induced by bile duct ligation (BDL). Our results showed that BDL mice elicited significant itch on fifth and seventh day after the procedure. This scratching behavior was inhibited by intraperitoneal (IP) treatment of mice with non-selective NOS inhibitor N-nitro-l-arginine methyl ester (l-NAME; 3mg/kg) and inducible NOS (iNOS) inhibitor aminoguanidine (AG; 100mg/kg). The inhibitory effects of l-NAME and AG were reversed by pretreatment with l-arginine (100mg/kg). Administration of l-NAME, AG and l-arginine per se, in BDL and SHAM mice did not produce scratching behaviors. In addition, intradermal injection of l-arginine at dose of 300 nmol/site significantly increased itch in BDL mice. Furthermore, nitrite levels in skin and serum of BDL animals significantly increased after 7 d of operation and administration of NOS inhibitors decreased this enhancement. l-arginine injection reversed the effects of NOS inhibitors on reduction of nitrite levels in the skin and serum of BDL mice. Finally, cutaneous iNOS expression increased in BDL mice 7 d after surgery. Taken together, our study showed for the first time that BDL, as a model of acute cholestasis in rodents, induces NO over-production by activating NOS enzymes, especially iNOS, which contribute to pruritus.

  3. Dual effect of nitric oxide in articular inflammatory pain in zymosan-induced arthritis in rats.

    PubMed

    da S Rocha, José C; Peixoto, Magno E B; Jancar, Sônia; de Q Cunha, Fernando; de A Ribeiro, Ronaldo; da Rocha, Francisco A C

    2002-06-01

    The contribution of nitric oxide (NO) to articular pain in arthritis induced by zymosan (1 mg, intra articular) in rats was assessed by measuring articular incapacitation (AI). Systemic treatment with the non-selective NO synthase (NOS) inhibitor L-NAME (10 - 100 mg kg(-1) i.p.) or with the selective iNOS inhibitors aminoguanidine (AG; 10 - 100 mg kg(-1) i.p.) or 1400W (0.5 - 1 mg kg(-1) s.c.) inhibited the AI induced by injection of zymosan 30 min later. Local (intra articular) treatment with the NOS inhibitors (L-NAME or AG, 0.1 - 1 micromol; 1400W, 0.01 (micromol) 30 min before zymosan also inhibited the AI. Systemic or local treatment with the NOS inhibitors (L-NAME; AG, 100 mg kg(-1) i.p. or 0.1 micromol joint(-1); 1400W, 1 mg kg(-1) s.c. or 0.01 micromol joint(-1)), 2 h after zymosan did not affect the subsequent AI. Local treatment with the NO donors SNP or SIN-1, 2 h after zymosan did inhibit AI. L-NAME and AG, given i.p. inhibited nitrite but not prostaglandin E(2) (PGE(2)) levels in the joints. L-NAME (100 mg kg(-1)) but not AG (100 mg kg(-1)) increased mean arterial blood pressure. Neither L-NAME, AG nor the NO donor SIN-1 altered articular oedema induced by zymosan. In conclusion, inhibitors of iNOS decrease pain in zymosan arthritis only when given before the zymosan. This was not due to inhibition of articular PGE(2) release or oedema. NO donors also promoted antinociception in zymosan arthritis without affecting oedema.

  4. Regorafenib Induces Rapid and Reversible Changes in Plasma Nitric Oxide and Endothelin-1

    PubMed Central

    Humphreys, Benjamin D.

    2012-01-01

    Background Hypertension is a toxicity of antiangiogenic therapies and a possible biomarker that identifies patients with superior cancer outcomes. Understanding its mechanism will aid in treatment and could lead to the development of other biomarkers for predicting toxicity and anticancer efficacy. Recent evidence implicates nitric oxide (NO) suppression and endothelin-1 (ET-1) stimulation as potential mechanisms leading to antiangiogenic therapy-induced hypertension. The aim of this study was to evaluate the effects of regorafenib, a novel broad-spectrum kinase inhibitor with activity against multiple targets, including vascular endothelial growth factor receptor 2 inhibition, on NO and ET-1 levels. Methods Regorafenib was administered to 32 subjects with gastrointestinal stromal tumor on a 3-week-on, 1-week-off basis. Plasma levels of NO and ET-1 were measured at baseline, 2, 4, and 6 weeks of therapy. Data analysis was by Wilcoxon rank-sum and paired t-tests. Results Twenty subjects (63%) developed regorafenib-induced hypertension. Two weeks after starting regorafenib therapy, plasma ET-1 levels increased (25% increase, P < 0.05) and NO was suppressed (20% decrease, P < 0.05). These normalized after 1-week washout but ET-1 rose again by 30% (P < 0.05) and NO fell by 50% (P < 0.05) after restarting regorafenib. Conclusions These findings indicate that regorafenib induces a coordinated and reversible suppression of NO and stimulation of ET-1. Whether NO and ET-1 might predict therapeutic efficacy in these patients requires further study. American Journal of Hypertension, advance online publication 12 July 2012. doi:10.1038/ajh.2012.97 PMID:22785409

  5. Minocycline attenuates experimental colitis in mice by blocking expression of inducible nitric oxide synthase and matrix metalloproteinases

    SciTech Connect

    Huang, T.-Y.; Chu, H.-C.; Lin, Y.-L.; Lin, C.-K.; Hsieh, T.-Y.; Chang, W.-K.; Chao, Y.-C.; Liao, C.-L.

    2009-05-15

    In addition to its antimicrobial activity, minocycline exerts anti-inflammatory effects in several disease models. However, whether minocycline affects the pathogenesis of inflammatory bowel disease has not been determined. We investigated the effects of minocycline on experimental colitis and its underlying mechanisms. Acute and chronic colitis were induced in mice by treatment with dextran sulfate sodium (DSS) or trinitrobenzene sulfonic acid (TNBS), and the effect of minocycline on colonic injury was assessed clinically and histologically. Prophylactic and therapeutic treatment of mice with minocycline significantly diminished mortality rate and attenuated the severity of DSS-induced acute colitis. Mechanistically, minocycline administration suppressed inducible nitric oxide synthase (iNOS) expression and nitrotyrosine production, inhibited proinflammatory cytokine expression, repressed the elevated mRNA expression of matrix metalloproteinases (MMPs) 2, 3, 9, and 13, diminished the apoptotic index in colonic tissues, and inhibited nitric oxide production in the serum of mice with DSS-induced acute colitis. In DSS-induced chronic colitis, minocycline treatment also reduced body weight loss, improved colonic histology, and blocked expression of iNOS, proinflammatory cytokines, and MMPs from colonic tissues. Similarly, minocycline could ameliorate the severity of TNBS-induced acute colitis in mice by decreasing mortality rate and inhibiting proinflammatory cytokine expression in colonic tissues. These results demonstrate that minocycline protects mice against DSS- and TNBS-induced colitis, probably via inhibition of iNOS and MMP expression in intestinal tissues. Therefore, minocycline is a potential remedy for human inflammatory bowel diseases.

  6. Nitric oxide in the hippocampal cortical area interacts with naloxone in inducing pain

    PubMed Central

    Hafeshjani, Zahra K.; Karami, Manizheh; Biglarnia, Masoomeh

    2012-01-01

    Objective: Role of nitric oxide (NO) in reversing morphine anti-nociception has been shown. However, the interaction between NO and naloxone-induced pain in the hippocampus is unknown. The present study aimed to investigate the involvement of molecule NO in naloxone-induced pain and its possible interaction with naloxone into cortical area 1 (CA1) of hippocampus. Materials and Methods: Male Wistar rats (250–350 g), provided by Pasteur Institute of Iran, were housed two per cage with food and water ad libitum. The animals’ skulls were cannulated bilaterally at coordinates adjusted for CA1 of hippocampus (AP: -3.8; L: ±1.8– 2.2: V: 3) by using stereotaxic apparatus. Each experimental group included 6–8 rats. To induce inflammation pain, the rats received subcutaneous (s.c.) injections of formalin (50 μL at 2.5%) once prior to testing. To evaluate the nociceptive effect of naloxone, the main narcotic antagonist of morphine (0.1–0.4 mg/kg) was injected intraperitoneally (i.p.) 10 min before injection of formalin. Injections of L-arginine, a precursor of NO, and NG-Nitro-L-arginine Methyl Ester (L-NAME), an inhibitor of NO synthase (NOS), intra-CA1, were conducted orderly prior to the administration of naloxone. The pain induction was analyzed by analysis of variance (ANOVA). Results: Naloxone at the lower doses caused a significant (P<0.01) pain in the naloxone-treated animals. However, pre-administration (1–2 min) of L-arginine (0.04, 0.08, 0.15, 0.3, 1.0, and 3.0 μg/rat, intra-CA1) reversed the response to naloxone. But, the response to L-arginine was blocked by pre-microinjection (1–2 min) of L-NAME (0.15, 0.3, 1.0, and 3.0 μg/rat), whilst, L-arginine or L-NAME alone did not induce pain behavior. Conclusion: NO in the rat hippocampal CA1 area is involved in naloxone-induced nociception. PMID:23087502

  7. Catalytic Intermediates of Inducible Nitric-oxide Synthase Stabilized by the W188H Mutation*

    PubMed Central

    Sabat, Joseph; Egawa, Tsuyoshi; Lu, Changyuan; Stuehr, Dennis J.; Gerfen, Gary J.; Rousseau, Denis L.; Yeh, Syun-Ru

    2013-01-01

    Nitric-oxide synthase (NOS) catalyzes nitric oxide (NO) synthesis via a two-step process: l-arginine (l-Arg) → N-hydroxy-l-arginine → citrulline + NO. In the active site the heme is coordinated by a thiolate ligand, which accepts a H-bond from a nearby tryptophan residue, Trp-188. Mutation of Trp-188 to histidine in murine inducible NOS was shown to retard NO synthesis and allow for transient accumulation of a new intermediate with a Soret maximum at 420 nm during the l-Arg hydroxylation reaction (Tejero, J., Biswas, A., Wang, Z. Q., Page, R. C., Haque, M. M., Hemann, C., Zweier, J. L., Misra, S., and Stuehr, D. J. (2008) J. Biol. Chem. 283, 33498–33507). However, crystallographic data showed that the mutation did not perturb the overall structure of the enzyme. To understand how the proximal mutation affects the oxygen chemistry, we carried out biophysical studies of the W188H mutant. Our stopped-flow data showed that the 420-nm intermediate was not only populated during the l-Arg reaction but also during the N-hydroxy-l-arginine reaction. Spectroscopic data and structural analysis demonstrated that the 420-nm intermediate is a hydroxide-bound ferric heme species that is stabilized by an out-of-plane distortion of the heme macrocycle and a cation radical centered on the tetrahydrobiopterin cofactor. The current data add important new insights into the previously proposed catalytic mechanism of NOS (Li, D., Kabir, M., Stuehr, D. J., Rousseau, D. L., and Yeh, S. R. (2007) J. Am. Chem. Soc. 129, 6943–6951). PMID:23269673

  8. Protective effect of angiotensin II-induced increase in nitric oxide in the renal medullary circulation.

    PubMed

    Zou, A P; Wu, F; Cowley, A W

    1998-01-01

    This study examined the effect of intravenous infusion of subpressor doses of angiotensin (Ang II) on renal medullary blood flow (MBF), medullary partial oxygen pressure (PO2), and nitric oxide (NO) concentration under normal conditions and during reduction of the medullary nitric oxide synthase (NOS) activity in anesthetized rats. With laser Doppler flowmetry and polarographic measurement of PO2 with microelectrodes, Ang II (5 ng/kg per minute) did not alter renal cortical and medullary blood flows or medullary PO2. N(omega)-nitro-L-arginine methyl ester (L-NAME) was infused into the renal medullary interstitial space at a dose of 1.4 microg/kg per minute, a dose that did not significantly alter basal levels of MBF or PO2. Intravenous infusion of Ang II at the same dose in the presence of L-NAME decreased MBF by 23% and medullary PO2 by 28%, but it had no effect on cortical blood flow or arterial blood pressure. An in vivo microdialysis-oxyhemoglobin NO trapping technique was used in other rats to determine tissue NO concentrations using the same protocol. Ang II infusion increased tissue NO concentrations by 85% in the renal cortex and 150% in the renal medulla. Renal medullary interstitial infusion of L-NAME (1.4 microg/kg per minute) reduced medullary NO concentrations and substantially blocked Ang II-induced increases in NO concentrations in the renal medulla, but not in the renal cortex. Tissue slices of the renal cortex and medulla were studied to determine the effects of Ang II and L-NAME on the nitrite/nitrate production. Ang II stimulated the nitrite/nitrate production predominately in the renal medulla, which was significantly attenuated by L-NAME. We conclude that small elevations of circulating Ang II levels increase medullary NO production and concentrations, which plays an important role in buffering the vasoconstrictor effects of this peptide and in maintaining a constancy of MBF.

  9. Activation of peroxynitrite by inducible nitric-oxide synthase: a direct source of nitrative stress.

    PubMed

    Maréchal, Amandine; Mattioli, Tony A; Stuehr, Dennis J; Santolini, Jérôme

    2007-05-11

    In mammals, nitric oxide (NO) is an essential biological mediator that is exclusively synthesized by nitric-oxide synthases (NOSs). However, NOSs are also directly or indirectly responsible for the production of peroxynitrite, a well known cytotoxic agent involved in numerous pathophysiological processes. Peroxynitrite reactivity is extremely intricate and highly depends on activators such as hemoproteins. NOSs present, therefore, the unique ability to both produce and activate peroxynitrite, which confers upon them a major role in the control of peroxynitrite bioactivity. We report here the first kinetic analysis of the interaction between peroxynitrite and the oxygenase domain of inducible NOS (iNOSoxy). iNOSoxy binds peroxynitrite and accelerates its decomposition with a second order rate constant of 22 x 10(4) m(-1)s(-1) at pH 7.4. This reaction is pH-dependent and is abolished by the binding of substrate or product. Peroxynitrite activation is correlated with the observation of a new iNOS heme intermediate with specific absorption at 445 nm. iNOSoxy modifies peroxynitrite reactivity and directs it toward one-electron processes such as nitration or one-electron oxidation. Taken together our results suggest that, upon binding to iNOSoxy, peroxynitrite undergoes homolytic cleavage with build-up of an oxo-ferryl intermediate and concomitant release of a NO(2)(.) radical. Successive cycles of peroxynitrite activation were shown to lead to iNOSoxy autocatalytic nitration and inhibition. The balance between peroxynitrite activation and self-inhibition of iNOSoxy may determine the contribution of NOSs to cellular oxidative stress.

  10. E3 ubiquitin ligase NKLAM positively regulates macrophage inducible nitric oxide synthase expression.

    PubMed

    Lawrence, Donald W; Gullickson, Gail; Kornbluth, Jacki

    2015-01-01

    Stimulated macrophages generate potent anti-microbial reactive oxygen and nitrogen species within their phagosomes. Previous studies have shown that the E3 ubiquitin ligase natural killer lytic-associated molecule (NKLAM) is a macrophage phagosomal protein that plays a role in macrophage anti-bacterial activity. In vivo, NKLAM-knockout (KO) mice produce less nitric oxide (NO) upon exposure to lipopolysaccharide (LPS) than wild type (WT) mice. In vitro, we found that NO production and inducible nitric oxide synthase (iNOS) protein were diminished in LPS-stimulated NKLAM-KO bone marrow-derived and splenic macrophages. Additionally, LPS-stimulated NKLAM-KO macrophages displayed defects in STAT1 tyrosine phosphorylation and production of interferon beta (IFNβ). The JAK/STAT pathway is critical for the production of IFNβ, which augments iNOS protein expression in mice. iNOS protein expression is also regulated by the transcription factor NFκB, thus we investigated whether NKLAM influences NFκB function. LPS-stimulated NKLAM-KO macrophages showed evidence of delayed nuclear translocation of the NFκB subunit p65. This was associated with a reduction in p65/DNA colocalization. The defect in p65 translocation was independent of IKBα degradation. NKLAM-KO macrophages also expressed less p65 and showed evidence of defective p65 phosphorylation at serine 536. Importantly, LPS-stimulated NKLAM-KO macrophages have diminished NFκB transcriptional activity as assessed by transfection of a luciferase reporter plasmid. Collectively, our data implicate NKLAM as a novel modulator of macrophage iNOS expression.

  11. Inhibition of inducible nitric oxide synthase expression by yuccaol C from Yucca schidigera roezl.

    PubMed

    Marzocco, Stefania; Piacente, Sonia; Pizza, Cosimo; Oleszek, Wieslaw; Stochmal, Anna; Pinto, Aldo; Sorrentino, Raffaella; Autore, Giuseppina

    2004-08-06

    Yucca schidigera extract finds wide commercial application in foods, cosmetics and pharmaceuticals. In a previous paper we have found as the main constituents of yucca bark, yuccaol A, B and C, new and very unusual spiro-derivatives made up of a C15 unit and a stilbenic portion closely related to resveratrol. This study was performed to examine whether yuccaol A, B or C (0.01-100 microM) could affect cytosolic inducible nitric oxide synthase (iNOS) protein expression and nitric oxide (NO) generation in vitro in Escherichia coli lipopolysaccharide (LPS)-activated J774.A1 macrophage cell line. NO production, detected as NO2-, increased significantly in LPS treated J774.A1 cells from 0.05 +/- 0.03 microM to 16.64 +/- 0.58 microM (P < 0.001). Yuccaol C (0.01-100 microM), added to the culture medium 1 h before LPS-stimulation, significantly (P < 0.001) and in a concentration related manner reduced NO release (P < 0.001) and iNOS protein expression (P < 0.05). In contrast, no inhibitory effect either on iNOS protein expression or on NO release was observed when yuccaol C was added after LPS stimulation. In contrast yuccaol A inhibited significantly (P < 0.001) only NO release at the highest concentration tested (100 microM) while yuccaol B did not reduce either NO release or iNOS expression. Yuccaol C was demonstrated to reduce iNOS protein expression via the transcription factor NF-kappaB. These results indicated that the empirical use of Y. schidigera as anti-inflammatory remedy could be addressed not only to the resveratrol content but also to the presence of yuccaol C.

  12. Structural requirements for human inducible nitric oxide synthase substrates and substrate analogue inhibitors.

    PubMed

    Grant, S K; Green, B G; Stiffey-Wilusz, J; Durette, P L; Shah, S K; Kozarich, J W

    1998-03-24

    Inducible nitric oxide synthase (iNOS; EC 1.14.13.39) catalyzes the NADPH-dependent oxidation of one of the free guanidino nitrogens of L-Arg to form nitric oxide and L-citrulline. Analogues of L-Arg and the inhibitor, L-N6-(1-iminoethyl)lysine, were used to define structural elements required for the binding and catalysis of compounds. L-Arg analogues with sequentially shorter methylene spacing between the guanidino group and the amino acid portion of the molecule were not iNOS substrates but were reversible inhibitors. L-Arg analogues such as agmatine with a hydroxyl substitution at the 2-amino position were substrates. Desaminoarginine was not a substrate but a reversible inhibitor. Desaminoarginine, agmatine, and argininic acid bound to the enzyme to give type I difference spectra similar to that of L-Arg. The amidino compounds L-N6-(1-iminoethyl)lysine, L-N5-(1-iminoethyl)ornithine, and N5-(1-iminoethyl)cadaverdine, but not N6-(1-iminoethyl)-6-aminocaproic acid, were NADPH-dependent, irreversible inactivators of iNOS. For both the L-Arg and L-N6-(1-iminoethyl)lysine analogues, the 2-amino group appeared to play an important role in catalytic events leading to either substrate turnover or mechanism-based inactivation. Inactivation of iNOS by L-N6-(1-iminoethyl)lysine was NADPH- and dioxygen-dependent, but low incorporation of radiolabel with DL--4, 5-3H]-N6-(1-iminoethyl)lysine indicates that the mechanism of enzyme inactivation is not covalent modification of the protein.

  13. Mas receptor is involved in the estrogen-receptor induced nitric oxide-dependent vasorelaxation.

    PubMed

    Sobrino, Agua; Vallejo, Susana; Novella, Susana; Lázaro-Franco, Macarena; Mompeón, Ana; Bueno-Betí, Carlos; Walther, Thomas; Sánchez-Ferrer, Carlos; Peiró, Concepción; Hermenegildo, Carlos

    2017-04-01

    The Mas receptor is involved in the angiotensin (Ang)-(1-7) vasodilatory actions by increasing nitric oxide production (NO). We have previously demonstrated an increased production of Ang-(1-7) in human umbilical vein endothelial cells (HUVEC) exposed to estradiol (E2), suggesting a potential cross-talk between E2 and the Ang-(1-7)/Mas receptor axis. Here, we explored whether the vasoactive response and NO-related signalling exerted by E2 are influenced by Mas. HUVEC were exposed to 10nM E2 for 24h in the presence or absence of the selective Mas receptor antagonist A779, and the estrogen receptor (ER) antagonist ICI182780 (ICI). E2 increased Akt and endothelial nitric oxide synthase (eNOS) mRNA and protein expression, measured by RT-PCR and Western blot, respectively. Furthermore, E2 increased Akt activity (determined by the levels of phospho-Ser(473)) and eNOS activity (by the enhanced phosphorylation of Ser(1177), the activated form), resulting in increased NO production, which was measured by the fluorescence probe DAF-2-FM. These signalling events were dependent on ER and Mas receptor activation, since they were abolished in the presence of ICI or A779. In ex-vivo functional experiments performed with a small-vessel myograph in isolated mesenteric vessels from wild-type mice pre-contracted with noradrenaline, the relaxant response to physiological concentrations of E2 was blocked by ICI and A779, to the same extent to that obtained in the vessels isolated from Mas-deficient. In conclusion, E2 induces NO production and vasodilation through mechanisms that require Mas receptor activation.

  14. Gomisin J from Schisandra chinensis induces vascular relaxation via activation of endothelial nitric oxide synthase.

    PubMed

    Park, Ji Young; Choi, Young Whan; Yun, Jung Wook; Bae, Jin Ung; Seo, Kyo Won; Lee, Seung Jin; Park, So Youn; Kim, Chi Dae

    2012-01-01

    Gomisin J (GJ) is a lignan contained in Schisandra chinensis (SC) which is a well-known medicinal herb for improvement of cardiovascular symptoms in Korean. Thus, the present study examined the vascular effects of GJ, and also determined the mechanisms involved. Exposure of rat thoracic aorta to GJ (1-30μg/ml) resulted in a concentration-dependent vasorelaxation, which was more prominent in the endothelium (ED)-intact aorta. ED-dependent relaxation induced by GJ was markedly attenuated by pretreatment with L-NAME, a nitric oxide synthase (NOS) inhibitor. In the intact endothelial cells of rat thoracic aorta, GJ also enhanced nitric oxide (NO) production. In studies using human coronary artery endothelial cells, GJ enhanced phosphorylation of endothelial NOS (eNOS) at Ser(1177) with increased cytosolic translocation of eNOS, and subsequently increased NO production. These effects of GJ were attenuated not only by calcium chelators including EGTA and BAPTA-AM, but also by LY294002, a PI3K/Akt inhibitor, indicating calcium- and PI3K/Akt-dependent activation of eNOS by GJ. Moreover, the levels of intracellular calcium were increased immediately after GJ administration, but Akt phosphorylation was started to increase at 20min of GJ treatment. Based on these results with the facts that ED-dependent relaxation occurred rapidly after GJ treatment, it was suggested that the ED-dependent vasorelaxant effects of GJ were mediated mainly by calcium-dependent activation of eNOS with subsequent production of endothelial NO.

  15. Interferon-γ-Induced Nitric Oxide Causes Intrinsic Intestinal Denervation in Trypanosoma cruzi-Infected Mice

    PubMed Central

    Arantes, Rosa M.E.; Marche, Homero H.F.; Bahia, Maria T.; Cunha, Fernando Q.; Rossi, Marcos A.; Silva, João S.

    2004-01-01

    In this study, the role of nitric oxide (NO) in neuronal destruction during acute-phase Trypanosoma cruzi infection was evaluated in male C57BL/6 (WT, wild-type) mice and knockout mice [inducible nitric oxide synthase (iNOS)−/− and interferon (IFN)−/−]. Selected animals were infected by intraperitoneal injection of 100 trypomastigote forms of the Y strain of T. cruzi. Others were injected intraperitoneally with an equal volume of saline solution and served as controls. Our findings support those of previous studies regarding myenteric denervation in acute-phase T. cruzi infection. In addition, we clearly demonstrate that, despite the fact that parasite nests and similar inflammatory infiltrate in the intestinal wall were more pronounced in infected iNOS−/− mice than in infected WT mice, the former presented no reduction in myenteric plexus neuron numbers. Neuronal nerve profile expression, as revealed by the general nerve marker PGP 9.5, was preserved in all knockout animals. Infected IFN−/− mice suffered no significant neuronal loss and there was no inflammatory infiltrate in the intestinal wall. On days 5 and 10 after infection, iNOS activity was greater in infected WT mice than in controls, whereas iNOS activity in infected knockout mice remained unchanged. These findings clearly demonstrate that neuronal damage does not occur in NO-impaired infected knockout mice, regardless of whether inflammatory infiltrate is present (iNOS−/−) or absent (IFN−/−). In conclusion, our observations strongly indicate that myenteric denervation in acute-phase T. cruzi infection is because of IFN-γ-elicited NO production resulting from iNOS activation in the inflammatory foci along the intestinal wall. PMID:15039223

  16. Immunolocalization of inducible nitric oxide synthase in synovium and cartilage in rheumatoid arthritis and osteoarthritis.

    PubMed

    Grabowski, P S; Wright, P K; Van 't Hof, R J; Helfrich, M H; Ohshima, H; Ralston, S H

    1997-06-01

    Nitric oxide has been implicated as a mediator of inflammatory arthritis, and recent work has shown that pro-inflammatory cytokines stimulate NO production in vitro by activation of the inducible nitric oxide synthase (iNOS) pathway. In order to identify the cellular sources of NO production within the joint, we have used immunohistochemical techniques to study the distribution of iNOS in synovium and cartilage from normal and diseased joints. iNOS was most strongly expressed in the synovial lining layer, subsynovium, vascular smooth muscle and chondrocytes from patients with rheumatoid arthritis (RA). Analysis of serial sections, coupled with double immunofluorescent staining, showed that the CD68+ macrophages in the synovial lining layer and, to a lesser extent, fibroblasts were the predominant source of iNOS within synovium, whereas T cells, B cells and neutrophils were negative. A similar pattern of iNOS staining was seen in osteoarthritis, but fewer cells were iNOS positive and the intensity of staining, particularly in cartilage, was much weaker than in RA. In contrast, no evidence of iNOS was detected in non-inflammatory synovium or in cartilage derived from normal joints (fractured neck of femur). In conclusion, these data support the hypothesis that synovium and cartilage are important sources of increased NO production in patients with inflammatory arthritis. Localization of iNOS at these sites within the inflamed joint raises the possibility that increased local production of NO may contribute to the pathogenesis of inflammatory arthritis by increasing synovial blood flow and by modulating cellular function within synovium and articular cartilage.

  17. Morphine-induced nitric oxide production in isolated, iris-ciliary bodies

    PubMed Central

    Dortch-Carnes, Juanita; Randall, Karen Russell

    2009-01-01

    Considerable evidence suggests that the nitric oxide (NO)/cGMP signaling pathway plays an integral role in opioid receptor-mediated responses in the cardiovascular and immune systems. Previous studies in our laboratory and others have shown that nitric oxide (NO) plays a role in morphine-induced reduction of intraocular pressure (IOP) and pupil diameter (PD) in the New Zealand white (NZW) rabbit. The present study is designed to determine the effect of morphine on NO production in the isolated, iris-ciliary body (ICB), site of aqueous humor production, as this effect could be associated with morphine-stimulated changes in aqueous humor dynamics and iris function. ICBs obtained from normal NZW rabbits were utilized in these experiments. In some experiments, ICB samples were treated with morphine (1, 10 and 100 μM) for 1 hour and later examined for changes in NO levels using a NO detection kit. In other experiments, tissue samples were pretreated with naloxone (non-selective opioid receptor antagonist), L-NAME (non-selective NO synthase inhibitor) or GSH (sulfhydryl reagent) for 30 minutes, followed by treatment with morphine (10 μM). Morphine caused a concentration-dependent increase in the release of NO from ICBs. Levels of NO detected in the incubation medium of ICB samples increased from 1.49 ± 0.19 (control) to 8.81 ± 2.20 μM/mg protein (morphine treated; 100 μM). Morphine-stimulated release of NO was significantly inhibited in tissues pretreated with 10 μM naloxone, L-NAME, or GSH. Results obtained from this study suggest that morphine stimulates NO release from the ICB through a mechanism that involves activation of NO-releasing opioid receptors. These results support the in vivo effects of morphine demonstrated in previous studies. PMID:19555685

  18. Trans Fatty Acids Induce Vascular Inflammation and Reduce Vascular Nitric Oxide Production in Endothelial Cells

    PubMed Central

    Iwata, Naomi G.; Pham, Matilda; Rizzo, Norma O.; Cheng, Andrew M.; Maloney, Ezekiel; Kim, Francis

    2011-01-01

    Intake of trans fatty acids (TFA), which are consumed by eating foods made from partially hydrogenated vegetable oils, is associated with a higher risk of cardiovascular disease. This relation can be explained by many factors including TFA's negative effect on endothelial function and reduced nitric oxide (NO) bioavailability. In this study we investigated the effects of three different TFA (2 common isomers of C18 found in partially hydrogenated vegetable oil and a C18 isomer found from ruminant-derived—dairy products and meat) on endothelial NF-κB activation and nitric oxide (NO) production. Human endothelial cells were treated with increasing concentrations of Elaidic (trans-C18:1 (9 trans)), Linoelaidic (trans-C18:2 (9 trans, 12 trans)), and Transvaccenic (trans-C18:1 (11 trans)) for 3 h. Both Elaidic and Linoelaidic acids were associated with increasing NF-κB activation as measured by IL-6 levels and phosphorylation of IκBα, and impairment of endothelial insulin signaling and NO production, whereas Transvaccenic acid was not associated with these responses. We also measured superoxide production, which has been hypothesized to be necessary in fatty acid-dependent activation of NF-κB. Both Elaidic acid and Linoelaidic acid are associated with increased superoxide production, whereas Transvaccenic acid (which did not induce inflammatory responses) did not increase superoxide production. We observed differential activation of endothelial superoxide production, NF-κB activation, and reduction in NO production by different C18 isomers suggesting that the location and number of trans double bonds effect endothelial NF-κB activation. PMID:22216328

  19. Nitric oxide exerts protective effects against bleomycin-induced pulmonary fibrosis in mice

    PubMed Central

    2014-01-01

    Background Increased expression of nitric oxide synthase (NOS) and an increase in plasma nitrite plus nitrate (NOx) have been reported in patients with pulmonary fibrosis, suggesting that nitric oxide (NO) plays an important role in its development. However, the roles of the entire NO and NOS system in the pathogenesis of pulmonary fibrosis still remain to be fully elucidated. The aim of the present study is to clarify the roles of NO and the NOS system in pulmonary fibrosis by using the mice lacking all three NOS isoforms. Methods Wild-type, single NOS knockout and triple NOS knockout (n/i/eNOS−/−) mice were administered bleomycin (BLM) intraperitoneally at a dose of 8.0 mg/kg/day for 10 consecutive days. Two weeks after the end of the procedure, the fibrotic and inflammatory changes of the lung were evaluated. In addition, we evaluated the effects of long-term treatment with isosorbide dinitrate, a NO donor, on the n/i/eNOS−/− mice with BLM-induced pulmonary fibrosis. Results The histopathological findings, collagen content and the total cell number in bronchoalveolar lavage fluid were the most severe/highest in the n/i/eNOS−/− mice. Long-term treatment with the supplemental NO donor in n/i/eNOS−/− mice significantly prevented the progression of the histopathological findings and the increase of the collagen content in the lungs. Conclusions These results provide the first direct evidence that a lack of all three NOS isoforms led to a deterioration of pulmonary fibrosis in a BLM-treated murine model. We speculate that the entire endogenous NO and NOS system plays an important protective role in the pathogenesis of pulmonary fibrosis. PMID:25092105

  20. The Arabidopsis Prohibitin Gene PHB3 Functions in Nitric Oxide–Mediated Responses and in Hydrogen Peroxide–Induced Nitric Oxide Accumulation[C][W

    PubMed Central

    Wang, Yong; Ries, Amber; Wu, Kati; Yang, Albert; Crawford, Nigel M.

    2010-01-01

    To discover genes involved in nitric oxide (NO) metabolism, a genetic screen was employed to identify mutants defective in NO accumulation after treatment with the physiological inducer hydrogen peroxide. In wild-type Arabidopsis thaliana plants, NO levels increase eightfold in roots after H2O2 treatment for 30 min. A mutant defective in H2O2-induced NO accumulation was identified, and the corresponding mutation was mapped to the prohibitin gene PHB3, converting the highly conserved Gly-37 to an Asp in the protein's SPFH domain. This point mutant and a T-DNA insertion mutant were examined for other NO-related phenotypes. Both mutants were defective in abscisic acid–induced NO accumulation and stomatal closure and in auxin-induced lateral root formation. Both mutants were less sensitive to salt stress, showing no increase in NO accumulation and less inhibition of primary root growth in response to NaCl treatment. In addition, light-induced NO accumulation was dramatically reduced in cotyledons. We found no evidence for impaired H2O2 metabolism or signaling in the mutants as H2O2 levels and H2O2-induced gene expression were unaffected by the mutations. These findings identify a component of the NO homeostasis system in plants and expand the function of prohibitin genes to include regulation of NO accumulation and NO-mediated responses. PMID:20068191

  1. The Arabidopsis Prohibitin Gene PHB3 Functions in Nitric Oxide-Mediated Responses and in Hydrogen Peroxide-Induced Nitric Oxide Accumulation.

    PubMed

    Wang, Yong; Ries, Amber; Wu, Kati; Yang, Albert; Crawford, Nigel M

    2010-01-01

    To discover genes involved in nitric oxide (NO) metabolism, a genetic screen was employed to identify mutants defective in NO accumulation after treatment with the physiological inducer hydrogen peroxide. In wild-type Arabidopsis thaliana plants, NO levels increase eightfold in roots after H(2)O(2) treatment for 30 min. A mutant defective in H(2)O(2)-induced NO accumulation was identified, and the corresponding mutation was mapped to the prohibitin gene PHB3, converting the highly conserved Gly-37 to an Asp in the protein's SPFH domain. This point mutant and a T-DNA insertion mutant were examined for other NO-related phenotypes. Both mutants were defective in abscisic acid-induced NO accumulation and stomatal closure and in auxin-induced lateral root formation. Both mutants were less sensitive to salt stress, showing no increase in NO accumulation and less inhibition of primary root growth in response to NaCl treatment. In addition, light-induced NO accumulation was dramatically reduced in cotyledons. We found no evidence for impaired H(2)O(2) metabolism or signaling in the mutants as H(2)O(2) levels and H(2)O(2)-induced gene expression were unaffected by the mutations. These findings identify a component of the NO homeostasis system in plants and expand the function of prohibitin genes to include regulation of NO accumulation and NO-mediated responses.

  2. Characterization of Calmodulin-Free Murine Inducible Nitric-Oxide Synthase

    PubMed Central

    Nagpal, Latika; Panda, Koustubh

    2015-01-01

    Nitric-Oxide Synthase (NOS), that produces the biological signal molecule Nitric-Oxide (NO), exists in three different isoforms called, neuronal (nNOS), endothelial (eNOS) and inducible (iNOS). All NOS isoforms require post-translational interaction with the calcium-binding protein, calmodulin (CaM) for manifesting their catalytic activity. However, CaM has been suggested to control the translational assembly of the enzyme as well, particularly in helping its inducible isoform, iNOS assume a stable, heme-replete, dimeric and active form. Expression of recombinant murine iNOS in E.coli in the absence of CaM has been previously shown to give extremely poor yield of the enzyme which was claimed to be absolutely heme-free, devoid of flavins, completely monomeric and catalytically inactive when compared to the heme-replete, active, dimeric iNOS, generated through co-expression with CaM. In contrast, we found that although iNOS expressed without CaM does produce significantly low amounts of the CaM-free enzyme, the iNOS thus produced, is not completely devoid of heme and is neither entirely monomeric nor absolutely bereft of catalytic activity as reported before. In fact, iNOS synthesized in the absence of CaM undergoes compromised heme incorporation resulting in extremely poor dimerization and activity compared to its counterpart co-expressed with CaM. Moreover, such CaM-free iNOS has similar flavin content and reductase activity as iNOS co-expressed with CaM, suggesting that CaM may not be as much required for the functional assembly of the iNOS reductase domain as its oxygenase domain. LC-MS/MS-based peptide mapping of the CaM-free iNOS confirmed that it had the same full-length sequence as the CaM-replete iNOS. Isothermal calorimetric measurements also revealed high affinity for CaM binding in the CaM-free iNOS and thus the possible presence of a CaM-binding domain. Thus CaM is essential but not indispensible for the assembly of iNOS and such CaM-free iNOS may help

  3. The role of nitric oxide in saline-induced natriuresis and diuresis in rats.

    PubMed

    Noonan, W T; Banks, R O

    1999-09-01

    This study was designed to determine to what extent nitric oxide (NO) mediates the natriuretic and diuretic responses to acute isotonic saline (0.9 gram % NaCl) volume expansion (SVE, 0.5 ml min-1 kg-1). Studies were performed on 49 pentobarbital anesthetized (65 mg/kg) female Sprague-Dawley rats with or without a NO synthase inhibitor, Nomega-nitro-L-arginine (LNA). Group 1 received saline at 27 microliter/min for 1 hr (baseline) and then SVE for 1 hr; Groups 2-4 received LNA at 10, 150, and 200 microgram kg-1 min-1, respectively, for 1 hr followed by LNA + SVE. To determine to what extent inhibition of NOS would reverse an ongoing SVE-induced natriuresis and diuresis, Group 5 was saline-volume-expanded for hours 1 and 2 whereas Group 6 was administered SVE during the first hour and then SVE + 150 microgram kg -1 min-1 LNA during the second hour. SVE caused a significant (P < 0.05) increase in the glomerular filtration rate (GFR) of Group 1 and the LNA-treated rats (Groups 2-4). This SVE-induced increase in the GFR occurred despite the fact that baseline GFR was significantly lower in the two groups of rats that were infused with the highest doses of LNA (Groups 3-4). SVE was also associated with similar increases in urine flow rate, sodium and potassium excretion, and total osmolar excretion in Groups 1-4. On the other hand, mean arterial pressure (MAP) was significantly higher in Group 2 during SVE + LNA and during the baseline as well as during the SVE periods in Groups 3-4; MAP was also significantly elevated in Group 6 during SVE + LNA. Thus, despite the fact that MAP was higher in LNA-treated rats, sodium and urine flow rates were the same as in Group 1 (i.e., there was no evidence of a pressure natriuresis or diuresis in these animals). Along these lines, there was a small but significant positive linear correlation coefficient (r = 0.41, P = 0.05) between sodium excretion values and corresponding MAP values in SVE control rats but not in Groups 3-4 during

  4. Expression of inducible nitric oxide synthase and nitric oxide production in the mud-dwelled air-breathing singhi catfish, Heteropneustes fossilis under condition of water shortage.

    PubMed

    Choudhury, Mahua G; Saha, Nirmalendu

    2012-12-01

    Nitric oxide (NO) is known to be an important regulator molecule for regulating the multiple signaling pathways and also to play diverse physiological functions in mammals including that of adaptation to various stresses. The present study reports on the production of nitric oxide (NO) and the expression of inducible nitric oxide synthase (iNOS) enzyme that produces NO from l-arginine in the freshwater air-breathing catfish (Heteropneustes fossilis) while dwelling inside the mud peat under semidry conditions. Desiccation stress, due to mud-dwelling for 2 weeks, led to significant increase of NO concentration in different tissues and in plasma of singhi catfish, and also the increase of NO efflux from the perfused liver with an accompanying increase of toxic ammonia level in different tissues. Mud-dwelling also resulted to induction of iNOS activity, expression of iNOS protein in different tissues after 7 days with further increase after 14 days, which otherwise was not detectable in control fish. Further, mud-dwelling also resulted to a significant expression of iNOS mRNA after 7 days with a more increase of mRNA level after 14 days, suggesting that the desiccation stress caused transcriptional regulation of iNOS gene. Immunocytochemical analysis indicated the zonal specific expression of iNOS protein in different tissues. Desiccation stress also led to activation and nuclear translocation of nuclear factor кB (NFкB) in hepatic cells. These results suggest that the activation of iNOS gene under desiccation-induced stresses such as high ammonia load was probably mediated through the activation of one of the major transcription factors, the NFкB. This is the first report of desiccation-induced induction of iNOS gene, iNOS protein expression leading to more generation of NO while living inside the mud peat under condition of water shortage in any air-breathing teleosts.

  5. Endothelial Nitric Oxide Synthase Deficient Mice Are Protected from Lipopolysaccharide Induced Acute Lung Injury

    PubMed Central

    Gross, Christine M.; Rafikov, Ruslan; Kumar, Sanjiv; Aggarwal, Saurabh; Ham III, P. Benson; Meadows, Mary Louise; Cherian-Shaw, Mary; Kangath, Archana; Sridhar, Supriya; Lucas, Rudolf; Black, Stephen M.

    2015-01-01

    Lipopolysaccharide (LPS) derived from the outer membrane of gram-negative bacteria induces acute lung injury (ALI) in mice. This injury is associated with lung edema, inflammation, diffuse alveolar damage, and severe respiratory insufficiency. We have previously reported that LPS-mediated nitric oxide synthase (NOS) uncoupling, through increases in asymmetric dimethylarginine (ADMA), plays an important role in the development of ALI through the generation of reactive oxygen and nitrogen species. Therefore, the focus of this study was to determine whether mice deficient in endothelial NOS (eNOS-/-) are protected against ALI. In both wild-type and eNOS-/- mice, ALI was induced by the intratracheal instillation of LPS (2 mg/kg). After 24 hours, we found that eNOS-/-mice were protected against the LPS mediated increase in inflammatory cell infiltration, inflammatory cytokine production, and lung injury. In addition, LPS exposed eNOS-/- mice had increased oxygen saturation and improved lung mechanics. The protection in eNOS-/- mice was associated with an attenuated production of NO, NOS derived superoxide, and peroxynitrite. Furthermore, we found that eNOS-/- mice had less RhoA activation that correlated with a reduction in RhoA nitration at Tyr34. Finally, we found that the reduction in NOS uncoupling in eNOS-/- mice was due to a preservation of dimethylarginine dimethylaminohydrolase (DDAH) activity that prevented the LPS-mediated increase in ADMA. Together our data suggest that eNOS derived reactive species play an important role in the development of LPS-mediated lung injury. PMID:25786132

  6. Hypoxia inducible factor signaling modulates susceptibility to mycobacterial infection via a nitric oxide dependent mechanism.

    PubMed

    Elks, Philip M; Brizee, Sabrina; van der Vaart, Michiel; Walmsley, Sarah R; van Eeden, Fredericus J; Renshaw, Stephen A; Meijer, Annemarie H

    2013-01-01

    Tuberculosis is a current major world-health problem, exacerbated by the causative pathogen, Mycobacterium tuberculosis (Mtb), becoming increasingly resistant to conventional antibiotic treatment. Mtb is able to counteract the bactericidal mechanisms of leukocytes to survive intracellularly and develop a niche permissive for proliferation and dissemination. Understanding of the pathogenesis of mycobacterial infections such as tuberculosis (TB) remains limited, especially for early infection and for reactivation of latent infection. Signaling via hypoxia inducible factor α (HIF-α) transcription factors has previously been implicated in leukocyte activation and host defence. We have previously shown that hypoxic signaling via stabilization of Hif-1α prolongs the functionality of leukocytes in the innate immune response to injury. We sought to manipulate Hif-α signaling in a well-established Mycobacterium marinum (Mm) zebrafish model of TB to investigate effects on the host's ability to combat mycobacterial infection. Stabilization of host Hif-1α, both pharmacologically and genetically, at early stages of Mm infection was able to reduce the bacterial burden of infected larvae. Increasing Hif-1α signaling enhanced levels of reactive nitrogen species (RNS) in neutrophils prior to infection and was able to reduce larval mycobacterial burden. Conversely, decreasing Hif-2α signaling enhanced RNS levels and reduced bacterial burden, demonstrating that Hif-1α and Hif-2α have opposing effects on host susceptibility to mycobacterial infection. The antimicrobial effect of Hif-1α stabilization, and Hif-2α reduction, were demonstrated to be dependent on inducible nitric oxide synthase (iNOS) signaling at early stages of infection. Our findings indicate that induction of leukocyte iNOS by stabilizing Hif-1α, or reducing Hif-2α, aids the host during early stages of Mm infection. Stabilization of Hif-1α therefore represents a potential target for therapeutic

  7. Dietary polyphenols generate nitric oxide from nitrite in the stomach and induce smooth muscle relaxation.

    PubMed

    Rocha, Bárbara S; Gago, Bruno; Barbosa, Rui M; Laranjinha, João

    2009-11-09

    Nitrite, considered a biological waste and toxic product, is being regarded as an important physiological molecule in nitric oxide (NO) biochemistry. Because the interaction of dietary phenolic compounds and nitrite would be kinetically (due to the high concentrations achieved) and thermodynamically (on basis of the redox potentials) feasible in the stomach, we have studied the potential reduction of nitrite by polyphenols present in several dietary sources. By measuring the time courses of *NO production in simulated gastric juice (pH 2), the efficiency of the compounds studied is as follows: Epicatechin-3-O-gallate>quercetin>procyanidin B8 dimer>oleuropein>procyanidin B2 dimer>chlorogenic acid>epicatechin>catechin>procyanidin B5 dimer. The initial rates of *NO production fall in a narrow range (ca. 1-5 microMs(-1)) but the distinct kinetics of the decay of *NO signals suggest that competition reactions for *NO are operative. The proof of concept that, in the presence of nitrite, phenol-containing dietary products induce a strong increase of *NO in the stomach was established in an in vivo experiment with healthy volunteers consuming lettuce, onions, apples, wine, tea, berries and cherries. Moreover, selected mixtures of oleuropein and catechin with low nitrite (1 microM) were shown to induce muscle relaxation of stomach strips in a structure-dependent way. Data presented here brings strong support to the concept that polyphenols consumed in a variety of dietary products, under gastric conditions, reduce nitrite to *NO that, in turn, may exert a biological impact as a local relaxant.

  8. Nitric oxide content and apoptosis rate in steroid-induced avascular necrosis of the femoral head

    PubMed Central

    BAI, RUI; LIU, WANLIN; ZHAO, AIQING; ZHAO, ZHENGQUN; JIANG, DIANMING

    2015-01-01

    The aim of the present study was to explore the effect on nitric oxide (NO) content and osteocyte apoptosis of steroid-induced avascular necrosis of the femoral head (SANFH) in an animal model of SANFH. A total of 40 Japanese white rabbits, 5 months of age and weighing 2.5±0.5 kg, were randomly divided into groups A (hormone + endotoxin group), B (endotoxin + normal saline group), C (normal saline + hormone group) and D (control group). Following the establishment of the model, a blood sample was taken from the heart of each animal and centrifuged; the levels of NO in the serum were detected. The bilateral femoral heads were conventionally dissected, fixed, decalcified and stained with hematoxylin and eosin. Subsequently, the empty bone lacunae were counted under an optical microscope. Changes in osteocyte morphology were observed using electron microscopy and osteocyte apoptosis was detected with a terminal deoxynucleotidyl transferase dUTP nick end labeling assay. The percentage of empty bone lacunae in group A was significantly higher compared with that in groups B, C and D (P<0.01); however, there was no significant difference in percentage among groups B, C and D. The NO content in group A was significantly higher compared with that in groups B, C and D (P<0.01); however, there was no significant difference in NO content among groups B, C and D. The osteocyte apoptosis index in group A was significantly higher compared with that in the other groups (P<0.01); there was no significant difference among groups B, C and D. NO content was positively correlated with osteocyte apoptosis index (r=0.707). Thus, the present study found that NO content and the osteocyte apoptosis index were increased in SANFH, and that they play an important role in SANFH. The content of NO was positively correlated with the osteocyte apoptosis index, indicating that NO induces apoptosis. PMID:26622359

  9. Roles of nitric oxide in inducible resistance of Escherichia coli to activated murine macrophages.

    PubMed

    Nunoshiba, T; DeRojas-Walker, T; Tannenbaum, S R; Demple, B

    1995-03-01

    Nitric oxide (NO.) is produced as a cytotoxic free radical through enzymatic oxidation of L-arginine in activated macrophages. Pure NO. gas was previously found to induce the Escherichia coli soxRS oxidative stress regulon, which is readily monitored by using a soxS'::lac fusion. The soxRS system includes antioxidant defenses, such as a superoxide dismutase and a DNA repair enzyme for oxidative damage, and protects E. coli from the cytotoxicity of NO.-generating macrophages. Previous experiments involved exposing E. coli to a bolus of NO. rather than the steadily generated gas expected of activated macrophages. We show here detectable induction of soxS transcription by NO. delivered at rates as low as 25 microM/h. Maximal induction was observed at 25 microM NO. per h under anaerobic conditions but at 125 microM/h aerobically. After incubation with murine macrophages, soxS expression was induced in the phagocytosed bacteria up to approximately 30-fold after an 8-h exposure. This in vivo induction was almost completely eliminated by the NO. synthase inhibitor NG-monomethyl-L-arginine. The inhibitor increased the survival of a delta soxRS strain but not that of wild-type E. coli after phagocytosis, which suggests that induction of the soxRS regulon by NO. can counteract most of the cytotoxic effects of NO. production by the macrophages. We show that the soxRS-regulated enzyme glucose-6-phosphate dehydrogenase is an important element of the defense against macrophages.

  10. Metal allergens induce nitric oxide production by mouse dermal fibroblasts via the hypoxia-inducible factor-2α-dependent pathway.

    PubMed

    Kuroishi, Toshinobu; Bando, Kanan; Endo, Yasuo; Sugawara, Shunji

    2013-09-01

    Nickel (Ni) has been shown to be one of the most frequent metal allergens. We have already reported a murine metal allergy model with pathogen-associated molecular patterns (PAMPs) as adjuvants. Interleukin (IL)-1β plays a critical role in our mouse model. Because nonimmune cells, including fibroblasts, play important roles in local allergic inflammation, we investigated whether Ni induces inflammatory responses in mouse dermal fibroblasts (MDF). We also analyzed the synergistic effects between Ni, PAMPs, and IL-1β. MDF stimulated with Ni produced a significantly higher amount of nitric oxide (NO) in a dose-dependent manner. NO production was augmented by costimulation with IL-1β but not with PAMPs. On the other hand, IL-1β or PAMPs induced a significantly higher amount of IL-6 production by MDF, but no augmentation was detected in the presence of Ni. A specific inhibitor for inducible nitric oxide synthase (iNOS) inhibited Ni-induced NO production. iNOS mRNA expression was significantly higher in MDF stimulated with Ni, IL-1β, or both. A specific inhibitor for hypoxia-inducible factor (HIF)-2α, but not HIF-1α, inhibited NO production. Another frequent metal allergen, cobalt, also induced iNOS expression and NO production by MDF via the HIF-2α-dependent pathway. The inhibitor for iNOS augmented ear swelling in Ni allergy mouse model. On the other hand, HIF-2α inhibitor attenuates allergic inflammation. These results indicate that metal allergens induce NO production in MDF via the HIF-2α-dependent pathway and IL-1β augments NO production, which suggests that the NO induced by metal allergens plays a pathological role in metal allergies.

  11. Nitric oxide in microgravity-induced orthostatic intolerance: relevance to spinal cord injury

    NASA Technical Reports Server (NTRS)

    Vaziri, N. D.; Purdy, R. E. (Principal Investigator)

    2003-01-01

    Prolonged exposure to microgravity results in cardiovascular deconditioning which is marked by orthostatic intolerance in the returning astronauts and recovering bed-ridden patients. Recent studies conducted in our laboratories at University of California, Irvine have revealed marked elevation of nitric oxide (NO) production in the kidney, heart, brain, and systemic arteries coupled with significant reduction of NO production in the cerebral arteries of microgravity-adapted animals. We have further demonstrated that the observed alteration of NO metabolism is primarily responsible for the associated cardiovascular deconditioning. Recovery from acute spinal cord injury (SCI) is frequently complicated by orthostatic intolerance that is due to the combined effects of the disruption of efferent sympathetic pathway and cardiovascular deconditioning occasioned by prolonged confinement to bed. In this presentation, I will review the nature of altered NO metabolism and its role in the pathogenesis of microgravity-induced cardiovascular deconditioning. The possible relevance of the new findings to orthostatic intolerance in patients with acute SCI and its potential therapeutic implications will be discussed.

  12. Inducible nitric oxide synthase is an endogenous neuroprotectant after traumatic brain injury in rats and mice.

    PubMed

    Sinz, E H; Kochanek, P M; Dixon, C E; Clark, R S; Carcillo, J A; Schiding, J K; Chen, M; Wisniewski, S R; Carlos, T M; Williams, D; DeKosky, S T; Watkins, S C; Marion, D W; Billiar, T R

    1999-09-01

    Nitric oxide (NO) derived from the inducible isoform of NO synthase (iNOS) is an inflammatory product implicated both in secondary damage and in recovery from brain injury. To address the role of iNOS in experimental traumatic brain injury (TBI), we used 2 paradigms in 2 species. In a model of controlled cortical impact (CCI) with secondary hypoxemia, rats were treated with vehicle or with 1 of 2 iNOS inhibitors (aminoguanidine and L-N-iminoethyl-lysine), administered by Alzet pump for 5 days and 1. 5 days after injury, respectively. In a model of CCI, knockout mice lacking the iNOS gene (iNOS(-/-)) were compared with wild-type (iNOS(+/+)) mice. Functional outcome (motor and cognitive) during the first 20 days after injury, and histopathology at 21 days, were assessed in both studies. Treatment of rats with either of the iNOS inhibitors after TBI significantly exacerbated deficits in cognitive performance, as assessed by Morris water maze (MWM) and increased neuron loss in vulnerable regions (CA3 and CA1) of hippocampus. Uninjured iNOS(+/+) and iNOS(-/-) mice performed equally well in both motor and cognitive tasks. However, after TBI, iNOS(-/-) mice showed markedly worse performance in the MWM task than iNOS(+/+) mice. A beneficial role for iNOS in TBI is supported.

  13. Inducible nitric oxide synthase in duodenum of children with Giardia lamblia infection.

    PubMed

    Mokrzycka, Małgorzata; Kolasa, Agnieszka; Kosierkiewicz, Anita; Wiszniewska, Barbara

    2010-01-01

    The investigation were performed on children with Giardia lamblia infection, diagnosed on the basis of positive stool tests for Giardia antigen (Elisa) or by microscopical detection of trophozoites in duodenal fluid. In duodenal biopsies morphological studies and immunohistochemical reaction for inducible nitric oxide synthase (iNOS) were performed. The control group was made up of duodenal tissue of children with excluded giardiasis and inflammation of the upper part of gastrointestinal tract. The duodenal biopsies from children without Giardia lamblia infection were found to have a high immunoreactivity for iNOS in enterocytes, the cells of intestinal crypts, endothelial cells of vessels and connective tissue cells of lamina propria. In children with giardiasis: in some biopsies the expression of iNOS was as high as in control group, in others was weaker detectable and the shortening of intestinal villi was seen. There were also duodenal biopsies with the lack of immunoreactivity for iNOS, with shorter villi and a large amount of mucus in the intestinal epithelium. Beside of goblet cells, also enterocytes were loaded with mucus. The pathological changes may cause malabsorption and also may have a negative influence on the defense of the intestinal wall against Giardia lamblia infection. The different morphological and immunohistochemical results in the duodenum of children with giardiasis can elucidate a variety of clinical symptoms from asymptomatic to severe infection.

  14. Role of nitric oxide in the radiation-induced bystander effect.

    PubMed

    Yakovlev, Vasily A

    2015-12-01

    Cells that are not irradiated but are affected by "stress signal factors" released from irradiated cells are called bystander cells. These cells, as well as directly irradiated ones, express DNA damage-related proteins and display excess DNA damage, chromosome aberrations, mutations, and malignant transformation. This phenomenon has been studied widely in the past 20 years, since its first description by Nagasawa and Little in 1992, and is known as the radiation-induced bystander effect (RIBE). Several factors have been identified as playing a role in the bystander response. This review will focus on one of them, nitric oxide (NO), and its role in the stimulation and propagation of RIBE. The hydrophobic properties of NO, which permit its diffusion through the cytoplasm and plasma membranes, allow this signaling molecule to easily spread from irradiated cells to bystander cells without the involvement of gap junction intercellular communication. NO produced in irradiated tissues mediates cellular regulation through posttranslational modification of a number of regulatory proteins. The best studied of these modifications are S-nitrosylation (reversible oxidation of cysteine) and tyrosine nitration. These modifications can up- or down-regulate the functions of many proteins modulating different NO-dependent effects. These NO-dependent effects include the stimulation of genomic instability (GI) and the accumulation of DNA errors in bystander cells without direct DNA damage.

  15. Inducible nitric oxide synthase in tangle-bearing neurons of patients with Alzheimer's disease

    PubMed Central

    1996-01-01

    In Alzheimer's disease (AD), affected neurons accumulate beta amyloid protein, components of which can induce mouse microglia to express the high-output isoform of nitric oxide synthase (NOS2) in vitro. Products of NOS2 can be neurotoxic. In mice, NOS2 is normally suppressed by transforming growth factor beta 1 (TGF-beta 1). Expression of TGF-beta 1 is decreased in brains from AD patients, a situation that might be permissive for accumulation of NOS2. Accordingly, we investigated the expression of NOS2 in patients with AD, using three monospecific antibodies: a previously described polyclonal and two new monoclonal antibodies. Neurofibrillary tangle-bearing neurons and neuropil threads contained NOS2 in brains from each of 11 AD patients ranging in age from 47 to 81 years. NOS2 was undetectable in brains from 6 control subjects aged 23-72 years, but was expressed in small amounts in 3 control subjects aged 77-87 years. Thus, human neurons can express NOS2 in vivo. The high-output pathway of NO production may contribute to pathogenesis in AD. PMID:8879214

  16. Nitric oxide alleviates oxidative damage induced by high temperature stress in wheat.

    PubMed

    Bavita, A; Shashi, B; Navtej, S B

    2012-05-01

    Effect of sodium nitroprusside (SNP), a donor of nitric oxide (NO) was examined in two wheat (Triticum aestivum L.) cultivars, C 306 (heat tolerant) and PBW 550 (comparatively heat susceptible) to study the extent of oxidative injury and activities of antioxidant enzyme in relation to high temperature (HT) stress. HT stress resulted in a marked decrease in membrane thermostability (MTS) and 2, 3, 5-triphenyl tetrazolium chloride (TTC) cell viability whereas content of lipid peroxide increased in both the cultivars. The tolerant cultivar C 306 registered less damage to cellular membranes compared to PBW 550 under HT stress. Activities of antioxidant enzymes viz, superoxide dismutase, catalase, ascorbate peroxidase, guaicol peroxidase and glutathione reductase increased with HT in both the cultivars. Following treatment with SNP, activities of all antioxidant enzymes further increased in correspondence with an increase in MTS and TTC. Apparently, lipid peroxide content was reduced by SNP more in shoots of heat tolerant cultivar C 306 indicating better protection over roots under HT stress. The up-regulation of the antioxidant system by NO possibly contributed to better tolerance against HT induced oxidative damage in wheat.

  17. Sigma 1 receptor agonists act as neuroprotective drugs through inhibition of inducible nitric oxide synthase.

    PubMed

    Vagnerova, Kamila; Hurn, Patricia D; Bhardwaj, Anish; Kirsch, Jeffrey R

    2006-08-01

    Postischemic administration of the sigma-1 agonists reduces ischemic brain injury; however, the mechanism is unclear. We hypothesized that the sigma-1 agonist (+)isoform of pentazocine (P(+)) reduces damage in part by ameliorating cell death mediated via inducible nitric oxide synthase (iNOS) and that the (-)isoform (P(-)) lacks this effect. We compared treatment with P(+) with or without the iNOS inhibitor aminoguanidine (AG) and also the effects of P(+) in iNOS deficient (iNOSKO) mice. A possible mechanism of neuroprotection is inhibition of iNOS expression. Male C57/Bl6 mice were subjected to transient middle cerebral artery occlusion (90 min) and drugs were administered with reperfusion: 1) P(+) with AG (P+/AG), 2) P(+), 3) P(-), 4) AG, or 5) placebo. iNOSKOs were treated with either P(+) or placebo. Infarction (triphenyltetrazolium chloride histology, 72 h) was reduced by P(+) treatment in striatum by 44% and in neocortex by 23% versus placebo (P < 0.05), a reduction comparable to AG effect. P(-) did not attenuate brain injury. There was no difference in P(+)/AG treatment compared with showed the same level of neuroprotection as P(+) alone. P(+) also did not provide further neuroprotection for iNOSKOs. We conclude that postischemic administration of P(+) reduces infarct volume in mice. Because AG provides no additional benefit to P(+) treatment and iNOSKOs do not benefit from P(+), we speculate that P(+) acts by suppressing cell death resulting from iNOS toxicity.

  18. Resolution of experimental lung injury by Monocyte-derived inducible nitric oxide synthase (iNOS)

    PubMed Central

    D’Alessio, Franco R.; Tsushima, Kenji; Aggarwal, Neil R.; Mock, Jason R.; Eto, Yoshiki; Garibaldi, Brian T.; Files, Daniel C.; Avalos, Claudia R.; Rodriguez, Jackie V.; Waickman, Adam T.; Reddy, Sekhar P.; Pearse, David B.; Sidhaye, Venkataramana K.; Hassoun, Paul M.; Crow, Michael T.; King, Landon S.

    2012-01-01

    While early events in the pathogenesis of acute lung injury (ALI) have been defined, little is known about mechanisms mediating resolution. To search for determinants of resolution, we exposed wild type (WT) mice to intratracheal lipopolysacaccharide (i.t. LPS) and assessed the response at intervals to day 10, when injury had resolved. Inducible nitric oxide synthase (iNOS) was significantly upregulated in the lung at day 4 after LPS. When iNOS−/− mice were exposed to i.t. LPS, early lung injury was attenuated, however recovery was markedly impaired compared to wild type (WT) mice. iNOS−/− mice had increased mortality and sustained increases in markers of lung injury. Adoptive transfer of WT (iNOS+/+) bone marrow-derived monocytes or direct adenoviral gene delivery of iNOS into injured iNOS−/− mice restored resolution of ALI. Irradiated bone marrow chimeras confirmed the protective effects of myeloid-derived iNOS, but not of epithelial iNOS. Alveolar macrophages exhibited sustained expression of co-signalling molecule CD86 in iNOS−/− mice compared to WT mice. Antibody-mediated blockade of CD86 in iNOS−/− mice improved survival and enhanced resolution of lung inflammation. Our findings show that monocyte-derived iNOS plays a pivotal role in mediating resolution of ALI by modulating lung immune responses, thus facilitating clearance of alveolar inflammation and promoting lung repair. PMID:22844117

  19. Excessive levels of nitric oxide in rat model of Parkinson’s disease induced by rotenone

    PubMed Central

    XIONG, ZHONG-KUI; LANG, JUAN; XU, GANG; LI, HAI-YU; ZHANG, YUN; WANG, LEI; SU, YAO; SUN, AI-JING

    2015-01-01

    Systemic rotenone models of Parkinson’s disease (PD) are highly reproducible and may provide evidence on the pathogenesis of PD. In the present study, male Sprague-Dawley rats (1-year-old) were subcutaneously administered with rotenone (1.5 mg/kg/day) for six days and observed for the following three weeks. Compared with the control rats, a significant decrease was observed in the body weight and a marked increase was observed in the areas under the behavioral scoring curves in the rotenone-treated rats. Immunohistochemical staining revealed that the abundance of nigral tyrosine hydroxylase (TH)-positive neurons was markedly reduced following rotenone treatment. ELISA and neurochemical assays demonstrated a significant increase in the levels of nitric oxide (NO) and NO synthase, whereas a marked decrease was observed in the thiol levels in the brains of the rotenone-treated rats. Thus, subacute rotenone treatment was found to induce behavioral deficits and the loss of nigral TH-positive neurons which may be associated with the excessive levels of NO in the rat brains. PMID:25574233

  20. Laser induced fluorescence measurements and modeling of nitric oxide in high-pressure premixed flames

    NASA Technical Reports Server (NTRS)

    Reisel, John R.; Laurendeau, Normand M.

    1994-01-01

    Laser-induced fluorescence (LIF) has been applied to the quantitative measurement of nitric oxide (NO) in premixed, laminar, high-pressure flames. Their chemistry was also studied using three current kinetics schemes to determine the predictive capabilities of each mechanism with respect to NO concentrations. The flames studied were low-temperature (1600 less than T less than 1850K) C2H6/O2/N2 and C2H6/O2/N2 flames, and high temperature (2100 less than T less than 2300K) C2H6/O2/N2 flames. Laser-saturated fluorescence (LSF) was initially used to measure the NO concentrations. However, while the excitation transition was well saturated at atmospheric pressure, the fluorescence behavior was basically linear with respect to laser power at pressures above 6 atm. Measurements and calculations demonstrated that the fluorescence quenching rate variation is negligible for LIF measurements of NO at a given pressure. Therefore, linear LIF was used to perform quantitative measurements of NO concentration in these high-pressure flames. The transportability of a calibration factor from one set of flame conditions to another also was investigated by considering changes in the absorption and quenching environment for different flame conditions. The feasibility of performing LIF measurements of (NO) in turbulent flames was studied; the single-shot detection limit was determined to be 2 ppm.

  1. Hydrogen peroxide induces a rapid production of nitric oxide in mung bean (Phaseolus aureus).

    PubMed

    Lum, H K; Butt, Y K C; Lo, S C L

    2002-03-01

    Nitric oxide (NO) has recently been identified as an important signaling molecule in plant immune response. The present study aims to investigate the signaling pathway that leads to NO production. Using the NO specific fluorescent dye DAF-2DA, we observed rapid production of NO in mung bean leaves after the addition of 10 mM hydrogen peroxide (H(2)O(2)). NO was probably produced by a NOS-like enzyme in plants, as the NO production was inhibited by l-NAME, a NOS inhibitor. The NOS-like activity in the total leaf protein preparation of mung bean (Phaseolus aureus) was elevated 8.3-fold after 10 mM H(2)O(2) treatment, as demonstrated using the chemiluminescence NOS assay. The NOS-like activity was BH(4) dependent: omitting BH(4) in the reaction mixture of NOS assay reduced the NOS activity by 76%. We also found that the H(2)O(2) induced NO production was mediated via calcium ion flux, as it was blocked in the presence of a calcium ion channel blocker, verapamil. Results from the present study identified H(2)O(2) as an upstream signal that leads to NO production in plants. H(2)O(2) and NO, besides acting as two independent signaling molecules in plant immune response, may interrelate to form an oxidative cell death (OCD) cycle.

  2. Inducible nitric oxide synthase (NOS II) is constitutive in human neutrophils.

    PubMed

    Cedergren, Jan; Follin, Per; Forslund, Tony; Lindmark, Maria; Sundqvist, Tommy; Skogh, Thomas

    2003-10-01

    The objective was to study the expression of inducible nitric oxide synthase (NOS II) in and NO production by human blood neutrophils and in in vivo exudated neutrophils. Cellular expression of NOS II was evaluated by flow cytometry in whole blood, in isolated blood neutrophils, and in neutrophils obtained by exudation in vivo into skin chambers. Neutrophil NOS II was also demonstrated by Western blotting. Uptake of 3H-labelled L-arginine was studied in vitro and NOS activity measured in a whole cell assay by the conversion of 3H-arginine to 3H-citrulline. In contrast to unseparated blood cells, NOS II was demonstrable both in isolated blood neutrophils and exudated cells. The failure to detect NOS II by flow cytometry in whole blood cells thus proved to be due to the quenching effect of hemoglobin. Western blotting revealed a 130 kD band corresponding to NOS II in isolated blood neutrophils, but detection was dependent on diisopropylfluorophosphate for proteinase inhibition. L-arginine was taken up by neutrophils, but enzymatic activity could not be demonstrated. We conclude that human neutrophils constitutively express NOS II, but that its demonstration by FITC-labelling is inhibited by hemoglobin-mediated quenching in whole blood samples.

  3. Contribution of myeloperoxidase and inducible nitric oxide synthase to pathogenesis of psoriasis

    PubMed Central

    Dilek, Aziz Ramazan; Taşkın, Yakup; Erkinüresin, Taşkın; Yalçın, Ömer; Saral, Yunus

    2016-01-01

    Introduction Histological changes of psoriasis include invasion of neutrophils into the epidermis and formation of Munro abscesses in the epidermis. Neutrophils are the predominant white blood cells in circulation when stimulated; they discharge the abundant myeloperoxidase (MPO) enzyme that uses hydrogen peroxide to oxidize chloride for killing ingested bacteria. Aim To investigate the contribution of neutrophils to the pathogenesis of psoriasis at the blood and tissue levels through inducible nitric oxide synthase (iNOS) and MPO. Material and methods A total of 50 adult patients with a chronic plaque form of psoriasis and 25 healthy controls were enrolled to this study. Serum MPO and iNOS levels were measured using ELISA method. Two biopsy specimens were taken in each patient from the center of the lesion and uninvolved skin. Immunohistochemistry was performed for MPO and iNOS on both normal and psoriasis vulgaris biopsies. Results While a significant difference between serum myeloperoxidase levels were detected, a similar statistical difference between participants in the serum iNOS levels was not found. In immunohistochemistry, intensely stained leukocytes with MPO and intensely staining with iNOS in psoriatic skin was observed. Conclusions Neutrophils in psoriasis lesions are actively producing MPO and this indirectly triggers the synthesis of iNOS. Targeting of MPO or synthesis of MPO in the lesion area may contribute to development of a new treatment option. PMID:28035220

  4. Nerve-induced release of nitric oxide in the rabbit gastrointestinal tract as measured by in vivo microdialysis

    PubMed Central

    Iversen, Henrik H; Celsing, Fredrik; Leone, Anna M; Gustafsson, Lars E; Wiklund, N Peter

    1997-01-01

    Nitric oxide (NO) has been suggested as a gastrointestinal neurotransmitter, mediating the gastric receptive relaxation and the relaxation in the peristaltic reflex. The aim of the present study was to measure nerve-induced NO formation in vivo in the gastrointestinal tract.Formation of the nitric oxide oxidation products nitrite and nitrate during vagal nerve stimulation were measured in the anaesthetized rabbit. Microdialysis probes were inserted into the wall of the stomach and proximal colon, and nitrite and nitrate in dialysate measured by capillary electrophoresis.During bilateral vagal nerve stimulation there was an increase in nitrite and nitrate formation at the level of the stomach and in nitrite formation at the level of the colon. This increase was inhibited by intravenous administration of the NO synthase inhibitor Nω-nitro-L-arginine methyl ester (L-NAME 30 mg kg−1). Furthermore, L-NAME significantly increased nerve-induced gastric and colonic contractions, as well as spontaneous colonic contractions.In summary, we present a new methodological procedure for quantification of small changes in nitric oxide formation in vivo. This study provides evidence that nitric oxide is released in the stomach and colonic wall during vagal nerve activity, at concentrations able to cause inhibition of smooth muscle contractions in vivo. PMID:9051311

  5. Dexmedetomidine Inhibits Phenylephrine-induced Contractions via Alpha-1 Adrenoceptor Blockade and Nitric Oxide Release in Isolated Rat Aortae

    PubMed Central

    Byon, Hyo-Jin; Ok, Seong-Ho; Lee, Soo Hee; Kang, Sebin; Cho, Youngil; Han, Jeong Yeol; Sohn, Ju-Tae

    2017-01-01

    The goal of this in vitro study was to examine the effect of the alpha-2 adrenoceptor agonist dexmedetomidine on phenylephrine (alpha-1 adrenoceptor agonist)-induced contraction in isolated rat aortae and to elucidate the associated cellular mechanisms, with a particular focus on alpha-1 adrenoceptor antagonism. Dexmedetomidine dose-response curves were generated in isolated endothelium-intact and endothelium-denuded rat aortae precontracted with phenylephrine or 5-hydroxytryptamine. Endothelium-denuded aortic rings were pretreated with either dexmedetomidine or the reversible alpha-1 adrenoceptor antagonist phentolamine, followed by post-treatment with the irreversible alpha-1 adrenoceptor blocker phenoxybenzamine. Control rings were treated with phenoxybenzamine alone. All rings were repeatedly washed with Krebs solution to remove all pretreatment drugs, including phenoxybenzamine, phentolamine and dexmedetomidine. Phenylephrine dose-response curves were then generated. The effect of rauwolscine on the dexmedetomidine-mediated change in phenylephrine-induced endothelial nitric oxide synthase (eNOS) phosphorylation in human umbilical vein endothelial cells was examined using western blotting. The magnitude of the dexmedetomidine-mediated inhibition of phenylephrine-induced contraction was higher in endothelium-intact aortae than in endothelium-denuded aortae or endothelium-intact aortae treated with Nω-nitro-L-arginine methyl ester. However, dexmedetomidine did not significantly alter 5-hydroxytryptamine-induced contraction. In further experiments, prazosin attenuated dexmedetomidine-induced contraction. Additionally, pretreatment with either dexmedetomidine plus phenoxybenzamine or phentolamine plus phenoxybenzamine produced greater phenylephrine-induced contraction than phenoxybenzamine alone, suggesting that dexmedetomidine protects aortae from the alpha-1 adrenoceptor blockade induced by phenoxybenzamine. Rauwolscine attenuated the dexmedetomidine

  6. Macrolactins from Marine-Derived Bacillus subtilis B5 Bacteria as Inhibitors of Inducible Nitric Oxide and Cytokines Expression

    PubMed Central

    Yan, Xia; Zhou, Yun-Xia; Tang, Xi-Xiang; Liu, Xiu-Xiu; Yi, Zhi-Wei; Fang, Mei-Juan; Wu, Zhen; Jiang, Fu-Quan; Qiu, Ying-Kun

    2016-01-01

    In order to find new natural products with anti-inflammatory activity, chemical investigation of a 3000-meter deep-sea sediment derived bacteria Bacillus subtilis B5 was carried out. A new macrolactin derivative was isolated and identified as 7,13-epoxyl-macrolactin A (1). Owing to the existence of the epoxy ring, 1 exhibited a significant inhibitory effect on the expression of inducible nitric oxide and cytokines, compared with previously isolated known macrolactins (2–5). Real-time Polymerase Chain Reaction (PCR) analysis showed that the new compound significantly inhibited the mRNA expressions of inducible nitric oxide synthase (iNOS), interleukin-1β (IL-1β), and interleukin-6 (IL-6) in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Reverse transcription-PCR analysis demonstrated that the new compound reduced the mRNA expression level of IL-1β in a concentration-dependent manner. PMID:27792158

  7. The role of nitric oxide and sulphydryls in gastric mucosal protection induced by sodium cromoglycate in rats.

    PubMed

    Ali, A T

    1995-09-01

    The role of endogenous nitric oxide and sulphydryls in gastric protection afforded by sodium cromoglycate against ethanol-induced gastric lesions was studied in rats. Drugs were administered either intraperitoneally (i.p.) or subcutaneously (s.c.) 30, 45 or 60 min before oral administration of ethanol. Administration of cromoglycate before ethanol dose-dependently inhibited ethanol-induced gastric lesions. Pretreatment with NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide biosynthesis, dose-dependently aggravated gastric lesions and reduced cromoglycate-induced gastric protection. The attenuating effect of L-NAME on gastric protection elicited by cromoglycate was reversible by pretreatment with L-arginine but not by D-arginine. On the other hand, ethanol-induced gastric lesions were found to be associated with a reduction of nonprotein sulphydryl content of glandular stomachs. Pretreatment with cromoglycate prevented non protein sulphydryl depletion and afforded protection. Pretreatment with N-ethylmaleimide, a sulphydryl blocker, caused dose-dependent enhancement of ethanol-induced gastric lesions and further depletion of non protein-sulphydryl. Treatment with N-ethylmaleimide before cromoglycate reduced the gastric protection that was associated with depletion of nonprotein sulphydryls. Furthermore, combined N-ethylmaleimide and L-NAME pretreatment caused a greater aggravation of ethanol-induced gastric lesions and significantly produced a higher reduction of the protective effects of cromoglycate. However, pretreatment with L-arginine only partially restored the protective effects of cromoglycate. These results suggest that the protective effects of cromoglycate may be dependent on the maintenance of a critical level of both endogenous nitric oxide and nonprotein sulphydryls in the gastric mucosa.

  8. The effect of acute aripiprazole treatment on chemically and electrically induced seizures in mice: The role of nitric oxide.

    PubMed

    Shafaroodi, Hamed; Oveisi, Simin; Hosseini, Mahsa; Niknahad, Hossein; Moezi, Leila

    2015-07-01

    Aripiprazole is an antipsychotic drug which acts through dopamine and serotonin receptors. Aripiprazole was noted to have antiseizure effects in a study on mice, while it induced seizures in a few human case reports. Dopaminergic and serotonergic systems relate to nitric oxide, and aripiprazole also has effects on dopamine and serotonin receptors. This study investigated the effects of aripiprazole on seizures and the potential role of nitric oxide in the process. The following three models were examined to explore the role of aripiprazole on seizures in mice: 1 - pentylenetetrazole administered intravenously, 2 - pentylenetetrazole administered intraperitoneally, and 3 - electroshock. Aripiprazole administration delayed clonic seizure in intravenous and intraperitoneal pentylenetetrazole models. In the electroshock-induced seizure model, tonic seizure and mortality protection percent were increased after aripiprazole administration. In intraperitoneal administration of pentylenetetrazole, aripiprazole effects on clonic seizure latency were significantly decreased when l-NAME - a nonselective nitric oxide synthase (NOS) inhibitor, 7-nitroindazole - a selective neuronal NOS (nNOS) inhibitor, or aminoguanidine - a selective inducible NOS (iNOS) inhibitor was injected before aripiprazole administration. In the intravenous pentylenetetrazole method, administration of l-NAME or aminoguanidine inhibited aripiprazole effects on clonic seizure threshold. Aminoguanidine or l-NAME administration decreased aripiprazole-induced protection against tonic seizures and death in the electroshock model. In both intravenous and intraperitoneal seizure models, aripiprazole and l-arginine coadministration delayed the onset of clonic seizures. Moreover, it increased protection against tonic seizures and death in intraperitoneal pentylenetetrazole and electroshock models. In conclusion, the release of nitric oxide via iNOS or nNOS may be involved in anticonvulsant properties of

  9. Protective actions of estrogen on angiotensin II-induced hypertension: role of central nitric oxide.

    PubMed

    Xue, Baojian; Singh, Minati; Guo, Fang; Hay, Meredith; Johnson, Alan Kim

    2009-11-01

    The present study tested the hypotheses that 1) nitric oxide (NO) is involved in attenuated responses to ANG II in female mice, and 2) there is differential expression of neuronal NO synthase (nNOS) in the subfornical organ (SFO) and paraventricular nucleus (PVN) in response to systemic infusions of ANG II in males vs. females. Aortic blood pressure (BP) was measured in conscious mice with telemetry implants. N(G)-nitro-l-arginine methyl ester (l-NAME; 100 microg x kg(.-1)day(-1)), an inhibitor of NOS, was administrated into the lateral cerebral ventricle for 14 days before and during ANG II pump implantation. Central infusion of l-NAME augmented the pressor effects of systemic ANG II in females (Delta21.5 + or - 2.2 vs. Delta9.2 + or - 1.5 mmHg) but not in males (Delta29.4 + or - 2.5 vs. Delta30.1 + or - 2.5 mmHg). Central administration of N(5)-(1-imino-3-butenyl)-l-ornithine (l-VNIO), a selective nNOS inhibitor, also significantly potentiated the increase in BP induced by ANG II in females (Delta17.5 + or - 3.2 vs. Delta9.2 + or - 1.5 mmHg). In gonadectomized mice, central l-NAME infusion did not affect the pressor response to ANG II in either males or females. Ganglionic blockade after ANG II infusion resulted in a greater reduction in BP in central l-NAME- or l-VNIO-treated females compared with control females. Western blot analysis of nNOS protein expression indicated that levels were approximately 12-fold higher in both the SFO and PVN of intact females compared with those in intact males. Seven days of ANG II treatment resulted in a further increase in nNOS protein expression only in intact females (PVN, to approximately 51-fold). Immunohistochemical studies revealed colocalization of nNOS and estrogen receptors in the SFO and PVN. These results suggest that NO attenuates the increase in BP induced by ANG II through reduced sympathetic outflow in females and that increased nNOS protein expression associated with the presence of female sex hormones plays a

  10. Monocyte-induced downregulation of nitric oxide synthase in cultured aortic endothelial cells.

    PubMed

    Marczin, N; Antonov, A; Papapetropoulos, A; Munn, D H; Virmani, R; Kolodgie, F D; Gerrity, R; Catravas, J D

    1996-09-01

    Since endothelium-dependent vasodilation is altered in atherosclerosis and enhanced monocyte/endothelial interactions are implicated in early atherosclerosis, we evaluated the effects of monocytes on the endothelial nitric oxide (NO) pathway by estimating release of biologically active NO from cultured endothelial cells and levels of constitutive NO synthase (ecNOS). NO release was estimated in a short-term bioassay using endothelial cell-induced cGMP accumulation in vascular smooth muscle (SM) cells. Exposure of SM cells to porcine aortic endothelial cells (PAECs) and human aortic endothelial cells (HAECs) produced large increases in SM cGMP content; this increase was prevented by NG-nitro-L-arginine methyl ester, the inhibitor of endothelial NOS. Confluent monolayers of PAECs and HAECs cocultured with monocytes also stimulated SM cGMP formation; however, NO release from these cultures was attenuated in a coculture time (2 to 48 hours)- and monocyte concentration (20 to 200 x 10(3) per well)-dependent manner. This effect of monocyte adhesion appeared to be selective for NO release since other biochemical pathways, such as atriopeptin-and isoproterenol-induced cyclic nucleotide accumulation within the endothelial cells, were not altered by monocytes. The effects of adherent monocytes on NO release were mimicked by monocyte-derived cytokines tumor necrosis factor (TNF)-alpha and interleukin (IL)-1 alpha. Furthermore, the conditioned medium of monocytes contained significant quantities of these cytokines. Conditioned medium, as well as monocytes physically separated from the endothelial cells, attenuated NO release, suggesting that soluble factors may mediate the effects of monocytes. An IL-1 beta neutralizing antibody fully prevented the NO dysfunction in response to directly adherent monocytes. Superoxide dismutase, catalase, 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron), and exogenous L-arginine failed to improve NO release, suggesting that oxidant stress-induced

  11. Induced RAW 264.7 macrophages express soluble and particulate nitric oxide synthase: inhibition by transforming growth factor-beta.

    PubMed

    Förstermann, U; Schmidt, H H; Kohlhaas, K L; Murad, F

    1992-02-13

    RAW 264.7 macrophages induced with lipopolysaccharide and interferon-gamma expressed nitric oxide (NO) synthase. Approximately two-thirds of the total induced NO synthase activity was found in the cytosolic fraction, whereas one-third was associated with the particulate fraction. Both enzymes formed L-citrulline in addition to NO-like material. NO and L-citrulline formation by both enzymes were calcium-independent and inhibited by NG-nitro-L-arginine and NG-methyl-L-arginine. Transforming growth factor-beta 1 prevented the induction of both enzymes.

  12. JS-K, a nitric oxide prodrug, induces cytochrome c release and caspase activation in HL-60 myeloid leukemia cells.

    PubMed

    Udupi, Vidya; Yu, Margaret; Malaviya, Swati; Saavedra, Joseph E; Shami, Paul J

    2006-10-01

    Nitric oxide (NO) induces differentiation and apoptosis in acute myelogenous leukemia (AML) cells. The NO prodrug O2-(2,4-dinitrophenyl)1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate, or JS-K, has potent antileukemic activity. JS-K induces apoptosis in HL-60 cells by a caspase-dependent mechanism. The purpose of this study was to determine the pathway through which JS-K induces apoptosis. We show that JS-K alters mitochondrial membrane potential (DeltaPsim) and induces cytochrome c release from mitochondria into the cytoplasm. Treatment with JS-K resulted in activation of Caspase (Casp) 9, Casp 3 and Casp 8. JS-K constitutes a promising lead for a new class of anti-leukemic agents.

  13. The effect of thalidomide on ethanol-induced gastric mucosal damage in mice: involvement of inflammatory cytokines and nitric oxide.

    PubMed

    Amirshahrokhi, Keyvan; Khalili, Ali-Reza

    2015-01-05

    Excessive ethanol ingestion causes gastric mucosal damage through the inflammatory and oxidative processes. The present study was aimed to evaluate the protective effect of thalidomide on ethanol-induced gastric mucosal damage in mice. The animals were pretreated with vehicle or thalidomide (30 or 60 mg/kg, orally), and one hour later, the gastric mucosal injury was induced by oral administration of acidified ethanol. The animals were euthanized one hour after ethanol ingestion, and gastric tissues were collected to biochemical analyzes. The gastric mucosal lesions were assessed by macroscopic and histopathological examinations. The results showed that treatment of mice with thalidomide prior to the administration of ethanol dose-dependently reduced the gastric ulcer index. Thalidomide pretreatment significantly reduced the levels of pro-inflammatory cytokines [tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6], malondialdehyde (MDA) and myeloperoxidase (MPO) activity. In addition, thalidomide significantly inhibited ethanol-induced nitric oxide (NO) overproduction in gastric tissue. Histological observations showed that ethanol-induced gastric mucosal damage was attenuated by thalidomide pretreatment. It seems that thalidomide as an anti-inflammatory agent may have a protective effect against alcohol-induced mucosal damage by inhibition of neutrophil infiltration and reducing the production of nitric oxide and inflammatory cytokines in gastric tissue.

  14. Upregulation of N-acetylaspartic acid resulting nitric oxide toxicity induces aspartoacylase mutations and protein interaction to cause pathophysiology seen in Canavan disease.

    PubMed

    Surendran, Sankar

    2010-12-01

    Aspartoacylase (ASPA) converts N-acetylaspartic acid into aspartate and acetate. In Canavan disease (CD), N-acetylaspartic acid (NAA) is found to be increased and over 65 mutations including IVS4+1 G → T, deletion of introns and exons have been reported in the ASPA gene. These changes lead to severe form or mild form of CD. The present study was aimed to understand mechanism in the cause of mutations in ASPA and pathophysiology seen in patients with CD. We have reported that elevated levels of NAA induce inducible nitric oxide (iNOS) to produce nitric oxide toxicity in CD. Nitric oxide toxicity has been shown to induce several mutations including base change G → T and deletion and enhances protein interaction in several genes. Therefore we hypothesize that upregulation of NAA stimulates NOS and the resulting nitric oxide toxicity induces ASPA mutations and protein interaction to result pathophysiological abnormalities seen in patients with CD.

  15. Endothelial surface glycocalyx can regulate flow-induced nitric oxide production in microvessels in vivo.

    PubMed

    Yen, Wanyi; Cai, Bin; Yang, Jinlin; Zhang, Lin; Zeng, Min; Tarbell, John M; Fu, Bingmei M

    2015-01-01

    Due to its unique location, the endothelial surface glycocalyx (ESG) at the luminal side of the microvessel wall may serve as a mechano-sensor and transducer of blood flow and thus regulate endothelial functions. To examine this role of the ESG, we used fluorescence microscopy to measure nitric oxide (NO) production in post-capillary venules and arterioles of rat mesentery under reduced (low) and normal (high) flow conditions, with and without enzyme pretreatment to remove heparan sulfate (HS) of the ESG and in the presence of an endothelial nitric oxide synthase (eNOS) inhibitor, NG-monomethyl-L-arginine (L-NMMA). Rats (SD, 250-300 g) were anesthetized. The mesentery was gently taken out from the abdominal cavity and arranged on the surface of a glass coverslip for the measurement. An individual post-capillary venule or arteriole was cannulated and loaded for 45 min with 5 μM 4, 5-Diaminofluorescein diacetate, a membrane permeable fluorescent indictor for NO, then the NO production was measured for ~10 min under a low flow (~300 μm/s) and for ~60 min under a high flow (~1000 μm/s). In the 15 min after switching to the high flow, DAF-2-NO fluorescence intensity increased to 1.27-fold of its baseline, DAF-2-NO continuously increased under the high flow, to 1.53-fold of its baseline in 60 min. Inhibition of eNOS by 1 mM L-NMMA attenuated the flow-induced NO production to 1.13-fold in 15 min and 1.30-fold of its baseline in 60 min, respectively. In contrast, no significant increase in NO production was observed after switching to the high flow for 60 min when 1 h pretreatment with 50 mU/mL heparanase III to degrade the ESG was applied. Similar NO production was observed in arterioles under low and high flows and under eNOS inhibition. Our results suggest that ESG participates in endothelial cell mechanosensing and transduction through its heparan sulfate to activate eNOS.

  16. A Cytokine Signalling Network for the Regulation of Inducible Nitric Oxide Synthase Expression in Rheumatoid Arthritis

    PubMed Central

    Dey, Poulami; Panga, Venugopal; Raghunathan, Srivatsan

    2016-01-01

    In rheumatoid arthritis (RA), nitric oxide (NO) is implicated in inflammation, angiogenesis and tissue destruction. The enzyme inducible nitric oxide synthase (iNOS) is responsible for the localised over-production of NO in the synovial joints affected by RA. The pro- and anti-inflammatory cytokines stimulate the synovial macrophages and the fibroblast-like synoviocytes to express iNOS. Therefore, the cytokine signalling network underlying the regulation of iNOS is essential to understand the pathophysiology of the disease. By using information from the literature, we have constructed, for the first time, the cytokine signalling network involved in the regulation of iNOS expression. Using the differential expression patterns obtained by re-analysing the microarray data on the RA synovium and the synovial macrophages available in the Gene Expression Omnibus (GEO) database, we aimed to establish the role played by the network genes towards iNOS regulation in the RA synovium. Our analysis reveals that the network genes belonging to interferon (IFN) and interleukin-10 (IL-10) pathways are always up-regulated in the RA synovium whereas the genes which are part of the anti-inflammatory transforming growth factor-beta (TGF-β) signalling pathway are mostly down-regulated. We observed a consistent up-regulation of the transcription factor signal transducers and activators of transcription 1 (STAT1) in the RA synovium and the macrophages. Interestingly, we found a consistent up-regulation of the iNOS interacting protein ras-related C3 botulinum toxin substrate 2 (RAC2) in the RA synovium as well as the macrophages. Importantly, we have constructed a model to explain the impact of IFN and IL-10 pathways on Rac2-iNOS interaction leading to over-production of NO and thereby causing chronic inflammation in the RA synovium. The interplay between STAT1 and RAC2 in the regulation of NO could have implications for the identification of therapeutic targets for RA. PMID:27626941

  17. Immunization with A91 peptide or copolymer-1 reduces the production of nitric oxide and inducible nitric oxide synthase gene expression after spinal cord injury.

    PubMed

    García, Elisa; Silva-García, Raúl; Mestre, Humberto; Flores, Nayeli; Martiñón, Susana; Calderón-Aranda, Emma S; Ibarra, Antonio

    2012-03-01

    Immunization with neurally derived peptides (INDP) boosts the action of an autoreactive immune response that has been shown to induce neuroprotection in several neurodegenerative diseases, especially after spinal cord (SC) injury. This strategy provides an environment that promotes neuronal survival and tissue preservation. The mechanisms by which this autoreactive response exerts its protective effects is not totally understood at the moment. A recent study showed that INDP reduces lipid peroxidation. Lipid peroxidation is a neurodegenerative phenomenon caused by the increased production of reactive nitrogen species such as nitric oxide (NO). It is possible that INDP could be interfering with NO production. To test this hypothesis, we examined the effect of INDP on the amount of NO produced by glial cells when cocultured with autoreactive T cells. We also evaluated the amount of NO and the expression of the inducible form of nitric oxide synthase (iNOS) at the injury site of SC-injured animals. The neural-derived peptides A91 and Cop-1 were used to immunize mice and rats with SC injury. In vitro studies showed that INDP significantly reduces the production of NO by glial cells. This observation was substantiated by in vivo experiments demonstrating that INDP decreases the amount of NO and iNOS gene expression at the site of injury. The present study provides substantial evidence on the inhibitory effect of INDP on NO production, helpingour understanding of the mechanisms through which protective autoimmunity promotes neuroprotection.

  18. Acute restraint stress induces specific changes in nitric oxide production and inflammatory markers in the rat hippocampus and striatum.

    PubMed

    Chen, Hsiao-Jou Cortina; Spiers, Jereme G; Sernia, Conrad; Lavidis, Nickolas A

    2016-01-01

    Chronic mild stress has been shown to cause hippocampal neuronal nitric oxide synthase (NOS) overexpression and the resultant nitric oxide (NO) production has been implicated in the etiology of depression. However, the extent of nitrosative changes including NOS enzymatic activity and the overall output of NO production in regions of the brain like the hippocampus and striatum following acute stress has not been characterized. In this study, outbred male Wistar rats aged 6-7 weeks were randomly allocated into 0 (control), 60, 120, or 240 min stress groups and neural regions were cryodissected for measurement of constitutive and inducible NOS enzymatic activity, nitrosative status, and relative gene expression of neuronal and inducible NOS. Hippocampal constitutive NOS activity increased initially but was superseded by the inducible isoform as stress duration was prolonged. Interestingly, hippocampal neuronal NOS and interleukin-1β mRNA expression was downregulated, while the inducible NOS isoform was upregulated in conjunction with other inflammatory markers. This pro-inflammatory phenotype within the hippocampus was further confirmed with an increase in the glucocorticoid-antagonizing macrophage migration inhibitory factor, Mif, and the glial surveillance marker, Ciita. This indicates that despite high levels of glucocorticoids, acute stress sensitizes a neuroinflammatory response within the hippocampus involving both pro-inflammatory cytokines and inducible NOS while concurrently modulating the immunophenotype of glia. Furthermore, there was a delayed increase in striatal inducible NOS expression while no change was found in other pro-inflammatory mediators. This suggests that short term stress induces a generalized increase in inducible NOS signaling that coincides with regionally specific increased markers of adaptive immunity and inflammation within the brain.

  19. A new and potent calmodulin antagonist, HF-2035, which inhibits vascular relaxation induced by nitric oxide synthase.

    PubMed

    Win, N H; Ishikawa, T; Saito, N; Kato, M; Yokokura, H; Watanabe, Y; Iida, Y; Hidaka, H

    1996-03-28

    HF-2035, 2-[N-(2-aminoethyl)-N-(2,4,5-trichlorobenzenesulfonyl)] amino-N-(4-chlorocinnamyl)-N-methylbenzylamine, was synthesized and its effects on calmodulin-dependent enzymes were investigated. HF-2035 inhibited calmodulin kinase I, calmodulin kinase II and myosin light-chain kinase with IC50 values of 1.3 microM, 1.6 microM and 68 microM, respectively. HF-2035 also inhibited the activity of recombinant rat neuronal nitric oxide synthase, one of the calmodulin-dependent enzymes, with a Ki of 0.78 microM. Partially purified nitric oxide synthase of rat brain was also inhibited by HF-2035 with an IC50 of 3.2 microM. Kinetic analysis indicated that this inhibitory effect of HF-2035 was competitive with respect to calmodulin. We examined the effects of HF-2035 on constitutive nitric oxide synthase in a bioassay using vascular strips of rabbit carotid artery with and without endothelium. HF-2035 inhibited acetylcholine- and calcium ionophore, A23187 (6S-[6 alpha (2S*,3S*),8 beta (R*),9 beta, 11 alpha]-5- (methylamino)-2-[[3,9,11-trimethyl-8-[1-methyl-2-oxo-2-(1H-pyrrol-2-yl)- ethyl]-1,7-dioxaspiro[5.5]undec-2-yl]methyl]-4-benzoxazol ecarboxylic acid)-induced relaxation of endothelium-intact strips with an ED50 of 1.5 +/- 0.5 microM and 2.8 +/- 1 microM, respectively. This compound, however, did not inhibit N-nitroso-N-morpholinoaminoacetonitrile (SIN-1A), an exogenous nitric oxide donor, -induced relaxation of endothelium-denuded strips. W-7 (N-(6-aminohexyl)-5-chloro-1- naphthalenesulfonamide) inhibited acetylcholine-induced relaxation with an ED50 of 46 +/- 7 microM, which was 30-fold less potent than HF-2035. HF-2035 was unable to inhibit the activity of the inducible form of nitric oxide synthase in isolated thoracic aorta of rat treated with Escherichia coli lipopolysaccharide. These findings suggest that HF-2035 is a new and potent calmodulin antagonist, and may be used as a mother compound to develop more selective inhibitors of constitutive nitric oxide

  20. Role of nitric oxide in the central interferon-alpha-induced inhibition of gastric acid secretion in rats.

    PubMed

    Czimmer, Jozsef; Király, Ágnes; Szabó, Imre Laszlo; Mózsik, Gyula; Sütő, Gabor

    2013-01-01

    Cytokines are known to play a key role in regulation of gastric functions. Interferon-alpha (IFN-α) has been published to impair gastric motility. Aims of this study were to clarify effect of IFN-α on gastric acid secretion (GAS) and determine role of nitric oxide (NO) in the process. Both subcutaneous (1000, 10000, 100 000 IU, s.c.) and intracisternal (10, 100, 1000 IU, i.c.) injections of IFN-α dose-dependently inhibited GAS induced by pylorus ligation in male SD rats in 2 hrs (370±40, 233±39, 208±50 micromol vs control 415±59 micromol and 481±50, 249±75, 141±25 micromol vs control 485±65 micromol, respectively). Central doses inducing same level inhibition were 100 times lower. NOS inhibitor L-NAME (3 mg/kg, i.v.) blocked the inhibitory effect of i.c. ED(50) dose 100 IU IFN-α (507±75 micromol/2 hrs), while L-arginine, the substrate of nitric oxide synthase (NOS) prevented L-NAME action (266±82 micromol/2 hrs). D-arginine failed to prevent L-NAME action on IFN-α-induced inhibition of GAS. Aminoguanidine, a selective inhibitor of inducible NOS (iNOS) failed to block IFN-α induced inhibition of GAS. Results suggest that IFN-α inhibits GAS centrally through nitric oxide pathways probably mediated by continuous isoform of NOS that can be important in regulation of GAS in healthy or pathological conditions.

  1. Protective Effect of Edaravone Against Cyclosporine-Induced Chronic Nephropathy Through Antioxidant and Nitric Oxide Modulating Pathways in Rats

    PubMed Central

    Sattarinezhad, Elahe; Panjehshahin, Mohammad Reza; Torabinezhad, Simin; Kamali-Sarvestani, Eskandar; Farjadian, Shirin; Pirsalami, Fatema; Moezi, Leila

    2017-01-01

    Background: Cyclosporine A (CsA) is an immunosuppressant with therapeutic indications in various immunological diseases; however, its use is associated with chronic nephropathy. Oxidative stress has a crucial role in CsA-induced nephrotoxicity. The present study evaluates the protective effect of edaravone on CsA-induced chronic nephropathy and investigates its antioxidant and nitric oxide modulating property. Methods: Male Sprague-Dawley rats (n=66) were distributed into nine groups, including a control (group 1) (n=7). Eight groups received CsA (15 mg/kg) for 28 days while being treated. The groups were categorized as: Group 2: Vehicle (n=10)Groups 3, 4, and 5: Edaravone (1, 5, and 10 mg/kg) (n=7 each)Group 6: Diphenyliodonium chloride, a specific endothelial nitric oxide synthase (eNOS) inhibitor (n=7)Group 7: Aminoguanidine, a specific inducible nitric oxide synthase (iNOS) inhibitor (n=7)Group 8: Edaravone (10 mg/kg) plus diphenyliodonium chloride (n=7)Group 9: Edaravone (10 mg/kg) plus aminoguanidine (n=7) Blood urea nitrogen and serum creatinine levels, malondialdehyde, superoxide dismutase, and glutathione reductase enzyme activities were measured using standard kits. Renal histopathological evaluations and measurements of eNOS and iNOS gene expressions by RT-PCR were also performed. Data were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s test (SPSS software version 18.0). Results: Edaravone (10 mg/kg) significantly attenuated CsA-induced oxidative stress, renal dysfunction, and kidney tissue injury. Aminoguanidine improved the renoprotective effect of edaravone. Edaravone reduced the elevated mRNA level of iNOS, but could not alter the level of eNOS mRNA significantly. Conclusion: Edaravone protects against CsA-induced chronic nephropathy using antioxidant property and probably through inhibiting iNOS gene expression. PMID:28360443

  2. Possible involvement of nitric oxide in morphine-induced miosis and reduction of intraocular pressure in rabbits.

    PubMed

    Bonfiglio, Vincenza; Bucolo, Claudio; Camillieri, Giovanni; Drago, Filippo

    2006-03-18

    The role of mu3 opioid receptors in morphine-induced intraocular pressure (IOP) lowering effect and miosis was evaluated in conscious, dark-adapted New Zealand white (NZW) rabbits using a masked-design study. IOP and pupil diameter (PD) measurements were taken at just before and 0.5, 1, 2, 4, 6 h after monolateral instillation of morphine (10, 50 and 100 microg/30 microl) as compared to vehicle administered in the contralateral eye. Morphine-induced ocular effects were challenged by a pre-treatment with the non-selective opioid receptor antagonist, naloxone (100 microg/30 microl), the nitric oxide synthase inhibitor, N(omega)-nitro-L-arginine methyl ester (L-NAME, 1%, 30 microl), or the non-selective mu3 opioid receptor inhibitor, reduced L-glutathione (GSH, 1%, 30 microl). Morphine induced a dose-dependent decrease in IOP and PD. Pre-treatment with naloxone totally prevented morphine-induced decrease in IOP and miosis. Ocular administration of L-NAME or GSH alone failed to affect IOP or PD of NZW rabbits. However, pre-treatment with either drugs significantly reduced, but not totally prevented ocular effects of morphine. These results suggest that biochemical mechanisms related to nitric oxide release are involved, at least in part, in morphine effects on the eye. Since the mu3 opioid receptor subtype is able to release nitric oxide and is sensitive to inactivation by GSH, it may be possible that mu3 opioid receptors are involved in morphine-induced miosis and reduction in IOP.

  3. Thymoquinone mitigate ischemia-reperfusion-induced liver injury in rats: a pivotal role of nitric oxide signaling pathway.

    PubMed

    Abd-Elbaset, Mohamed; Arafa, El-Shaimaa A; El Sherbiny, Gamal A; Abdel-Bakky, Mohamed S; Elgendy, Abdel Nasser A M

    2017-01-01

    Oxidative and nitrosative stress-induced endothelial cell damage play an essential role in the pathogenesis of hepatic ischemia-reperfusion (IR) injury. IR is associated with reduced eNOS expression and exacerbated by superimposed stress. NOSTRIN induces intracellular endothelial nitric oxide synthase (eNOS) translocation and inducible nitric oxide synthase (iNOS) increases nitric oxide (NO) production. Our aim was to assess hepatic expression of iNOS, eNOS, and NOSTRIN in IR with or without N-acetylcysteine (NAC) or thymoquinone (TQ) pretreatment and to compare their hepatoprotective effects. Surgical induction of IR was performed by occlusion of hepatic pedicle for 30 min with mini-clamp and reperfused for 30 min. The effects of TQ (20 mg/kg/day) or NAC (300 mg/kg/day) administered orally for 10 days were evaluated by serum ALT and AST, oxidative stress parameters, NO production, and histopathological analysis. Also, localization and expression of iNOS, eNOS, and NOSTRIN were assessed by immunofluorescence. TQ or NAC pretreatment significantly decreased elevated serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), and myeloperoxidase (MPO) activities, malondialdehyde (MDA) level, and NO production. In addition, they restored the depleted GSH content and alleviated histopathological changes. Furthermore, they up-regulated eNOS and down-regulated iNOS and NOSTRIN expressions. TQ exerts its hepatoprotective effect, at least in part, by nitric oxide signaling pathway through modulation of iNOS, eNOS, and NOSTRIN expressions as well as suppression of oxidative stress.

  4. IL-6 Improves the Nitric Oxide-Induced Cytotoxic CD8+ T Cell Dysfunction in Human Chagas Disease.

    PubMed

    Sanmarco, Liliana Maria; Visconti, Laura Marina; Eberhardt, Natalia; Ramello, Maria Cecilia; Ponce, Nicolás Eric; Spitale, Natalia Beatriz; Vozza, Maria Lola; Bernardi, Germán Andrés; Gea, Susana; Minguez, Angel Ramón; Aoki, Maria Pilar

    2016-01-01

    Reactive oxygen and nitrogen species are important microbicidal agents and are also involved in lymphocyte unresponsiveness during experimental infections. Many of the biological effects attributed to nitric oxide are mediated by peroxynitrites, which induce the nitration of immune cells, among others. Our group has demonstrated that nitric oxide is involved in the suppressive activity of myeloid-derived suppressor cells in Trypanosoma cruzi-infected mice, with a higher number of CD8+ T cells suffering surface-nitration compared to uninfected controls. Studying the functional and phenotypic features of peripheral CD8+ T cells from chagasic patients and human cells experimentally infected with T. cruzi, we found that different regulatory mechanisms impaired the effector functions of T cytotoxic population from seropositive patients. Peripheral leukocytes from chagasic patients showed increased nitric oxide production concomitant with increased tyrosine nitration of CD8+ T cells. Additionally, this cytotoxic population exhibited increased apoptotic rate, loss of the TCRζ-chain, and lower levels of CD107a, a marker of degranulation. Strikingly, IL-6 stimulation of in vitro-infected peripheral blood mononuclear cells obtained from healthy donors, blunted T. cruzi-induced nitration of CD3+CD8+ cells, and increased their survival. Furthermore, the treatment of these cultures with an IL-6 neutralizing antibody increased the percentage of T. cruzi-induced CD8+ T cell nitration and raised the release of nitric oxide. The results suggest that the under-responsiveness of cytotoxic T cell population observed in the setting of long-term constant activation of the immune system could be reverted by the pleiotropic actions of IL-6, since this cytokine improves its survival and effector functions.

  5. IL-6 Improves the Nitric Oxide-Induced Cytotoxic CD8+ T Cell Dysfunction in Human Chagas Disease

    PubMed Central

    Sanmarco, Liliana Maria; Visconti, Laura Marina; Eberhardt, Natalia; Ramello, Maria Cecilia; Ponce, Nicolás Eric; Spitale, Natalia Beatriz; Vozza, Maria Lola; Bernardi, Germán Andrés; Gea, Susana; Minguez, Angel Ramón; Aoki, Maria Pilar

    2016-01-01

    Reactive oxygen and nitrogen species are important microbicidal agents and are also involved in lymphocyte unresponsiveness during experimental infections. Many of the biological effects attributed to nitric oxide are mediated by peroxynitrites, which induce the nitration of immune cells, among others. Our group has demonstrated that nitric oxide is involved in the suppressive activity of myeloid-derived suppressor cells in Trypanosoma cruzi-infected mice, with a higher number of CD8+ T cells suffering surface-nitration compared to uninfected controls. Studying the functional and phenotypic features of peripheral CD8+ T cells from chagasic patients and human cells experimentally infected with T. cruzi, we found that different regulatory mechanisms impaired the effector functions of T cytotoxic population from seropositive patients. Peripheral leukocytes from chagasic patients showed increased nitric oxide production concomitant with increased tyrosine nitration of CD8+ T cells. Additionally, this cytotoxic population exhibited increased apoptotic rate, loss of the TCRζ-chain, and lower levels of CD107a, a marker of degranulation. Strikingly, IL-6 stimulation of in vitro-infected peripheral blood mononuclear cells obtained from healthy donors, blunted T. cruzi-induced nitration of CD3+CD8+ cells, and increased their survival. Furthermore, the treatment of these cultures with an IL-6 neutralizing antibody increased the percentage of T. cruzi-induced CD8+ T cell nitration and raised the release of nitric oxide. The results suggest that the under-responsiveness of cytotoxic T cell population observed in the setting of long-term constant activation of the immune system could be reverted by the pleiotropic actions of IL-6, since this cytokine improves its survival and effector functions. PMID:28066435

  6. Nitric oxide and DOPAC-induced cell death: from GSH depletion to mitochondrial energy crisis.

    PubMed

    Nunes, Carla; Barbosa, Rui M; Almeida, Leonor; Laranjinha, João

    2011-09-01

    The molecular mechanisms inherent to cell death associated with Parkinson's disease are not clearly understood. Diverse pathways, sequence of events and models have been explored in several studies. Recently, we have proposed an integrative mechanism, encompassing the interaction of nitric oxide (•NO) and a major dopamine metabolite, dihydroxyphenylacetic (DOPAC), leading to a synergistic mitochondrial dysfunction and cell death that may be operative in PD. In this study, we have studied the sequence of events underlying the mechanisms of cell death in PC12 cells exposed to •NO and DOPAC in terms of: a) free radical production; b) modulation by glutathione (GSH); c) energetic status and d) outer membrane mitochondria permeability. Using Electron Paramagnetic Resonance (EPR) it is shown the early production of oxygen free radicals followed by a depletion of GSH reflected by an increase of GSSG/GSH ratio in the cells treated with the mixture of •NO/DOPAC, as compared with the cells individually exposed to each of the stimulus. Glutathione ethyl ester (GSH-EE) and N-acetylcysteine (NAC) may rescue cells from death, increasing GSH content and preventing ATP loss in cells treated with the mixture DOPAC/•NO but failed to exert similar effects in the cells challenged only with •NO. The depletion of GSH is accompanied by a decreased activity of mitochondrial complex I. At a later stage, the concerted action of DOPAC and •NO include a rise in the ratio Bax/Bcl-2, an observation not evident when cells were exposed only to •NO. The results support a free radical-induced pathway leading to cell death involving the concerted action of DOPAC and •NO and the critical role of GSH in maintaining a functional mitochondria.

  7. Theoretical studies on the binding of rhenium(I) complexes to inducible nitric oxide synthase.

    PubMed

    Oliveira, Bruno L; Moreira, Irina S; Fernandes, Pedro A; Ramos, Maria J; Santos, Isabel; Correia, João D G

    2013-09-01

    Considering our interest in the design of innovative radiometal-based complexes for in vivo imaging of nitric oxide synthase (NOS), we have recently introduced a set of M(CO)3-complexes (M=(99m)Tc, Re) containing a pendant N(ω)-NO2-L-arginine moiety, a known inhibitor of the enzyme. Enzymatic assays with purified inducible NOS have shown that the non-radioactive surrogates with 3-(Re1; Ki=84 μM) or 6-carbon linkers (Re2; Ki=6 μM) are stronger inhibitors than the respective metal-free conjugates L1 (Ki=178 μM) and L2 (Ki=36 μM), with Re2 displaying the highest inhibitory potency. Aiming to rationalize the experimental results we have performed a molecular docking study combined with molecular dynamics (MD) simulations and free energy perturbation (FEP) calculations. The higher inhibitory potency of Re2 arises from the stronger electrostatic interactions observed between the "Re(CO)3" core and the residues Arg260 and Arg382. This interaction is only possible due to the higher flexibility of its C6-carbon spacer, which links the N(ω)-NO2-L-arginine moiety and the "Re(CO)3" organometallic core. Furthermore, FEP calculations were carried out and the resultant relative binding energies (ΔΔGbind(calc)=0.690±0.028 kcal/mol,Re1/L1 and 1.825±0.318 kcal/mol, Re2/L2) are in accordance with the experimental results (ΔΔGbind(exp)=0.461±0.009 kcal/mol,Re1/L1 and 1.129±0.210 kcal/mol, Re2/L2); there is an energetic penalty for the transformation of the Re complexes into the ligands and this penalization is higher for the pair Re2/L2.

  8. Exenatide induces aortic vasodilation increasing hydrogen sulphide, carbon monoxide and nitric oxide production

    PubMed Central

    2014-01-01

    Background It has been reported that GLP-1 agonist exenatide (exendin-4) decreases blood pressure. The dose-dependent vasodilator effect of exendin-4 has previously been demonstrated, although the precise mechanism is not thoroughly described. Here we have aimed to provide in vitro evidence for the hypothesis that exenatide may decrease central (aortic) blood pressure involving three gasotransmitters, namely nitric oxide (NO) carbon monoxide (CO), and hydrogen sulphide (H2S). Methods We determined the vasoactive effect of exenatide on isolated thoracic aortic rings of adult rats. Two millimetre-long vessel segments were placed in a wire myograph and preincubated with inhibitors of the enzymes producing the three gasotransmitters, with inhibitors of reactive oxygen species formation, prostaglandin synthesis, inhibitors of protein kinases, potassium channels or with an inhibitor of the Na+/Ca2+-exchanger. Results Exenatide caused dose-dependent relaxation of rat thoracic aorta, which was evoked via the GLP-1 receptor and was mediated mainly by H2S but also by NO and CO. Prostaglandins and superoxide free radical also play a part in the relaxation. Inhibition of soluble guanylyl cyclase significantly diminished vasorelaxation. We found that ATP-sensitive-, voltage-gated- and calcium-activated large-conductance potassium channels are also involved in the vasodilation, but that seemingly the inhibition of the KCNQ-type voltage-gated potassium channels resulted in the most remarkable decrease in the rate of vasorelaxation. Inhibition of the Na+/Ca2+-exchanger abolished most of the vasodilation. Conclusions Exenatide induces vasodilation in rat thoracic aorta with the contribution of all three gasotransmitters. We provide in vitro evidence for the potential ability of exenatide to lower central (aortic) blood pressure, which could have relevant clinical importance. PMID:24693878

  9. Nitric Oxide Induction of Parkin Translocation in PTEN-induced Putative Kinase 1 (PINK1) Deficiency

    PubMed Central

    Han, Ji-Young; Kang, Min-Ji; Kim, Kyung-Hee; Han, Pyung-Lim; Kim, Hyun-Seok; Ha, Ji-Young; Son, Jin H.

    2015-01-01

    The failure to trigger mitophagy is implicated in the pathogenesis of familial Parkinson disease that is caused by PINK1 or Parkin mutations. According to the prevailing PINK1-Parkin signaling model, mitophagy is promoted by the mitochondrial translocation of Parkin, an essential PINK1-dependent step that occurs via a previously unknown mechanism. Here we determined that critical concentrations of NO was sufficient to induce the mitochondrial translocation of Parkin even in PINK1 deficiency, with apparent increased interaction of full-length PINK1 accumulated during mitophagy, with neuronal nitric oxide synthase (nNOS). Specifically, optimum levels of NO enabled PINK1-null dopaminergic neuronal cells to regain the mitochondrial translocation of Parkin, which appeared to be significantly suppressed by nNOS-null mutation. Moreover, nNOS-null mutation resulted in the same mitochondrial electron transport chain (ETC) enzyme deficits as PINK1-null mutation. The involvement of mitochondrial nNOS activation in mitophagy was further confirmed by the greatly increased interactions of full-length PINK1 with nNOS, accompanied by mitochondrial accumulation of phospho-nNOS (Ser1412) during mitophagy. Of great interest is that the L347P PINK1 mutant failed to bind to nNOS. The loss of nNOS phosphorylation and Parkin accumulation on PINK1-deficient mitochondria could be reversed in a PINK1-dependent manner. Finally, non-toxic levels of NO treatment aided in the recovery of PINK1-null dopaminergic neuronal cells from mitochondrial ETC enzyme deficits. In summary, we demonstrated the full-length PINK1-dependent recruitment of nNOS, its activation in the induction of Parkin translocation, and the feasibility of NO-based pharmacotherapy for defective mitophagy and ETC enzyme deficits in Parkinson disease. PMID:25716315

  10. Prolactin induces regional vasoconstriction through the beta2-adrenergic and nitric oxide mechanisms.

    PubMed

    Molinari, Claudio; Grossini, Elena; Mary, David A S G; Uberti, Francesca; Ghigo, Ezio; Ribichini, Flavio; Surico, Nicola; Vacca, Giovanni

    2007-08-01

    Prolactin has been associated with many effects and has been implicated in the pathogenesis of pregnancy-related hypertensive disorders, although little is known about its vascular effects. The present study was designed to determine the primary effect of prolactin on regional vascular beds and the mechanisms involved. In 37 anesthetized pigs, the infusion of 0.17 mug/kg min of prolactin at constant heart rate and arterial pressure decreased coronary, mesenteric, renal, and iliac blood flow. This response was graded in further five pigs by increasing the infused dose of the hormone between 0.017 and 1 mug/kg min. In 22 of the 37 pigs, blockade of cholinergic receptors (five pigs) and of alpha-adrenoceptors (five pigs) did not affect the prolactin-induced vascular response, which was abolished by blockade of beta(2)-adrenoceptors (five pigs) and by blockade of vascular nitric oxide (NO) synthase (seven pigs). In 15 of the 37 pigs the increases in measured blood flows caused by iv infusion of isoproterenol (five pigs) and by intraarterial administration of acetylcholine (five pigs) and of sodium nitroprusside (five pigs) were significantly reduced by infusion of prolactin. Moreover, the treatment of porcine aortic endothelial cells by prolactin caused a reduction of NO production and of the phosphorylation of ERK, Akt, and p38, which was prevented by the concomitant treatment by the beta(2)-adrenergic agonist albuterol. The present study showed that iv infusion of prolactin primarily caused coronary, mesenteric, renal, and iliac vasoconstriction. These effects were brought about by the inhibition of a vasodilatory beta(2)-adrenergic receptor-mediated effect related to the NO intracellular pathway.

  11. Participation of neuronal nitric oxide synthase in experimental neuropathic pain induced by sciatic nerve transection.

    PubMed

    Chacur, M; Matos, R J B; Alves, A S; Rodrigues, A C; Gutierrez, V; Cury, Y; Britto, L R G

    2010-04-01

    Nerve injury leads to a neuropathic pain state that results from central sensitization. This phenomenom is mediated by NMDA receptors and may involve the production of nitric oxide (NO). In this study, we investigated the expression of the neuronal isoform of NO synthase (nNOS) in the spinal cord of 3-month-old male, Wistar rats after sciatic nerve transection (SNT). Our attention was focused on the dorsal part of L3-L5 segments receiving sensory inputs from the sciatic nerve. SNT resulted in the development of neuropathic pain symptoms confirmed by evaluating mechanical hyperalgesia (Randall and Selitto test) and allodynia (von Frey hair test). Control animals did not present any alteration (sham-animals). The selective inhibitor of nNOS, 7-nitroindazole (0.2 and 2 microg in 50 microL), blocked hyperalgesia and allodynia induced by SNT. Immunohistochemical analysis showed that nNOS was increased (48% by day 30) in the lumbar spinal cord after SNT. This increase was observed near the central canal (Rexed's lamina X) and also in lamina I-IV of the dorsal horn. Real-time PCR results indicated an increase of nNOS mRNA detected from 1 to 30 days after SNT, with the highest increase observed 1 day after injury (1469%). Immunoblotting confirmed the increase of nNOS in the spinal cord between 1 and 15 days post-lesion (20%), reaching the greatest increase (60%) 30 days after surgery. The present findings demonstrate an increase of nNOS after peripheral nerve injury that may contribute to the increase of NO production observed after peripheral neuropathy.

  12. The Role of Nitric Oxide in Doxorubicin-Induced Cardiotoxicity: Experimental Study

    PubMed Central

    Bahadır, Ayşenur; Kurucu, Nilgün; Kadıoğlu, Mine; Yenilme, Engin

    2014-01-01

    Objective: We evaluated the myocardial damage in rats treated with doxorubicin (DOX) alone and in combination with nitric oxide synthase (NOS) inhibitors. Materials and Methods: Twenty-four male Sprague Dawley rats (12 weeks old, weighing 262±18 g) were randomly assigned into 4 groups (n=6). Group I was the control group. In Group II, rats were treated with intraperitoneal (ip) injections of 3 mg/kg DOX once a week for 5 weeks. In Group III, rats received weekly ip injections of 30 mg/kg L-NAME (nonspecific NOS inhibitor) 30 min before DOX injections for 5 weeks. In Group IV, rats received weekly ip injections of 3 mg/kg L-NIL (inducible NOS inhibitor) 30 min before DOX injections for 5 weeks. Rats were weighed 2 times a week. At the end of 6 weeks, hearts were excised and then fixed for light and electron microscopy evaluation and tissue lipid peroxidation (malondialdehyde). Blood samples were also obtained for measuring plasma lipid peroxidation. Results: Weight loss was observed in Group II, Group III, and Group IV. Weight loss was statistically significant in the DOX group. Findings of myocardial damage were significantly higher in animals treated with DOX only than in the control group. Histopathological findings of cardiotoxicity in rats treated with DOX in combination with L-NAME and L-NIL were not significantly different compared with the control group. The level of plasma malondialdehyde in the DOX group (9.3±3.4 µmol/L) was higher than those of all other groups. Conclusion: Our results showed that DOX cardiotoxicity was significantly decreased when DOX was given with NO synthase inhibitors. PMID:24764732

  13. Ultraviolet-B-induced flavonoid accumulation in Betula pendula leaves is dependent upon nitrate reductase-mediated nitric oxide signaling.

    PubMed

    Zhang, Ming; Dong, Ju-Fang; Jin, Hai-Hong; Sun, Li-Na; Xu, Mao-Jun

    2011-08-01

    Nitric oxide (NO) is an important signaling molecule involved in many physiological processes in plants. Nitric oxide generation and flavonoid accumulation are two early reactions of plants to ultraviolet-B (UV-B) irradiation. However, the source of UV-B-triggered NO generation and the role of NO in UV-B-induced flavonoid accumulation are not fully understood. In order to evaluate the origin of UV-B-triggered NO generation, we examined the responses of nitrate reductase (NR) activity and the expression levels of NIA1 and NIA2 genes in leaves of Betula pendula Roth (silver birch) seedlings to UV-B irradiation. The data show that UV-B irradiation stimulates NR activity and induces up-regulation of NIA1 but does not affect NIA2 expression during UV-B-triggered NO generation. Pretreatment of the leaves with NR inhibitors tungstate (TUN) and glutamine (Gln) abolishes not only UV-B-triggered NR activities but also UV-B-induced NO generation. Furthermore, application of TUN and Gln suppresses UV-B-induced flavonoid production in the leaves and the suppression of NR inhibitors on UV-B-induced flavonoid production can be reversed by NO via its donor sodium nitroprusside. Together, the data indicate that NIA1 in the leaves of silver birch seedlings is sensitive to UV-B and the UV-B-induced up-regulation of NIA1 may lead to enhancement of NR activity. Furthermore, our results demonstrate that NR is involved in UV-B-triggered NO generation and NR-mediated NO generation is essential for UV-B-induced flavonoid accumulation in silver birch leaves.

  14. An octamer motif is required for activation of the inducible nitric oxide synthase promoter in pancreatic beta-cells.

    PubMed

    Darville, Martine I; Terryn, Sara; Eizirik, Décio L

    2004-03-01

    Nitric oxide, generated by the inducible form of nitric oxide synthase (iNOS), is a potential mediator of cytokine-induced beta-cell dysfunction in type 1 diabetes mellitus. We have previously shown that cytokine-induced iNOS expression is cycloheximide (CHX) sensitive and requires nuclear factor-kappa B (NF-kappa B) activation. In the present study, we show that an octamer motif located 20 bp downstream of the proximal NF-kappa B binding site in the rat iNOS promoter is critical for IL-1 beta and interferon-gamma induction of promoter activity in rat primary beta-cells and insulin-producing RINm5F cells. In gel shift assays, the octamer motif bound constitutively the transcription factor Oct1. Neither Oct1 nor NF-kappa B binding activities were blocked by CHX, suggesting that other factor(s) synthesized in response to IL-1 beta contribute to iNOS promoter induction. The high mobility group (HMG)-I(Y) protein also bound the proximal iNOS promoter region. HMG-I(Y) binding was decreased in cells treated with CHX and HMG-I(Y) silencing by RNA interference reduced IL-1 beta-induced iNOS promoter activity. These results suggest that Oct1, NF-kappa B, and HMG-I(Y) cooperate for transactivation of the iNOS promoter in pancreatic beta-cells.

  15. Danggui Buxue Tang, Chinese Herbal Decoction Containing Astragali Radix and Angelicae Sinensis Radix, Induces Production of Nitric Oxide in Endothelial Cells: Signaling Mediated by Phosphorylation of Endothelial Nitric Oxide Synthase.

    PubMed

    Gong, Amy G W; Lau, K M; Zhang, Laura M L; Lin, H Q; Dong, Tina T X; Tsim, Karl W K

    2016-03-01

    Danggui Buxue Tang, an ancient Chinese herbal decoction containing Astragali Radix and Angelicae Sinensis Radix at the weight ratio of 5:1, is used to mitigate menopausal syndromes in women. The pharmacological properties of Danggui Buxue Tang have been illustrated in bone development, blood enhancement, and immune stimulation. Here, we extended the possible pharmacological role of Danggui Buxue Tang in cardiovascular function. In cultured human umbilical vein endothelial cells, the application of Danggui Buxue Tang induced the release of nitric oxide and the phosphorylation of endothelial nitric oxide synthase and Akt kinase in time- and dose-dependent manners. The robust activation of nitric oxide signaling, however, required the boiling of Astragali Radix and Angelicae Sinensis Radix together, i.e., as Danggui Buxue Tang instead of other herbal extracts. The Danggui Buxue Tang-induced phosphorylation of endothelial nitric oxide synthase and Akt kinase in human umbilical vein endothelial cells were fully blocked by treatment with an endothelial nitric oxide synthase inhibitor (L-NAME), a PI3K/Akt inhibitor (LY294002), and a Ca(2+) chelator (BAPTA-AM). In parallel, the blockage of endothelial nitric oxide synthase and Akt activation subsequently fully abolished the Danggui Buxue Tang-induced nitric oxide production.

  16. Nitric oxide mediates angiogenesis induced in vivo by platelet-activating factor and tumor necrosis factor-alpha.

    PubMed Central

    Montrucchio, G.; Lupia, E.; de Martino, A.; Battaglia, E.; Arese, M.; Tizzani, A.; Bussolino, F.; Camussi, G.

    1997-01-01

    We evaluated the role of an endogenous production of nitric oxide (NO) in the in vitro migration of endothelial cells and in the in vivo angiogenic response elicited by platelet-activating factor (PAF), tumor necrosis factor-alpha (TNF), and basic fibroblast growth factor (bFGF). The NO synthase inhibitor, N omega-nitro-L-arginine-methyl ester (L-NAME), but not its enantiomer D-NAME, prevented chemotaxis of endothelial cells induced in vitro by PAF and by TNF. The motogenic activity of TNF was also inhibited by WEB 2170, a specific PAF-receptor antagonist. In contrast, chemotaxis induced by bFGF was not prevented by L-NAME or by WEB 2170. Angiogenesis was studied in vivo in a murine model in which Matrigel was used as a vehicle for the delivery of mediators. In this model, the angiogenesis induced by PAF and TNF was inhibited by WEB 2170 and L-NAME but not by D-NAME. In contrast, angiogenesis induced by bFGF was not affected by L-NAME or by WEB 2170. TNF, but not bFGF, induced PAF synthesis within Matrigel. These results suggest that NO mediates the angiogenesis induced by PAF as well as that induced by TNF, which is dependent on the production of PAF. In contrast, the angiogenic effect of bFGF appears to be both PAF and NO independent. Images Figure 3 Figure 4 PMID:9250168

  17. Elevation of Inducible Nitric Oxide Synthase and Cyclooxygenase-2 Expression in the Mouse Brain after Chronic Nonylphenol Exposure

    PubMed Central

    Zhang, Yan-Qiu; Mao, Zhen; Zheng, Yuan-Lin; Han, Bao-Ping; Chen, Ling-Tong; Li, Jing; Li, Fei

    2008-01-01

    The present study was performed to investigate the effects of chronic administration of nonylphenol (NP) on the expression of inflammation-related genes in the brains of mice. NP was given orally by gavages at 0, 50, 100, and 200 mg/kg/d. The expression of inflammatory enzymes, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), was evaluated by immunohistochemistry and immunoblotting assays. The nitric oxide (NO) level and nitric oxide synthase (NOS) activity were also measured by biochemical analyses. The results showed that NP at a high dose (200 mg/kg/d) significantly increased the expression of iNOS and COX-2 in both the hippocampus and cortex. In parallel with the increase in iNOS expression, the NO level was significantly greater at the dose of 200 mg/kg/d, compared to the control. The activity of NOS was also increased in the brain of mice at the dose of 100 and 200 mg/kg/d. These findings demonstrate that NP may have the potential to induce the chronic inflammation or cause neurotoxicity in the mouse brain. PMID:19325730

  18. Nitric oxide induces cotyledon senescence involving co-operation of the NES1/MAD1 and EIN2-associated ORE1 signalling pathways in Arabidopsis

    PubMed Central

    Du, Jing; Li, Manli; Kong, Dongdong; Wang, Lei; Lv, Qiang; Wang, Jinzheng; Bao, Fang; Gong, Qingqiu; Xia, Jinchan; He, Yikun

    2014-01-01

    After germination, cotyledons undertake the major role in supplying nutrients to the pre-photoautorophy angiosperm seedlings until they senesce. Like other senescence processes, cotyledon senescence is a programmed degenerative process. Nitric oxide can induce premature cotyledon senescence in Arabidopsis thaliana, yet the underlying mechanism remains elusive. A screen for genetic mutants identified the nes1 mutant, in which cotyledon senescence was accelerated by nitric oxide. Map-based cloning revealed that NES1 is allelic to a previously reported mitotic checkpoint family gene, MAD1. The nes1/mad1 mutants were restored to the wild type, in response to nitric oxide, by transforming them with pNES1::NES1. Ectopic expression of NES1 in the wild type delayed nitric oxide-mediated cotyledon senescence, confirming the repressive role of NES1. Moreover, two positive regulators of leaf senescence, the ethylene signalling component EIN2 and the transcription factor ORE1/AtNAC2/ANAC092, were found to function during nitric oxide-induced senescence in cotyledons. The block of ORE1 function delayed senescence and ectopic expression induced the process, revealing the positive role of ORE1. EIN2 was required to induce ORE1. Furthermore, the genetic interaction analysis between NES1 and ORE1 showed that the ore1 loss-of-function mutants were epistatic to nes1, suggesting the dominant role of ORE1 and the antagonistic role of NES1 during nitric oxide-induced cotyledon senescence in Arabidopsis. PMID:24336389

  19. Attenuation of acute nitrogen mustard-induced lung injury, inflammation and fibrogenesis by a nitric oxide synthase inhibitor

    SciTech Connect

    Malaviya, Rama; Venosa, Alessandro; Hall, LeRoy; Gow, Andrew J.; Sinko, Patrick J.; Laskin, Jeffrey D.; Laskin, Debra L.

    2012-12-15

    Nitrogen mustard (NM) is a toxic vesicant known to cause damage to the respiratory tract. Injury is associated with increased expression of inducible nitric oxide synthase (iNOS). In these studies we analyzed the effects of transient inhibition of iNOS using aminoguanidine (AG) on NM-induced pulmonary toxicity. Rats were treated intratracheally with 0.125 mg/kg NM or control. Bronchoalveolar lavage fluid (BAL) and lung tissue were collected 1 d–28 d later and lung injury, oxidative stress and fibrosis assessed. NM exposure resulted in progressive histopathological changes in the lung including multifocal lesions, perivascular and peribronchial edema, inflammatory cell accumulation, alveolar fibrin deposition, bronchiolization of alveolar septal walls, and fibrosis. This was correlated with trichrome staining and expression of proliferating cell nuclear antigen (PCNA). Expression of heme oxygenase (HO)-1 and manganese superoxide dismutase (Mn-SOD) was also increased in the lung following NM exposure, along with levels of protein and inflammatory cells in BAL, consistent with oxidative stress and alveolar-epithelial injury. Both classically activated proinflammatory (iNOS{sup +} and cyclooxygenase-2{sup +}) and alternatively activated profibrotic (YM-1{sup +} and galectin-3{sup +}) macrophages appeared in the lung following NM administration; this was evident within 1 d, and persisted for 28 d. AG administration (50 mg/kg, 2 ×/day, 1 d–3 d) abrogated NM-induced injury, oxidative stress and inflammation at 1 d and 3 d post exposure, with no effects at 7 d or 28 d. These findings indicate that nitric oxide generated via iNOS contributes to acute NM-induced lung toxicity, however, transient inhibition of iNOS is not sufficient to protect against pulmonary fibrosis. -- Highlights: ► Nitrogen mustard (NM) induces acute lung injury and fibrosis. ► Pulmonary toxicity is associated with increased expression of iNOS. ► Transient inhibition of iNOS attenuates acute

  20. Contribution of polyamines metabolism and GABA shunt to chilling tolerance induced by nitric oxide in cold-stored banana fruit.

    PubMed

    Wang, Yansheng; Luo, Zisheng; Mao, Linchun; Ying, Tiejin

    2016-04-15

    Effect of exogenous nitric oxide (NO) on polyamines (PAs) catabolism, γ-aminobutyric acid (GABA) shunt, proline accumulation and chilling injury of banana fruit under cold storage was investigated. Banana fruit treated with NO sustained lower chilling injury index than the control. Notably elevated nitric oxide synthetase activity and endogenous NO level were observed in NO-treated banana fruit. PAs contents in treated fruit were significantly higher than control fruit, due to the elevated activities of arginine decarboxylase and ornithine decarboxylase. NO treatment increased the activities of diamine oxidase, polyamine oxidase and glutamate decarboxylase, while reduced GABA transaminase activity to lower levels compared with control fruit, which resulted the accumulation of GABA. Besides, NO treatment upregulated proline content and significantly enhanced the ornithine aminotransferase activity. These results indicated that the chilling tolerance induced by NO treatment might be ascribed to the enhanced catabolism of PAs, GABA and proline.

  1. Dopamine inhibits lipopolysaccharide-induced nitric oxide production through the formation of dopamine quinone in murine microglia BV-2 cells.

    PubMed

    Yoshioka, Yasuhiro; Sugino, Yuta; Tozawa, Azusa; Yamamuro, Akiko; Kasai, Atsushi; Ishimaru, Yuki; Maeda, Sadaaki

    2016-02-01

    Dopamine (DA) has been suggested to modulate functions of glial cells including microglial cells. To reveal the regulatory role of DA in microglial function, in the present study, we investigated the effect of DA on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in murine microglial cell line BV-2. Pretreatment with DA for 24 h concentration-dependently attenuated LPS-induced NO production in BV-2 cells. The inhibitory effect of DA on LPS-induced NO production was not inhibited by SCH-23390 and sulpiride, D1-like and D2-like DA receptor antagonists, respectively. In addition, pretreatment with (-)-(6aR,12bR)-4,6,6a,7,8,12b-Hexahydro-7-methylindolo[4,3-a]phenanthridin (CY 208-243) and bromocriptine, D1-like and D2-like DA receptor agonists, respectively, did not affect the LPS-induced NO production. N-Acetylcysteine, which inhibits DA oxidation, completely inhibited the effect of DA. Tyrosinase, which catalyzes the oxidation of DA to DA quionone (DAQ), accelerated the inhibitory effect of DA on LPS-induced NO production. These results suggest that DA attenuates LPS-induced NO production through the formation of DAQ in BV-2 cells.

  2. Strigolactones are required for nitric oxide to induce root elongation in response to nitrogen and phosphate deficiencies in rice.

    PubMed

    Sun, Huwei; Bi, Yang; Tao, Jinyuan; Huang, Shuangjie; Hou, Mengmeng; Xue, Ren; Liang, Zhihao; Gu, Pengyuan; Yoneyama, Koichi; Xie, Xiaonan; Shen, Qirong; Xu, Guohua; Zhang, Yali

    2016-07-01

    The response of the root system architecture to nutrient deficiencies is critical for sustainable agriculture. Nitric oxide (NO) is considered a key regulator of root growth, although the mechanisms remain unknown. Phenotypic, cellular and genetic analyses were undertaken in rice to explore the role of NO in regulating root growth and strigolactone (SL) signalling under nitrogen-deficient and phosphate-deficient conditions (LN and LP). LN-induced and LP-induced seminal root elongation paralleled NO production in root tips. NO played an important role in a shared pathway of LN-induced and LP-induced root elongation via increased meristem activity. Interestingly, no responses of root elongation were observed in SL d mutants compared with wild-type plants, although similar NO accumulation was induced by sodium nitroprusside (SNP) application. Application of abamine (the SL inhibitor) reduced seminal root length and pCYCB1;1::GUS expression induced by SNP application in wild type; furthermore, comparison with wild type showed lower SL-signalling genes in nia2 mutants under control and LN treatments and similar under SNP application. Western blot analysis revealed that NO, similar to SL, triggered proteasome-mediated degradation of D53 protein levels. Therefore, we presented a novel signalling pathway in which NO-activated seminal root elongation under LN and LP conditions, with the involvement of SLs.

  3. NG-methyl-L-arginine inhibits tumor necrosis factor-induced hypotension: implications for the involvement of nitric oxide.

    PubMed Central

    Kilbourn, R G; Gross, S S; Jubran, A; Adams, J; Griffith, O W; Levi, R; Lodato, R F

    1990-01-01

    Clinical assessment of the activity of tumor necrosis factor (TNF) against human cancer has been limited by a dose-dependent cardiovascular toxicity, most frequently hypotension. TNF is also thought to mediate the vascular collapse resulting from bacterial endotoxin. The present studies address the mechanism by which TNF causes hypotension and provide evidence for elevated production of nitric oxide, a potent vasodilator initially characterized as endothelium-derived relaxing factor. Nitric oxide is synthesized by several cell types, including endothelial cells and macrophages, from the guanidino nitrogen of L-arginine; the enzymatic pathway is competitively inhibited by NG-methyl-L-arginine. We found that hypotension induced in pentobarbital-anesthetized dogs by TNF (10 micrograms/kg, i.v., resulting in a fall in mean systemic arterial pressure from 124.7 +/- 7 to 62.0 +/- 22.9 mmHg; 1 mmHg = 133 Pa) was completely reversed within 2 min following administration of NG-methyl-L-arginine (4.4 mg/kg, i.v.). In contrast, NG-methyl-L-arginine failed to reverse the hypotensive response to an equivalent depressor dose of nitroglycerin, a compound that acts by forming nitric oxide by a nonenzymatic, arginine-independent mechanism. The effect of NG-methyl-L-arginine on TNF-induced hypotension was antagonized, and the hypotension restored, by administration of excess L-arginine (100 mg/kg, i.v.). Our findings suggest that excessive nitric oxide production mediates the hypotensive effect of TNF. PMID:2333306

  4. Signaling Mechanisms in the Nitric Oxide Donor- and Amphetamine-Induced Dopamine Release in Mesencephalic Primary Cultured Neurons.

    PubMed

    Salum, Cristiane; Schmidt, Fanny; Michel, Patrick P; Del-Bel, Elaine; Raisman-Vozari, Rita

    2016-01-01

    Previous research has shown that nitric oxide (NO) synthase inhibitors prevent rodents' sensorimotor gating impairments induced by dopamine releasing drugs, such as amphetamine (Amph) and methylphenidate. The mechanisms of this effect have not been entirely understood. In the present work, we investigated some possible mechanisms by which the NO donor, NOC-12 (3-ethyl-3-(ethylaminoethyl)-1-hydroxy-2-oxo-1-triazene), influence spontaneous and Amph-induced dopamine release, using rat mesencephalic primary cultured neurons preparations. Our results showed that NOC-12 increased dopamine release in a concentration-dependent manner and potentiated the Amph-induced one. Dopamine release induced by NOC-12 was disrupted by N-acetyl-L-cystein (NAC-a free radical scavenger) and MK-801, a NMDA (N-methyl-D-aspartate) non-competitive antagonist, and was concentration dependently affected by oxadiazolo[4,3]quinoxalin-1-one, an inhibitor of the soluble guanylate cyclase (sGC). In contrast, dopamine released by Amph was facilitated by NAC and by MK-801 and not affected by nifedipine (a L-type-Ca(+2) channel blocker), which enhanced NOC-12-induced dopamine release. The present work demonstrates that DA release induced by NOC-12 is partially dependent on sGC and on NMDA activation, and is modulated by L-type Ca(+2) channel and the antioxidant NAC. This mechanism differs from the Amph-induced one, which appears not to depend on L-type Ca(+2) channel and seems to be facilitated by NMDA channel blocking and by NAC. These results suggest that Amph and NOC-12 induce dopamine release through complementary pathways, which may explain the potentiation of Amph-induced dopamine release by NOC-12. These findings contribute to understand the involvement of NO in dopamine-related neuropsychiatric and neurodegenerative diseases.

  5. Nitric oxide induced by polyamines involves antioxidant systems against chilling stress in tomato (Lycopersicon esculentum Mill.) seedling*#

    PubMed Central

    Diao, Qian-Nan; Song, Yong-Jun; Shi, Dong-Mei; Qi, Hong-Yan

    2016-01-01

    Polyamines (PAs) and nitric oxide (NO) are vital signals in modulating plant response to abiotic stress. However, to our knowledge, studies on the relationship between NO and PAs in response to cold stress in tomato are limited. Accordingly, in this study, we investigated the effects of putrescine (Put) and spermidine (Spd) on NO generation and the function of Spd-induced NO in the tolerance of tomato seedling under chilling stress. Spd increased NO release via the nitric oxide synthase (NOS)-like and nitrate reductase (NR) enzymatic pathways in the seedlings, whereas Put had no such effect. Moreover, H2O2 might act as an upstream signal to stimulate NO production. Both exogenous NO donor (sodium nitroprusside (SNP)) and Spd enhanced chilling tolerance in tomato, thereby protecting the photosynthetic system from damage. Compared to chilling treatment alone, Spd enhanced the gene expressions of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX), and their enzyme activities in tomato leaves. However, a scavenger or inhibitor of NO abolished Spd-induced chilling tolerance and blocked the increased expression and activity due to Spd of these antioxidant enzymes in tomato leaves under chilling stress. The results showed that NO induced by Spd plays a crucial role in tomato’s response to chilling stress. PMID:27921397

  6. Nitric oxide and heat shock protein 90 co-regulate temperature-induced bleaching in the soft coral Eunicea fusca

    NASA Astrophysics Data System (ADS)

    Ross, Cliff

    2014-06-01

    Coral bleaching represents a complex physiological process that is affected not only by environmental conditions but by the dynamic internal cellular biology of symbiotic dinoflagellates ( Symbiodinium spp.) and their cnidarian hosts. Recently, nitric oxide (NO) has emerged as a key molecule involved with the expulsion of Symbiodinium from host cnidarian cells. However, the site of production remains under debate, and the corresponding signaling pathways within and between host and endosymbiont remain elusive. In this study, using freshly isolated Symbiodinium from the soft coral Eunicea fusca, I demonstrate that thermally induced stress causes an upregulation in Symbiodinium heat shock protein 90 (Hsp90). In turn, Hsp90 shows a concomitant ability to enhance the activity of a constitutively expressed isoform of NO synthase. The resulting production of NO constitutes a signaling molecule capable of inducing Symbiodinium expulsion. Using nitric oxide synthase (NOS) and Hsp90 polyclonal antibodies, thermal stress-induced Hsp90 was shown to co-immunoprecipitate with a constitutive isoform of NOS. The specific blocking of Hsp90 activity, with the Hsp90 inhibitor geldanamycin, was capable of inhibiting NO production implicating the involvement of a coordinated regulatory system. These results have strong evolutionary implications for Hsp90-NOS chaperone complexes among biological kingdoms and provide evidence for a new functional role in symbiotic associations.

  7. Reactive nitrogen species scavenging, rather than nitric oxide inhibition, protects from articular cartilage damage in rat zymosan-induced arthritis

    PubMed Central

    Bezerra, Mirna Marques; Brain, Susan D; Greenacre, Stan; Jerônimo, Selma Maria Bezerra; de Melo, Liana Batista; Keeble, Julie; da Rocha, Francisco Airton Castro

    2003-01-01

    The contribution of nitric oxide (NO) and peroxynitrite (PN) to inflammation in a zymosan-induced (1 mg, intra-articular, i.art.) rat model of arthritis was assessed by histopathology and by measuring the glycosaminoglycan (GAG) content of the articular cartilage. Progression of the chronic synovitis in zymosan-induced arthritis (ZYA) was associated with increased nitrite and nitrotyrosine (3-NT) levels in the joint exudates that paralleled a progressive loss of the GAG content. An increase in 3-NT was also observed after i.art. PN. The nonselective nitric oxide synthase (NOS) inhibitor L-NG-nitroarginine methyl ester (25–75 mg kg−1day−1) or the selective inducible NOS inhibitor aminoguanidine (50–100 mg kg−1day−1) given 1 h before (prophylactic) or 3 days after (therapeutic) injection of the zymosan ameliorated the synovitis, but worsened the GAG loss, as measured at the end of the experiment (day 7). The PN scavenger uric acid (100–250 mg kg−1 i.p. four times daily) given prophylactically until the end of the experiment (day 14), in a dose compatible with its PN scavenging activity, significantly decreased both the synovitis and the GAG loss. In conclusion, PN formation is associated with cartilage damage in addition to proinflammatory activity in ZYA. NOS inhibitors and a PN scavenger were able to reduce the cellular infiltration, while displaying opposite effects on cartilage homeostasis either by enhancing or ameliorating the damage, respectively. PMID:14662723

  8. Nitric oxide functions as a signal in ultraviolet-B-induced baicalin accumulation in Scutellaria baicalensis suspension cultures.

    PubMed

    Zhang, Jin-Jie; Li, Xue-Qin; Sun, Jun-Wei; Jin, Song-Heng

    2014-03-18

    Stress induced by ultraviolet-B (UV-B) irradiation stimulates the accumulation of various secondary metabolites in plants. Nitric oxide (NO) serves as an important secondary messenger in UV-B stress-induced signal transduction pathways. NO can be synthesized in plants by either enzymatic catalysis or an inorganic nitrogen pathway. The effects of UV-B irradiation on the production of baicalin and the associated molecular pathways in plant cells are poorly understood. In this study, nitric oxide synthase (NOS) activity, NO release and the generation of baicalin were investigated in cell suspension cultures of Scutellaria baicalensis exposed to UV-B irradiation. UV-B irradiation significantly increased NOS activity, NO release and baicalin biosynthesis in S. baicalensis cells. Additionally, exogenous NO supplied by the NO donor, sodium nitroprusside (SNP), led to a similar increase in the baicalin content as the UV-B treatment. The NOS inhibitor, Nω-nitro-l-arginine (LNNA), and NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) partially inhibited UV-B-induced NO release and baicalin accumulation. These results suggest that NO is generated by NOS or NOS-like enzymes and plays an important role in baicalin biosynthesis as part of the defense response of S. baicalensis cells to UV-B irradiation.

  9. Ghrelin counteracts salt-induced hypertension via promoting diuresis and renal nitric oxide production in Dahl rats.

    PubMed

    Aoki, Hirotaka; Nakata, Masanori; Dezaki, Katsuya; Lu, Ming; Gantulga, Darambazar; Yamamoto, Keiji; Shimada, Kazuyuki; Kario, Kazuomi; Yada, Toshihiko

    2013-01-01

    Ghrelin is the endogenous ligand for the growth hormone-secretagogue receptor expressed in various tissues including the heart, blood vessels and kidney. This study sought to determine the effects of long-term treatment with ghrelin (10 nmol/kg, twice a day, intraperitoneally) on the hypertension induced by high salt (8.0% NaCl) diet in Dahl salt-sensitive hypertensive (DS) rats. Systolic blood pressure (SBP) was measured by a tail cuff method. During the treatment period for 3 weeks, high salt diet increased blood pressure compared to normal salt (0.3% NaCl) diet, and this hypertension was partly but significantly (P<0.01) attenuated by simultaneous treatment with ghrelin. Ghrelin significantly increased urine volume and tended to increase urine Na⁺ excretion. Furthermore, ghrelin increased urine nitric oxide (NO) excretion and tended to increase renal neuronal nitric oxide synthase (nNOS) mRNA expression. Ghrelin did not alter the plasma angiotensin II level and renin activity, nor urine catecholamine levels. Furthermore, ghrelin prevented the high salt-induced increases in heart thickness and plasma ANP mRNA expression. These results demonstrate that long-term ghrelin treatment counteracts salt-induced hypertension in DS rats primarily through diuretic action associated with increased renal NO production, thereby exerting cardio-protective effects.

  10. Nitric oxide induced by polyamines involves antioxidant systems against chilling stress in tomato (Lycopersicon esculentum Mill.) seedling.

    PubMed

    Diao, Qian-Nan; Song, Yong-Jun; Shi, Dong-Mei; Qi, Hong-Yan

    Polyamines (PAs) and nitric oxide (NO) are vital signals in modulating plant response to abiotic stress. However, to our knowledge, studies on the relationship between NO and PAs in response to cold stress in tomato are limited. Accordingly, in this study, we investigated the effects of putrescine (Put) and spermidine (Spd) on NO generation and the function of Spd-induced NO in the tolerance of tomato seedling under chilling stress. Spd increased NO release via the nitric oxide synthase (NOS)-like and nitrate reductase (NR) enzymatic pathways in the seedlings, whereas Put had no such effect. Moreover, H2O2 might act as an upstream signal to stimulate NO production. Both exogenous NO donor (sodium nitroprusside (SNP)) and Spd enhanced chilling tolerance in tomato, thereby protecting the photosynthetic system from damage. Compared to chilling treatment alone, Spd enhanced the gene expressions of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX), and their enzyme activities in tomato leaves. However, a scavenger or inhibitor of NO abolished Spd-induced chilling tolerance and blocked the increased expression and activity due to Spd of these antioxidant enzymes in tomato leaves under chilling stress. The results showed that NO induced by Spd plays a crucial role in tomato's response to chilling stress.

  11. Altered Nitric Oxide Bioavailability Contributes to Diesel Exhaust Inhalation‐Induced Cardiovascular Dysfunction in Man

    PubMed Central

    Langrish, Jeremy P.; Unosson, Jon; Bosson, Jenny; Barath, Stefan; Muala, Ala; Blackwell, Scott; Söderberg, Stefan; Pourazar, Jamshid; Megson, Ian L.; Treweeke, Andrew; Sandström, Thomas; Newby, David E.; Blomberg, Anders; Mills, Nicholas L.

    2013-01-01

    Background Diesel exhaust inhalation causes cardiovascular dysfunction including impaired vascular reactivity, increased blood pressure, and arterial stiffness. We investigated the role of nitric oxide (NO) bioavailability in mediating these effects. Methods and Results In 2 randomized double‐blind crossover studies, healthy nonsmokers were exposed to diesel exhaust or filtered air. Study 1: Bilateral forearm blood flow was measured during intrabrachial infusions of acetylcholine (ACh; 5 to 20 μg/min) and sodium nitroprusside (SNP; 2 to 8 μg/min) in the presence of the NO clamp (NO synthase inhibitor NG‐monomethyl‐l‐arginine (l‐NMMA) 8 μg/min coinfused with the NO donor SNP at 90 to 540 ng/min to restore basal blood flow). Study 2: Blood pressure, arterial stiffness, and cardiac output were measured during systemic NO synthase inhibition with intravenous l‐NMMA (3 mg/kg). Following diesel exhaust inhalation, plasma nitrite concentrations were increased (68±48 versus 41±32 nmol/L; P=0.006) despite similar l‐NMMA–induced reductions in basal blood flow (−20.6±14.7% versus −21.1±14.6%; P=0.559) compared to air. In the presence of the NO clamp, ACh and SNP caused dose‐dependent vasodilatation that was not affected by diesel exhaust inhalation (P>0.05 for both). Following exposure to diesel exhaust, l‐NMMA caused a greater increase in blood pressure (P=0.048) and central arterial stiffness (P=0.007), but reductions in cardiac output and increases in systemic vascular resistance (P>0.05 for both) were similar to those seen with filtered air. Conclusions Diesel exhaust inhalation disturbs normal vascular homeostasis with enhanced NO generation unable to compensate for excess consumption. We suggest the adverse cardiovascular effects of air pollution are, in part, mediated through reduced NO bioavailability. Clinical Trial Registration URL: http://www.ClinicalTrials.gov. Unique identifiers: NCT00845767 and NCT01060930. PMID:23525434

  12. Contribution of nitric oxide in the contraction-induced rapid vasodilation in young and older adults.

    PubMed

    Casey, Darren P; Walker, Branton G; Ranadive, Sushant M; Taylor, Jennifer L; Joyner, Michael J

    2013-08-15

    We tested the hypothesis that reduced nitric oxide (NO) bioavailability contributes to the attenuated peak and total vasodilation following single-muscle contractions in older adults. Young (n = 10; 24 ± 2 yr) and older (n = 10; 67 ± 2 yr) adults performed single forearm contractions at 10, 20, and 40% of maximum during saline infusion (control) and NO synthase (NOS) inhibition via N(G)-monomethyl-l-arginine. Brachial artery diameters and velocities were measured using Doppler ultrasound and forearm vascular conductance (FVC; in ml·min(-1)·100 mmHg(-1)) was calculated from blood flow (ml/min) and blood pressure (mmHg). Peak and total vasodilator responses [change (Δ) in FVC from baseline] were attenuated in older adults at all intensities (P < 0.05). NOS inhibition reduced the peak ΔFVC at 10% (88 ± 12 vs. 52 ± 9 ml·min(-1)·100 mmHg(-1)), 20% (125 ± 13 vs. 83 ± 13 ml·min(-1)·100 mmHg(-1)), and 40% (207 ± 26 vs. 133 ± 20 ml·min(-1)·100 mmHg(-1)) in young subjects, (P < 0.05 for all) and in older adults at 10% (59 ± 5 vs. 47 ± 7 ml·min(-1)·100 mmHg(-1), P < 0.05) and 20% (88 ± 9 vs. 68 ± 9 ml·min(-1)·100 mmHg(-1), P < 0.05), but not 40% (128 ± 12 vs. 105 ± 11 ml·min(-1)·100 mmHg(-1), P = 0.11). The relative (%) reduction in peak ΔFVC due to NOS inhibition was greater in young vs. older adults at 20% (-36 ± 5 vs. -23 ± 5%, P < 0.05) and 40% (-35 ± 6 vs. -16 ± 7%, P < 0.05). The reduction in the total vasodilator response (area under the curve) with NOS inhibition was also greater in young vs. older adults at all intensities. Our data suggest that contraction-induced rapid vasodilation is mediated in part by NO, and that the contribution of NO is greater in young adults.

  13. Comparisons of Laser-Saturated, Laser-Induced, and Planar Laser-Induced Fluorescence Measurements of Nitric Oxide in a Lean Direct-Injection Spray Flame

    NASA Astrophysics Data System (ADS)

    Cooper, Clayton S.; Ravikrishna, Rayavarapu V.; Laurendeau, Normand M.

    1998-07-01

    We report quantitative, spatially resolved laser-saturated fluorescence (LSF), linear laser-induced fluorescence (LIF), and planar laser-induced fluorescence (PLIF) measurements of nitric oxide (NO) concentration in a preheated, lean direct-injection spray flame at atmospheric pressure. The spray is produced by a hollow-cone, pressure-atomized nozzle supplied with liquid heptane, and the overall equivalence ratio is unity. NO is excited by means of the Q 2 ( 26 . 5 ) transition of the (0, 0) band. LSF and LIF detection are performed in a 2-nm region centered on the (0, 1) band. PLIF detection is performed in a broad 70-nm region with a peak transmission at 270 nm. Quantitative radial NO profiles obtained by LSF are presented and analyzed so as to correct similar LIF and PLIF profiles. Excellent agreement is achieved among the three fluorescence methodologies.

  14. Potential of Inducible Nitric Oxide Synthase as a Therapeutic Target for Allergen-Induced Airway Hyperresponsiveness: A Critical Connection to Nitric Oxide Levels and PARP Activity

    PubMed Central

    Ghonim, Mohamed A.; Pyakurel, Kusma; Mishra, Anil

    2016-01-01

    Although expression of inducible NO synthase (iNOS) in the lungs of asthmatics and associated nitrosative damage are established, iNOS failed as a therapeutic target for blocking airway hyperresponsiveness (AHR) and inflammation in asthmatics. This dichotomy calls for better strategies with which the enzyme is adequately targeted. Here, we confirm iNOS expression in the asthmatic lung with concomitant protein nitration and poly(ADP-ribose) polymerase (PARP) activation. We show, for the first time, that iNOS is highly expressed in peripheral blood mononuclear cells (PBMCs) of asthmatics with uncontrolled disease, which did not correspond to protein nitration. Selective iNOS inhibition with L-NIL protected against AHR upon acute, but not chronic, exposure to ovalbumin or house dust mite (HDM) in mice. Supplementation of NO by nitrite administration significantly blocked AHR in chronically HDM-exposed mice that were treated with L-NIL. Protection against chronic HDM exposure-induced AHR by olaparib-mediated PARP inhibition may be associated with the partial but not the complete blockade of iNOS expression. Indeed, L-NIL administration prevented olaparib-mediated protection against AHR in chronically HDM-exposed mice. Our study suggests that the amount of iNOS and NO are critical determinants in the modulation of AHR by selective iNOS inhibitors and renews the potential of iNOS as a therapeutic target for asthma. PMID:27524861

  15. The role of constitutive nitric-oxide synthase in ultraviolet B light-induced nuclear factor κB activity.

    PubMed

    Tong, Lingying; Wu, Shiyong

    2014-09-19

    NF-κB is a transcription factor involved in many signaling pathways that also plays an important role in UV-induced skin tumorigenesis. UV radiation can activate NF-κB, but the detailed mechanism remains unclear. In this study, we provided evidence that the activation of constitutive nitric-oxide synthase plays a role in regulation of IκB reduction and NF-κB activation in human keratinocyte HaCaT cells in early phase (within 6 h) post-UVB. Treating the cells with l-NAME, a selective inhibitor of constitutive nitric-oxide synthase (cNOS), can partially reverse the IκB reduction and inhibit the DNA binding activity as well as nuclear translocation of NF-κB after UVB radiation. A luciferase reporter assay indicates that UVB-induced NF-κB activation is totally diminished in cNOS null cells. The cNOS-mediated reduction of IκB is likely due to the imbalance of nitric oxide/peroxynitrite because treating the cells with lower (50 μm), but not higher (100-500 μm), concentration of S-nitroso-N-acetylpenicillamine (SNAP) can reverse the effect of l-NAME in partial restore IκB level post-UVB. Our data also showed that NF-κB activity was required for maintaining a stable IκB kinase α subunit (IKKα) level because treating the cells with NF-κB or cNOS inhibitors could reduce IKKα level upon UVB radiation. In addition, our data demonstrated that although NF-κB protects cells from UVB-induced death, its pro-survival activity was likely neutralized by the pro-death activity of peroxynitrite after UVB radiation.

  16. Diclofenac enhances proinflammatory cytokine-induced nitric oxide production through NF-{kappa}B signaling in cultured astrocytes

    SciTech Connect

    Kakita, Hiroki; Aoyama, Mineyoshi Hussein, Mohamed Hamed; Kato, Shin; Suzuki, Satoshi; Ito, Tetsuya; Togari, Hajime; Asai, Kiyofumi

    2009-07-01

    Recently, the number of reports of encephalitis/encephalopathy associated with influenza virus has increased. In addition, the use of a non-steroidal anti-inflammatory drug, diclofenac sodium (DCF), is associated with a significant increase in the mortality rate of influenza-associated encephalopathy. Activated astrocytes are a source of nitric oxide (NO), which is largely produced by inducible NO synthase (iNOS) in response to proinflammatory cytokines. Therefore, we investigated whether DCF enhances nitric oxide production in astrocytes stimulated with proinflammatory cytokines. We stimulated cultured rat astrocytes with three cytokines, interleukin-1{beta}, tumor necrosis factor-{alpha} and interferon-{gamma}, and then treated the astrocytes with DCF or acetaminophen (N-acetyl-p-aminophenol: APAP). iNOS and NO production in astrocyte cultures were induced by proinflammatory cytokines. The addition of DCF augmented NO production, but the addition of APAP did not. NF-{kappa}B inhibitors SN50 and MG132 inhibited iNOS gene expression in cytokine-stimulated astrocytes with or without DCF. Similarly, NF-{kappa}B p65 Stealth small interfering RNA suppressed iNOS gene expression in cytokine-stimulated astrocytes with or without DCF. LDH activity and DAPI staining showed that DCF induces cell damage in cytokine-stimulated astrocytes. An iNOS inhibitor, L-NMMA, inhibited the cytokine- and DCF-induced cell damage. In conclusion, this study demonstrates that iNOS and NO are induced in astrocyte cultures by proinflammatory cytokines. Addition of DCF further augments NO production. This effect is mediated via NF-{kappa}B signaling and leads to cell damage. The enhancement of DCF on NO production may explain the significant increase in the mortality rate of influenza-associated encephalopathy in patients treated with DCF.

  17. Nitric Oxide Mediates 5-Aminolevulinic Acid-Induced Antioxidant Defense in Leaves of Elymus nutans Griseb. Exposed to Chilling Stress.

    PubMed

    Fu, Juanjuan; Chu, Xitong; Sun, Yongfang; Miao, Yanjun; Xu, Yuefei; Hu, Tianming

    2015-01-01

    Nitric oxide (NO) and 5-aminolevulinic acid (ALA) are both extremely important signalling molecules employed by plants to control many aspects of physiology. In the present study, the role of NO in ALA-induced antioxidant defense in leaves of two sources of Elymus nutans Griseb. (Damxung, DX and Zhengdao, ZD) was investigated. Chilling stress enhanced electrolyte leakage, accumulation of malondialdehyde (MDA), hydrogen peroxide (H2O2) and superoxide radical in two E. nutans, which were substantially alleviated by exogenous ALA and NO application. Pretreatment with NO scavenger PTIO or NOS inhibitor L-NNA alone and in combination with ALA induced enhancements in electrolyte leakage and the accumulation of MDA, H2O2 and superoxide radical in leaves of DX and ZD exposed to chilling stress, indicating that the inhibition of NO biosynthesis reduced the chilling resistance of E. nutans and the ALA-enhanced chilling resistance. Further analyses showed that ALA and NO enhanced antioxidant defense and activated plasma membrane (PM) H+-ATPase and decreased the accumulation of ROS induced by chilling stress. A pronounced increase in nitric oxide synthase (NOS) activity and NO release by exogenous ALA treatment was found in chilling-resistant DX plants exposed to chilling stress, while only a little increase was observed in chilling-sensitive ZD. Furthermore, inhibition of NO accumulation by PTIO or L-NNA blocked the protective effect of exogenous ALA, while both exogenous NO treatment and inhibition of endogenous NO accumulation did not induce ALA production. These results suggested that NO might be a downstream signal mediating ALA-induced chilling resistance in E. nutans.

  18. Impaired acetylcholine-induced cutaneous vasodilation in young smokers: roles of nitric oxide and prostanoids.

    PubMed

    Fujii, Naoto; Reinke, Maggie C; Brunt, Vienna E; Minson, Christopher T

    2013-03-01

    Cigarette smoking attenuates acetylcholine (ACh)-induced cutaneous vasodilation in humans, but the underlying mechanisms are unknown. We tested the hypothesis that smokers have impaired nitric oxide (NO)- and cyclooxygenase (COX)-dependent cutaneous vasodilation to ACh infusion. Twelve young smokers, who have smoked more than 5.2 ± 0.7 yr with an average daily consumption of 11.4 ± 1.2 cigarettes, and 12 nonsmokers were tested. Age, body mass index, and resting mean arterial pressure were similar between the groups. Cutaneous vascular conductance (CVC) was evaluated as laser-Doppler flux divided by mean arterial pressure, normalized to maximal CVC (local heating to 43.0°C plus sodium nitroprusside administration). We evaluated the increase in CVC from baseline to peak (CVCΔpeak) and area under the curve of CVC (CVCAUC) during a bolus infusion (1 min) of 137.5 μM ACh at four intradermal microdialysis sites: 1) Ringer (control), 2) 10 mM N(G)-nitro-l-arginine methyl ester (l-NAME; NO synthase inhibitor), 3) 10 mM ketorolac (COX inhibitor), and 4) combination of l-NAME + ketorolac. CVCΔpeak and CVCAUC at the Ringer site in nonsmokers were greater than in smokers (CVCΔpeak, 42.9 ± 5.1 vs. 22.3 ± 3.5%max, P < 0.05; and CVCAUC, 8,085 ± 1,055 vs. 3,145 ± 539%max·s, P < 0.05). In nonsmokers, CVCΔpeak and CVCAUC at the l-NAME site were lower than the Ringer site (CVCΔpeak, 29.5 ± 6.2%max, P < 0.05; and CVCAUC, 5,377 ± 1,109%max·s, P < 0.05), but in smokers, there were no differences between the Ringer and l-NAME sites (CVCΔpeak, 16.8 ± 4.3%max, P = 0.11; and CVCAUC, 2,679 ± 785%max·s, P = 0.30). CVCΔpeak and CVCAUC were reduced with ketorolac in nonsmokers (CVCΔpeak, 13.3 ± 3.6%max, P < 0.05; and CVCAUC, 1,967 ± 527%max·s, P < 0.05) and smokers (CVCΔpeak, 7.8 ± 1.8%max, P < 0.05; and CVCAUC, 1,246 ± 305%max·s, P < 0.05) and at the combination site in nonsmokers (CVCΔpeak, 15.9 ± 3.1%max, P < 0.05; and CVCAUC, 2,660 ± 512%max·s, P < 0

  19. Nitric Oxide-induced Activation of the Type 1 Ryanodine Receptor Is Critical for Epileptic Seizure-induced Neuronal Cell Death.

    PubMed

    Mikami, Yoshinori; Kanemaru, Kazunori; Okubo, Yohei; Nakaune, Takuya; Suzuki, Junji; Shibata, Kazuki; Sugiyama, Hiroki; Koyama, Ryuta; Murayama, Takashi; Ito, Akihiro; Yamazawa, Toshiko; Ikegaya, Yuji; Sakurai, Takashi; Saito, Nobuhito; Kakizawa, Sho; Iino, Masamitsu

    2016-09-01

    Status epilepticus (SE) is a life-threatening emergency that can cause neurodegeneration with debilitating neurological disorders. However, the mechanism by which convulsive SE results in neurodegeneration is not fully understood. It has been shown that epileptic seizures produce markedly increased levels of nitric oxide (NO) in the brain, and that NO induces Ca(2+) release from the endoplasmic reticulum via the type 1 ryanodine receptor (RyR1), which occurs through S-nitrosylation of the intracellular Ca(2+) release channel. Here, we show that through genetic silencing of NO-induced activation of the RyR1 intracellular Ca(2+) release channel, neurons were rescued from seizure-dependent cell death. Furthermore, dantrolene, an inhibitor of RyR1, was protective against neurodegeneration caused by SE. These results demonstrate that NO-induced Ca(2+) release via RyR is involved in SE-induced neurodegeneration, and provide a rationale for the use of RyR1 inhibitors for the prevention of brain damage following SE.

  20. Effect of the nitric oxide donor and the nitric oxide synthase inhibitor on the liver of rats with chronic hepatitis induced by dimethylnitrosamine.

    PubMed

    Lukivskaya, O; Lis, R; Zwierz, K; Buko, V

    2004-01-01

    The present study was designed to examine the effects of the donor of nitric oxide (NO), NaNO(2) and the inhibitor of NO synthase, N(omega)-nitro-L-arginine (L-NNA), on the development of dimethylnitrosamine (DMNA)-induced chronic hepatitis in rats. L-NNA decreased rat survival and enhanced the severity of hepatic encephalopathy in the DMNA-treated animals. The aggravation of the morphological signs of hepatitis, the activation of serum alanine aminotransferase and cytosolic superoxide dismutase activities and the increase in the liver malondialdehyde content were observed in this group. The treatment with NaNO(2) improved liver morphology, decreased serum marker enzyme activities, lowered the activities of alpha-D-mannosidase and N-acetyl-beta-D-glucosaminidase compared to the DMNA-treated group. The results of the morphological and biochemical studies suggest that L-NNA increased DMNA-induced liver damage, whereas NaNO(2) partially prevented the development of chronic hepatitis. It is proposed that the opposite effects of L-NNA and NaNO(2) are partially explained by a modulation of the free radical-dependent processes in the liver.

  1. MHz-rate nitric oxide planar laser-induced fluorescence imaging in a Mach 10 hypersonic wind tunnel.

    PubMed

    Jiang, Naibo; Webster, Matthew; Lempert, Walter R; Miller, Joseph D; Meyer, Terrence R; Ivey, Christopher B; Danehy, Paul M

    2011-02-01

    Nitric oxide planar laser-induced fluorescence (NO PLIF) imaging at repetition rates as high as 1 MHz is demonstrated in the NASA Langley 31 in. Mach 10 hypersonic wind tunnel. Approximately 200 time-correlated image sequences of between 10 and 20 individual frames were obtained over eight days of wind tunnel testing spanning two entries in March and September of 2009. The image sequences presented were obtained from the boundary layer of a 20° flat plate model, in which transition was induced using a variety of different shaped protuberances, including a cylinder and a triangle. The high-speed image sequences captured a variety of laminar and transitional flow phenomena, ranging from mostly laminar flow, typically at a lower Reynolds number and/or in the near wall region of the model, to highly transitional flow in which the temporal evolution and progression of characteristic streak instabilities and/or corkscrew-shaped vortices could be clearly identified.

  2. The AC-Stark Effect in Nitric Oxide Induced by Rapidly Swept Continuous Wave Quantum Cascade Lasers

    SciTech Connect

    Duxbury, Geoffrey; Kelly, James F.; Blake, Thomas A.; Langford, Nigel

    2012-05-07

    A large AC Stark effect has been observed when nitric oxide, at low pressure in a long optical path (100 m) Herriot cell, is subjected to infrared radiation from a rapidly swept, continuous wave infrared quantum cascade laser. As the frequency sweep rate of the laser is increased, an emission signal induced by rapid passage, occurs after the laser frequency has passed through the resonance of a molecular absorption line. At very high sweep rates a laser field-induced splitting of the absorptive part of the signal is observed, due to the AC Stark effect. This splitting is related to the Autler-Townes mixing of the hyperfine transitions, which lie within the lambda doublet components of the transition, under the Doppler broadened envelope.

  3. An aqueous extract of Ilex paraguariensis reduces carrageenan-induced edema and inhibits the expression of cyclooxygenase-2 and inducible nitric oxide synthase in animal models of inflammation.

    PubMed

    Schinella, Guillermo; Neyret, Elisa; Cónsole, Gloria; Tournier, Horacio; Prieto, José M; Ríos, José-Luis; Giner, Rosa María

    2014-08-01

    Mate (Ilex paraguariensis) is a highly popular herbal beverage in South America due to its high content of caffeine. Its hypolipidemic and antioxidant properties are of increasing interest in the treatment of cardiovascular disorders and for weight control. In the present study, we show for the first time both the local and systemic anti-inflammatory effects of an aqueous extract of mate in three classic in vivo models, namely acute and chronic 12-O-tetradecanoylphorbol 13-acetate-induced mouse ear edema and acute carrageenan-induced mouse paw edema. Caffeine, rutin, chlorogenic acid, 3,5-dicafeoyl quinic acid, and 4,5-dicafeoyl quinic acid, accompanied by a complex mixture of other simple phenolic acids, were identified in the extract by HPLC-UV analyses. In the acute edema model, mate extract applied topically (1 mg/ear) halved the 12-O-tetradecanoylphorbol 13-acetate-induced acute edema (50 %) and almost suppressed neutrophil infiltration (93 %), while in the 12-O-tetradecanoylphorbol 13-acetate-induced subchronic inflammation, the edema was significantly reduced by 62 % (1 mg/ear/day × seven doses). The oral administration of the mate extract (250 mg/kg) significantly reduced the carrageenan-induced edema at all time points, an effect which was accompanied by a 43 % and 53 % reduction of the expression of cyclooxygenase-2 and inducible nitric oxide synthase, respectively. Histological analyses confirmed a reduction of epithelium thickness, dermis with mild inflammation, hair follicles with some secretory cells of sebaceous glands, and hypodermic adipocytes. In conclusion, mate is endowed with in vivo preventative or therapeutic anti-inflammatory effects in both local and systemic inflammatory processes.

  4. Inhibition of neuronal (type 1) nitric oxide synthase prevents hyperaemia and hippocampal lesions resulting from kainate-induced seizures.

    PubMed

    Montécot, C; Rondi-Reig, L; Springhetti, V; Seylaz, J; Pinard, E

    1998-06-01

    The possible roles for nitric oxide produced by neurons in epileptic conditions have been investigated from two different aspects: microcirculation and delayed damage. Our aim was to determine whether the selective inhibition of neuronal (type 1) nitric oxide synthase by 7-nitroindazole, during seizures induced by systemic kainate, modifies hippocampal blood flow and oxygen supply and influences the subsequent hippocampal damage. Experiments were performed in conscious Wistar rats whose electroencephalogram was recorded. 7-Nitroindazole (25 mg/kg, i.p.) or its vehicle was injected 30 min before kainate administration (10 mg/kg, i.p.) and then twice at 1-h intervals. Kainate triggered typical limbic seizures evolving into status epilepticus, identified by uninterrupted electroencephalographic spike activity. The seizures were stopped by diazepam (5 mg/kg, i.p.) after 1 h of status epilepticus. Three types of experiments were performed in vehicle- and 7-nitroindazole-treated rats. (1) Hippocampal nitric oxide synthase activity was measured under basal conditions, at 1 h after the onset of the status epilepticus and at 24 h after its termination (n = 4-6 per group). (2) Hippocampal blood flow and tissue partial pressure of oxygen were measured simultaneously by mass spectrometry for the whole duration of the experiment, while systemic variables and body temperature were monitored (n = 6 per group). (3) Hippocampal damage was revealed by Cresyl Violet staining and evaluated with a lesion score seven days after status epilepticus (n = 12 per group). Hippocampal nitric oxide synthase activity was not significantly modified during status epilepticus or the following day in vehicle-treated rats. In contrast, it was inhibited by 57% in 7-nitroindazole-treated rats, both in basal conditions and after 1 h of status epilepticus, but was not different from its basal level 24 h later. 7-Nitroindazole significantly decreased basal hippocampal blood flow and tissue partial

  5. Hypoxia-induced nitric oxide production and tumour perfusion is inhibited by pegylated arginine deiminase (ADI-PEG20)

    PubMed Central

    Burrows, Natalie; Cane, Gaelle; Robson, Mathew; Gaude, Edoardo; J. Howat, William; Szlosarek, Peter W.; Pedley, R. Barbara; Frezza, Christian; Ashcroft, Margaret; Maxwell, Patrick H.

    2016-01-01

    The hypoxic tumour microenvironment represents an aggressive, therapy-resistant compartment. As arginine is required for specific hypoxia-induced processes, we hypothesised that arginine-deprivation therapy may be useful in targeting hypoxic cancer cells. We explored the effects of the arginine-degrading agent ADI-PEG20 on hypoxia-inducible factor (HIF) activation, the hypoxia-induced nitric oxide (NO) pathway and proliferation using HCT116 and UMUC3 cells and xenografts. The latter lack argininosuccinate synthetase (ASS1) making them auxotrophic for arginine. In HCT116 cells, ADI-PEG20 inhibited hypoxic-activation of HIF-1α and HIF-2α, leading to decreased inducible-nitric oxide synthase (iNOS), NO-production, and VEGF. Interestingly, combining hypoxia and ADI-PEG20 synergistically inhibited ASS1. ADI-PEG20 inhibited mTORC1 and activated the unfolded protein response providing a mechanism for inhibition of HIF and ASS1. ADI-PEG20 inhibited tumour growth, impaired hypoxia-associated NO-production, and decreased vascular perfusion. Expression of HIF-1α/HIF-2α/iNOS and VEGF were reduced, despite an increased hypoxic tumour fraction. Similar effects were observed in UMUC3 xenografts. In summary, ADI-PEG20 inhibits HIF-activated processes in two tumour models with widely different arginine biology. Thus, ADI-PEG20 may be useful in the clinic to target therapy-resistant hypoxic cells in ASS1-proficient tumours and ASS1-deficient tumours. PMID:26972697

  6. Uncovering the vasorelaxant effect induced by Vale do São Francisco red wine: a role for nitric oxide.

    PubMed

    Luciano, Melissa N; Ribeiro, Thaís P; França-Silva, Maria S; do Nascimento, Roberto J B; de Jesus Oliveira, Eduardo; França, Karime C; Antunes, Alessandra A; Nakao, Lia S; Aita, Carlos A M; Braga, Valdir A; de Medeiros, Isac A

    2011-06-01

    The aim of this study was to investigate the mechanisms underlying the vasorelaxant effect induced by the polyphenolic compounds found in red wine from Vale do São Francisco. In phenylephrine (10 μM) precontracted mesenteric artery rings, the red wine caused a concentration-dependent relaxation (maximum response to phenylephrine 10 μM = 87.5% ± 6.5%, n = 10). After endothelium removal, the vasorelaxant effect elicited by red wine was attenuated (28.4% ± 4.9%, n = 10). In addition, the vasorelaxant effect induced by red wine in rings pretreated with 100 μM of N(w)-nitro-l-arginine methyl ester and 10 μM of 1H-[1,2,4] oxadiazolo-[4,3-a]-quinoxalin-1-one was attenuated (23.4% ± 5.1%, n = 7 and 11.8% ± 2.7%, n = 6, respectively). Pretreatment with atropine did not affect the vasorelaxant effect induced by red wine (81% ± 3.9%, n = 6). Furthermore, in rabbit aortic endothelial cell line, red wine 100 and 300 μg/mL caused concentration-dependent increases in nitric oxide levels (58 ± 1; 82 ± 7.9; Δ% of fluorescence, n = 5, respectively). In conclusion, we suggest that the alcohol free-lyophilized red wine induces an endothelium-dependent vasorelaxant effect due, at least in part, to a secondary increase in the concentration of nitric oxide and that this effect might be associated with phenolic compounds found in the red wine.

  7. As(III) inhibits ultraviolet radiation-induced cyclobutane pyrimidine dimers repair via generation of nitric oxide in human keratinocytes

    PubMed Central

    Ding, Wei; Hudson, Laurie G.; Sun, Xi; Feng, Changjian; Liu, Ke Jian

    2008-01-01

    Inorganic arsenic enhances skin tumor formation when combined with other carcinogens including ultraviolet radiation (UVR). The inhibition of DNA damage repair by arsenic has been hypothesized to contribute to the co-carcinogenic activities of arsenic observed in vivo. Cyclobutane pyrimidine dimers (CPDs) are an important mutagenic UVR photoproduct and implicated in the genesis of non-melanoma skin cancer. The current study demonstrates that low concentrations of arsenite (As(III)) inhibit UVR-induced CPDs repair in a human keratinocyte cell line via nitric oxide (NO) and inducible nitric oxide synthase (iNOS). Following As(III) treatment, NO production and iNOS expression are elevated. Little is known about regulation of iNOS by As(III) and further investigations indicated that p38 mitogen-activated protein kinase (p38 MAPK) and NF-κB are required for As(III) induction of iNOS expression. This As(III)-stimulated signaling cascade was involved in inhibition of UVR-induced CPDs repair as disruption of p38 MAPK activity and NF-κB nuclear translocation counteracted the effects of As(III) on CPD repair. Selective inhibition of iNOS ameliorated As(III) inhibition of CPDs repair thereby suggesting that iNOS is a downstream mediator of As(III) activity. These findings provide evidence that an As(III) stimulated signal transduction cascade culminating in elevated iNOS expression and NO generation is an underlying mechanism for inhibition of UVR-induced DNA damage repair by arsenic. PMID:18621123

  8. Hypoxia-induced nitric oxide production and tumour perfusion is inhibited by pegylated arginine deiminase (ADI-PEG20).

    PubMed

    Burrows, Natalie; Cane, Gaelle; Robson, Mathew; Gaude, Edoardo; Howat, William J; Szlosarek, Peter W; Pedley, R Barbara; Frezza, Christian; Ashcroft, Margaret; Maxwell, Patrick H

    2016-03-14

    The hypoxic tumour microenvironment represents an aggressive, therapy-resistant compartment. As arginine is required for specific hypoxia-induced processes, we hypothesised that arginine-deprivation therapy may be useful in targeting hypoxic cancer cells. We explored the effects of the arginine-degrading agent ADI-PEG20 on hypoxia-inducible factor (HIF) activation, the hypoxia-induced nitric oxide (NO) pathway and proliferation using HCT116 and UMUC3 cells and xenografts. The latter lack argininosuccinate synthetase (ASS1) making them auxotrophic for arginine. In HCT116 cells, ADI-PEG20 inhibited hypoxic-activation of HIF-1α and HIF-2α, leading to decreased inducible-nitric oxide synthase (iNOS), NO-production, and VEGF. Interestingly, combining hypoxia and ADI-PEG20 synergistically inhibited ASS1. ADI-PEG20 inhibited mTORC1 and activated the unfolded protein response providing a mechanism for inhibition of HIF and ASS1. ADI-PEG20 inhibited tumour growth, impaired hypoxia-associated NO-production, and decreased vascular perfusion. Expression of HIF-1α/HIF-2α/iNOS and VEGF were reduced, despite an increased hypoxic tumour fraction. Similar effects were observed in UMUC3 xenografts. In summary, ADI-PEG20 inhibits HIF-activated processes in two tumour models with widely different arginine biology. Thus, ADI-PEG20 may be useful in the clinic to target therapy-resistant hypoxic cells in ASS1-proficient tumours and ASS1-deficient tumours.

  9. Chronic wheel running reduces maladaptive patterns of methamphetamine intake: regulation by attenuation of methamphetamine-induced neuronal nitric oxide synthase.

    PubMed

    Engelmann, Alexander J; Aparicio, Mark B; Kim, Airee; Sobieraj, Jeffery C; Yuan, Clara J; Grant, Yanabel; Mandyam, Chitra D

    2014-03-01

    We investigated whether prior exposure to chronic wheel running (WR) alters maladaptive patterns of excessive and escalating methamphetamine intake under extended access conditions, and intravenous methamphetamine self-administration-induced neurotoxicity. Adult rats were given access to WR or no wheel (sedentary) in their home cage for 6 weeks. A set of WR rats were injected with 5-bromo-2'-deoxyuridine (BrdU) to determine WR-induced changes in proliferation (2-h old) and survival (28-day old) of hippocampal progenitors. Another set of WR rats were withdrawn (WRw) or continued (WRc) to have access to running wheels in their home cages during self-administration days. Following self-administration [6 h/day], rats were tested on the progressive ratio (PR) schedule. Following PR, BrdU was injected to determine levels of proliferating progenitors (2-h old). WRc rats self-administered significantly less methamphetamine than sedentary rats during acquisition and escalation sessions, and demonstrated reduced motivation for methamphetamine seeking. Methamphetamine reduced daily running activity of WRc rats compared with that of pre-methamphetamine days. WRw rats self-administered significantly more methamphetamine than sedentary rats during acquisition, an effect that was not observed during escalation and PR sessions. WR-induced beneficial effects on methamphetamine self-administration were not attributable to neuroplasticity effects in the hippocampus and medial prefrontal cortex, but were attributable to WR-induced inhibition of methamphetamine-induced increases in the number of neuronal nitric oxide synthase expressing neurons and apoptosis in the nucleus accumbens shell. Our results demonstrate that WR prevents methamphetamine-induced damage to forebrain neurons to provide a beneficial effect on drug-taking behavior. Importantly, WR-induced neuroprotective effects are transient and continued WR activity is necessary to prevent compulsive methamphetamine intake.

  10. Stimulation of calcium-sensing receptors induces endothelium-dependent vasorelaxations via nitric oxide production and activation of IKCa channels

    PubMed Central

    Greenberg, Harry Z.E.; Shi, Jian; Jahan, Kazi S.; Martinucci, Matthew C.; Gilbert, Steven J.; Vanessa Ho, W.-S.; Albert, Anthony P.

    2016-01-01

    Stimulation of vascular calcium-sensing receptors (CaSRs) is reported to induce both constrictions and relaxations. However, cellular mechanisms involved in these responses remain unclear. The present study investigates the effect of stimulating CaSRs on vascular contractility and focuses on the role of the endothelium, nitric oxide (NO) and K+ channels in these responses. In wire myography studies, increasing [Ca2 +]o from 1 mM to 6 mM induced concentration-dependent relaxations of methoxamine pre-contracted rabbit mesenteric arteries. [Ca2 +]o-induced relaxations were dependent on a functional endothelium, and were inhibited by the negative allosteric CaSR modulator Calhex-231. [Ca2 +]o-induced relaxations were reduced by inhibitors of endothelial NO synthase, guanylate cyclase, and protein kinase G. CaSR activation also induced NO production in freshly isolated endothelial cells (ECs) in experiments using the fluorescent NO indicator DAF-FM. Pre-treatment with inhibitors of large (BKCa) and intermediate (IKCa) Ca2 +-activated K+ channels (iberiotoxin and charybdotoxin), and Kv7 channels (linopirdine) also reduced [Ca2 +]o-induced vasorelaxations. Increasing [Ca2 +]o also activated IKCa currents in perforated-patch recordings of isolated mesenteric artery ECs. These findings indicate that stimulation of CaSRs induces endothelium-dependent vasorelaxations which are mediated by two separate pathways involving production of NO and activation of IKCa channels. NO stimulates PKG leading to BKCa activation in vascular smooth muscle cells, whereas IKCa activity contributes to endothelium-derived hyperpolarisations. PMID:26772767

  11. Diethylcarbamazine activity against Brugia malayi microfilariae is dependent on inducible nitric-oxide synthase and the cyclooxygenase pathway

    PubMed Central

    McGarry, Helen F; Plant, Leigh D; Taylor, Mark J

    2005-01-01

    Background Diethylcarbamazine (DEC) has been used for many years in the treatment of human lymphatic filariasis. Its mode of action is not well understood, but it is known to interact with the arachidonic acid pathway. Here we have investigated the contribution of the nitric oxide and cyclooxygenase (COX) pathways to the activity of DEC against B. malayi microfilariae in mice. Methods B. malayi microfilariae were injected intravenously into mice and parasitaemia was measured 24 hours later. DEC was then administered to BALB/c mice with and without pre-treatment with indomethacin or dexamethasone and the parasitaemia monitored. To investigate a role for inducible nitric oxide in DEC's activity, DEC and ivermectin were administered to microfilaraemic iNOS-/- mice and their background strain (129/SV). Western blot analysis was used to determine any effect of DEC on the production of COX and inducible nitric-oxide synthase (iNOS) proteins. Results DEC administered alone to BALB/c mice resulted in a rapid and profound reduction in circulating microfilariae within five minutes of treatment. Microfilarial levels began to recover after 24 hours and returned to near pre-treatment levels two weeks later, suggesting that the sequestration of microfilariae occurs independently of parasite killing. Pre-treatment of animals with dexamethasone or indomethacin reduced DEC's efficacy by almost 90% or 56%, respectively, supporting a role for the arachidonic acid and cyclooxygenase pathways in vivo. Furthermore, experiments showed that treatment with DEC results in a reduction in the amount of COX-1 protein in peritoneal exudate cells. Additionally, in iNOS-/- mice infected with B. malayi microfilariae, DEC showed no activity, whereas the efficacy of another antifilarial drug, ivermectin, was unaffected. Conclusion These results confirm the important role of the arachidonic acid metabolic pathway in DEC's mechanism of action in vivo and show that in addition to its effects on the 5

  12. Effects of exercise training on stress-induced vascular reactivity alterations: role of nitric oxide and prostanoids

    PubMed Central

    Bruder-Nascimento, Thiago; Silva, Samuel T.; Boer, Patrícia A.; Cordellini, Sandra

    2015-01-01

    Background: Physical exercise may modify biologic stress responses. Objective: To investigate the impact of exercise training on vascular alterations induced by acute stress, focusing on nitric oxide and cyclooxygenase pathways. Method: Wistar rats were separated into: sedentary, trained (60-min swimming, 5 days/week during 8 weeks, carrying a 5% body-weight load), stressed (2 h-immobilization), and trained/stressed. Response curves for noradrenaline, in the absence and presence of L-NAME or indomethacin, were obtained in intact and denuded aortas (n=7-10). Results: None of the procedures altered the denuded aorta reactivity. Intact aortas from stressed, trained, and trained/stressed rats showed similar reduction in noradrenaline maximal responses (sedentary 3.54±0.15, stressed 2.80±0.10*, trained 2.82±0.11*, trained/stressed 2.97± 0.21*, *P<0.05 relate to sedentary). Endothelium removal and L-NAME abolished this hyporeactivity in all experimental groups, except in trained/stressed rats that showed a partial aorta reactivity recovery in L-NAME presence (L-NAME: sedentary 5.23±0,26#, stressed 5.55±0.38#, trained 5.28±0.30#, trained/stressed 4.42±0.41, #P<0.05 related to trained/stressed). Indomethacin determined a decrease in sensitivity (EC50) in intact aortas of trained rats without abolishing the aortal hyporeactivity in trained, stressed, and trained/stressed rats. Conclusions: Exercise-induced vascular adaptive response involved an increase in endothelial vasodilator prostaglandins and nitric oxide. Stress-induced vascular adaptive response involved an increase in endothelial nitric oxide. Beside the involvement of the endothelial nitric oxide pathway, the vascular response of trained/stressed rats involved an additional mechanism yet to be elucidated. These findings advance on the understanding of the vascular processes after exercise and stress alone and in combination. PMID:26083604

  13. Polyphosphoester-based cationic nanoparticles serendipitously release integral biologically-active components to serve as novel degradable inducible nitric oxide synthase inhibitors.

    PubMed

    Shen, Yuefei; Zhang, Shiyi; Zhang, Fuwu; Loftis, Alexander; Pavía-Sanders, Adriana; Zou, Jiong; Fan, Jingwei; Taylor, John-Stephen A; Wooley, Karen L

    2013-10-18

    A degradable polyphosphoester (PPE)-based cationic nanoparticle (cSCK), which is integrated constructed as a novel degradable drug device, demonstrates surprisingly efficient inhibition of inducible nitric oxide synthase (iNOS) transcription, and eventually inhibits nitric oxide (NO) over-production, without loading of any specific therapeutic drugs. This system may serve as a promising anti-inflammatory agent toward the treatment of acute lung injury.

  14. Nitric oxide synthase mediates PC12 differentiation induced by the surface topography of nanostructured TiO2

    PubMed Central

    2013-01-01

    Background Substrate nanoscale topography influences cell proliferation and differentiation through mechanisms that are at present poorly understood. In particular the molecular mechanism through which cells 'sense’ and adapt to the substrate and activate specific intracellular signals, influencing cells survival and behavior, remains to be clarified. Results To characterize these processes at the molecular level we studied the differentiation of PC12 cells on nanostructured TiO2 films obtained by supersonic cluster beam deposition. Our findings indicate that, in PC12 cells grown without Nerve Growth Factor (NGF), the roughness of nanostructured TiO2 triggers neuritogenesis by activating the expression of nitric oxide synthase (NOS) and the phospho-extracellular signal-regulated kinase 1/2 (pERK1/2) signaling. Differentiation is associated with an increase in protein nitration as observed in PC12 cells grown on flat surfaces in the presence of NGF. We demonstrate that cell differentiation and protein nitration induced by topography are not specific for PC12 cells but can be regarded as generalized effects produced by the substrate on different neuronal-like cell types, as shown by growing the human neuroblastoma SH-SY5Y cell line on nanostructured TiO2. Conclusion Our data provide the evidence that the nitric oxide (NO) signal cascade is involved in the differentiation process induced by nanotopography, adding new information on the mechanism and proteins involved in the neuritogenesis triggered by the surface properties. PMID:24119372

  15. Safranal of Crocus sativus L. inhibits inducible nitric oxide synthase and attenuates asthma in a mouse model of asthma.

    PubMed

    Bukhari, Syed Imran; Pattnaik, Bijay; Rayees, Sheikh; Kaul, Sanjana; Dhar, Manoj K

    2015-04-01

    The present study involves evaluation of antioxidant potential of Crocus sativus and its main constituents, safranal (SFN) and crocin (CRO), in bronchial epithelial cells, followed antiinflammatory potential of the active constituent safranal, in a murine model of asthma. To investigate the antioxidizing potential of Crocus sativus and its main constituents in bronchial epithelial cells, the stress was induced in these cells by a combination of different cytokines that resulted in an increase in nitric oxide production (NO), induced nitric oxide synthase (iNOS) levels, peroxynitrite ion generation, and cytochrome c release. Treatment with saffron and its constituents safranal and crocin resulted in a decrease of NO, iNOS levels, peroxynitrite ion generation, and prevented cytochrome c release. However, safranal significantly reduced oxidative stress in bronchial epithelial cells via iNOS reduction besides preventing apoptosis in these cells. In the murine model of asthma study, antiinflammatory role of safranal was characterized by increased airway hyper-responsiveness, airway cellular infiltration, and epithelial cell injury. Safranal pretreatment to these allergically inflamed mice lead to a significant decrease in airway hyper-responsiveness and airway cellular infiltration to the lungs. It also reduced iNOS production, bronchial epithelial cell apoptosis, and Th2 type cytokine production in the lungs.

  16. Inducible Nitric Oxide Inhibitors Block NMDA Antagonist-Stimulated Motoric Behaviors and Medial Prefrontal Cortical Glutamate Efflux

    PubMed Central

    Bergstrom, Hadley C.; Darvesh, Altaf S.; Berger, S. P.

    2015-01-01

    Nitric oxide (NO) plays a critical role in the motoric and glutamate releasing action of N-methyl-D-aspartate (NMDA)-antagonist stimulants. Earlier studies utilized neuronal nitric oxide synthase inhibitors (nNOS) for studying the neurobehavioral effects of non-competitive NMDA-antagonist stimulants such as dizocilpine (MK-801) and phencyclidine (PCP). This study explores the role of the inducible nitric oxide synthase inhibitors (iNOS) aminoguanidine (AG) and (-)-epigallocatechin-3-gallate (EGCG) in NMDA-antagonist induced motoric behavior and prefrontal cortical glutamate efflux. Adult male rats were administered a dose range of AG, EGCG, or vehicle prior to receiving NMDA antagonists MK-801, PCP, or a conventional psychostimulant (cocaine) and tested for motoric behavior in an open arena. Glutamate in the medial prefrontal cortex (mPFC) was measured using in vivo microdialysis after a combination of AG or EGCG prior to MK-801. Acute administration of AG or EGCG dose-dependently attenuated the locomotor and ataxic properties of MK-801 and PCP. Both AG and EGCG were unable to block the motoric effects of cocaine, indicating the acute pharmacologic action of AG and EGCG is specific to NMDA antagonism and not generalizable to all stimulant class drugs. AG and EGCG normalized MK-801-stimulated mPFC glutamate efflux. These data demonstrate that AG and EGCG attenuates NMDA antagonist-stimulated motoric behavior and cortical glutamate efflux. Our results suggest that EGCG-like polyphenol nutraceuticals (contained in “green tea” and chocolate) may be clinically useful in protecting against the adverse behavioral dissociative and cortical glutamate stimulating effects of NMDA antagonists. Medications that interfere with NMDA antagonists such as MK-801 and PCP have been proposed as treatments for schizophrenia. PMID:26696891

  17. Regulation of p53 by activated protein kinase C-delta during nitric oxide-induced dopaminergic cell death.

    PubMed

    Lee, Sung-Jin; Kim, Dong-Chan; Choi, Bo-Hwa; Ha, Hyunjung; Kim, Kyong-Tai

    2006-01-27

    Selective cell death of dopaminergic neurons in the substantia nigra is the major cause of Parkinson disease. Current evidence suggests that this cell death could be mediated by nitric oxide by-products such as nitrate and peroxynitrite. Because protein kinase C (PKC)-delta is implicated in apoptosis of various cell types, we studied its roles and activation mechanisms in nitric oxide (NO)-induced apoptosis of SN4741 dopaminergic cells. When cells were treated with sodium nitroprusside (SNP), a NO donor, endogenous PKC-delta was nitrated and activated. Immunoprecipitation revealed that p53 co-immunoprecipitated with PKC-delta and was phosphorylated at the 15th serine residue in SNP-treated cells. An in vitro kinase assay revealed that p53 was directly phosphorylated by SNP-activated PKC-delta. The p53 Ser-15 phosphorylation was suppressed in SNP-treated cells when the NO-mediated activation of PKC-delta was inhibited by rottlerin or (-)-epigallocatechin gallate. Within 3 h of p53 phosphorylation, its protein levels increased because of decreased ubiquitin-dependent proteosomal proteolysis, whereas the protein levels of MDM2, ubiquitin-protein isopeptide ligase, were down-regulated in a p53 phosphorylation-dependent fashion. Taken together, these results demonstrate that nitration-mediated activation of PKC-delta induces the phosphorylation of the Ser-15 residue in p53, which increases its protein stability, thereby contributing to the nitric oxide-mediated apoptosis-like cell death pathway. These findings may be expanded to provide new insight into the cellular mechanisms of Parkinson disease.

  18. Plasma membrane calcium ATPase 4b inhibits nitric oxide generation through calcium-induced dynamic interaction with neuronal nitric oxide synthase.

    PubMed

    Duan, Wenjuan; Zhou, Juefei; Li, Wei; Zhou, Teng; Chen, Qianqian; Yang, Fuyu; Wei, Taotao

    2013-04-01

    The activation and deactivation of Ca(2+)- and calmodulindependent neuronal nitric oxide synthase (nNOS) in the central nervous system must be tightly controlled to prevent excessive nitric oxide (NO) generation. Considering plasma membrane calcium ATPase (PMCA) is a key deactivator of nNOS, the present investigation aims to determine the key events involved in nNOS deactivation of by PMCA in living cells to maintain its cellular context. Using time-resolved Förster resonance energy transfer (FRET), we determined the occurrence of Ca(2+)-induced protein-protein interactions between plasma membrane calcium ATPase 4b (PMCA4b) and nNOS in living cells. PMCA activation significantly decreased the intracellular Ca(2+) concentrations ([Ca(2+)]i), which deactivates nNOS and slowdowns NO synthesis. Under the basal [Ca(2+)]i caused by PMCA activation, no protein-protein interactions were observed between PMCA4b and nNOS. Furthermore, both the PDZ domain of nNOS and the PDZ-binding motif of PMCA4b were essential for the protein-protein interaction. The involvement of lipid raft microdomains on the activity of PMCA4b and nNOS was also investigated. Unlike other PMCA isoforms, PMCA4 was relatively more concentrated in the raft fractions. Disruption of lipid rafts altered the intracellular localization of PMCA4b and affected the interaction between PMCA4b and nNOS, which suggest that the unique lipid raft distribution of PMCA4 may be responsible for its regulation of nNOS activity. In summary, lipid rafts may act as platforms for the PMCA4b regulation of nNOS activity and the transient tethering of nNOS to PMCA4b is responsible for rapid nNOS deactivation.

  19. Nitric Oxide Plays a Key Role in Ovariectomy-Induced Apoptosis in Anterior Pituitary: Interplay between Nitric Oxide Pathway and Estrogen

    PubMed Central

    Quinteros, Fernanda A.; Duvilanski, Beatriz H.; Cabilla, Jimena P.

    2016-01-01

    Changes in the estrogenic status produce deep changes in pituitary physiology, mainly because estrogens (E2) are one of the main regulators of pituitary cell population. Also, E2 negatively regulate pituitary neuronal nitric oxide synthase (nNOS) activity and expression and may thereby modulate the production of nitric oxide (NO), an important regulator of cell death and survival. Little is known about how ovary ablation affects anterior pituitary cell remodelling and molecular mechanisms that regulate this process have not yet been elucidated. In this work we used freshly dispersed anterior pituitaries as well as cell cultures from ovariectomized female rats in order to study whether E2 deficiency induces apoptosis in the anterior pituitary cells, the role of NO in this process and effects of E2 on the NO pathway. Our results showed that cell activity gradually decreases after ovariectomy (OVX) as a consequence of cell death, which is completely prevented by a pan-caspase inhibitor. Furthermore, there is an increase of fragmented nuclei and DNA cleavage thereby presenting the first direct evidence of the existence of apoptosis in the anterior pituitary gland after OVX. NO production and soluble guanylyl cyclase (sGC) expression in anterior pituitary cells increased concomitantly to the apoptosis. Inhibition of both, NO synthase (NOS) and sGC activities prevented the drop of cell viability after OVX, showing for the first time that increased NO levels and sGC activity observed post-OVX play a key role in the induction of apoptosis. Conversely, E2 and prolactin treatments decreased nNOS expression and activity in pituitary cells from OVX rats in a time- and E2 receptor-dependent manner, thus suggesting interplay between NO and E2 pathways in anterior pituitary. PMID:27611913

  20. Nitric oxide induces ataxia telangiectasia mutated (ATM) protein-dependent γH2AX protein formation in pancreatic β cells.

    PubMed

    Oleson, Bryndon J; Broniowska, Katarzyna A; Schreiber, Katherine H; Tarakanova, Vera L; Corbett, John A

    2014-04-18

    In this study, the effects of cytokines on the activation of the DNA double strand break repair factors histone H2AX (H2AX) and ataxia telangiectasia mutated (ATM) were examined in pancreatic β cells. We show that cytokines stimulate H2AX phosphorylation (γH2AX formation) in rat islets and insulinoma cells in a nitric oxide- and ATM-dependent manner. In contrast to the well documented role of ATM in DNA repair, ATM does not appear to participate in the repair of nitric oxide-induced DNA damage. Instead, nitric oxide-induced γH2AX formation correlates temporally with the onset of irreversible DNA damage and the induction of apoptosis. Furthermore, inhibition of ATM attenuates cytokine-induced caspase activation. These findings show that the formation of DNA double strand breaks correlates with ATM activation, irreversible DNA damage, and ATM-dependent induction of apoptosis in cytokine-treated β cells.

  1. Endotoxin-induced nitric oxide production rescues airway growth and maturation in atrophic fetal rat lung explants

    SciTech Connect

    Rae, C.; Cherry, J.I.; Land, F.M.; Land, S.C. . E-mail: s.c.land@dundee.ac.uk

    2006-10-13

    Inflammation induces premature maturation of the fetal lung but the signals causing this effect remain unclear. We determined if nitric oxide (NO) synthesis, evoked by Escherichia coli lipopolysaccharide (LPS, 2 {mu}g ml{sup -1}), participated in this process. Fetal rat lung airway surface complexity rose 2.5-fold over 96 h in response to LPS and was associated with increased iNOS protein expression and activity. iNOS inhibition by N6-(1-iminoethyl)-L-lysine-2HCl (L-NIL) abolished this and induced airway atrophy similar to untreated explants. Surfactant protein-C (SP-C) expression was also induced by LPS and abolished by L-NIL. As TGF{beta} suppresses iNOS activity, we determined if feedback regulation modulated NO-dependent maturation. LPS induced TGF{beta}1 release and SMAD4 nuclear translocation 96 h after treatment. Treatment of explants with a blocking antibody against TGF{beta}1 sustained NO production and airway morphogenesis whereas recombinant TGF{beta}1 antagonized these effects. Feedback regulation of NO synthesis by TGF{beta} may, thus, modulate airway branching and maturation of the fetal lung.

  2. Mechanisms of nitric-oxide-induced increase of free cytosolic Ca2+ concentration in Nicotiana plumbaginifolia cells.

    PubMed

    Lamotte, Olivier; Courtois, Cécile; Dobrowolska, Grazyna; Besson, Angélique; Pugin, Alain; Wendehenne, David

    2006-04-15

    In this study, we investigated a role for nitric oxide (NO) in mediating the elevation of the free cytosolic Ca(2+) concentration ([Ca(2+)](cyt)) in plants using Nicotiana plumbaginifolia cells expressing the Ca(2+) reporter apoaequorin. Hyperosmotic stress induced a fast increase of [Ca(2+)](cyt) which was strongly reduced by pretreating cell suspensions with the NO scavenger carboxy PTIO, indicating that NO mediates [Ca(2+)](cyt) changes in plant cells challenged by abiotic stress. Accordingly, treatment of transgenic N. plumbaginifolia cells with the NO donor diethylamine NONOate was followed by a transient increase of [Ca(2+)](cyt) sensitive to plasma membrane Ca(2+) channel inhibitors and antagonist of cyclic ADP ribose. We provided evidence that NO might activate plasma membrane Ca(2+) channels by inducing a rapid and transient plasma membrane depolarization. Furthermore, NO-induced elevation of [Ca(2+)](cyt) was suppressed by the kinase inhibitor staurosporine, suggesting that NO enhances [Ca(2+)](cyt) by promoting phosphorylation-dependent events. This result was further supported by the demonstration that the NO donor induced the activation of a 42-kDa protein kinase which belongs to SnRK2 families and corresponds to Nicotiana tabacum osmotic-stress-activated protein kinase (NtOSAK). Interestingly, NtOSAK was activated in response to hyperosmotic stress through a NO-dependent process, supporting the hypothesis that NO also promotes protein kinase activation during physiological processes.

  3. Distinctive expression patterns of hypoxia-inducible factor-1α and endothelial nitric oxide synthase following hypergravity exposure

    PubMed Central

    Yoon, Gun; Oh, Choong Sik; Kim, Hyun-Soo

    2016-01-01

    This study was designed to examine the expression of hypoxia-inducible factor-1α (HIF-1α) and the level and activity of endothelial nitric oxide synthase (eNOS) in the hearts and livers of mice exposed to hypergravity. Hypergravity-induced hypoxia and the subsequent post-exposure reoxygenation significantly increased cardiac HIF-1α levels. Furthermore, the levels and activity of cardiac eNOS also showed significant increase immediately following hypergravity exposure and during the reoxygenation period. In contrast, the expression of phosphorylated Akt (p-Akt) and phosphorylated extracellular signal-regulated kinase (p-ERK) showed significant elevation only during the reoxygenation period. These data raise the possibility that the increase in cardiac HIF-1α expression induced by reoxygenation involves a cascade of signaling events, including activation of the Akt and ERK pathways. In the liver, HIF-1α expression was significantly increased immediately after hypergravity exposure, indicating that hypergravity exposure to causes hepatocellular hypoxia. The hypergravity-exposed livers showed significantly higher eNOS immunoreactivity than did those of control mice. Consistent with these results, significant increases in eNOS activity and nitrate/nitrite levels were also observed. These findings suggest that hypergravity-induced hypoxia plays a significant role in the upregulation of hepatic eNOS. PMID:27191892

  4. Effects of plant-derived polyphenols on TNF-alpha and nitric oxide production induced by advanced glycation endproducts.

    PubMed

    Chandler, Dave; Woldu, Ameha; Rahmadi, Anton; Shanmugam, Kirubakaran; Steiner, Nicole; Wright, Elise; Benavente-García, Obdulio; Schulz, Oliver; Castillo, Julián; Münch, Gerald

    2010-07-01

    Advanced glycation endproducts (AGEs) accumulate on protein deposits including the beta-amyloid plaques in Alzheimer's disease. AGEs interact with the "receptor for advanced glycation endproducts", and transmit their signals using intracellular reactive oxygen species as second messengers. Ultimately, AGEs induce the expression of a variety of pro-inflammatory markers including the tumor necrosis factor (TNF-alpha) and inducible nitric oxide (NO) synthase. Antioxidants that act intracellularly, including polyphenols, have been shown to scavenge these "signaling" reactive oxygen species, and thus perform in an anti-inflammatory capacity. This study tested the pure compounds apigenin and diosmetin as well as extracts from silymarin, uva ursi (bearberry) and green olive leaf for their ability to attenuate AGE-induced NO and TNF-alpha production. All five tested samples inhibited BSA-AGE-induced NO production in a dose-dependent manner. Apigenin and diosmetin were most potent, and exhibited EC(50) values approximately 10 microM. In contrast, TNF-alpha expression was only reduced by apigenin, diosmetin and silymarin; not by the bearberry and green olive leaf extracts. In addition, the silymarin and bearberry extracts caused significant cell death at concentrations >or=10 microg/mL and >or=50 microg/mL, respectively. In conclusion, we suggest that plant-derived polyphenols might offer therapeutic opportunities to delay the progression of AGE-mediated and receptor for advanced glycation endproducts-mediated neuro-inflammatory diseases including Alzheimer's disease.

  5. Nitric oxide synthase inhibitor, aminoguanidine reduces intracerebroventricular colchicine induced neurodegeneration, memory impairments and changes of systemic immune responses in rats.

    PubMed

    Sil, Susmita; Ghosh, Tusharkanti; Ghosh, Rupsa; Gupta, Pritha

    2017-02-15

    Intracerebroventricular (i.c.v.) injection of colchicine induces neurodegeneration, memory impairments and changes of some systemic immune responses in rats. Though the role of cox 2 in these colchicine induced changes have been evaluated, the influence of nitric oxide synthase (NOS) remains to be studied. The present study was designed to assess the role of NOS on the i.c.v. colchicine induced neurodegeneration, memory impairments and changes of some systemic immune responses by inhibiting its activity with aminoguanidine. In the present study the impairments of working and reference memories, neurodegeneration (chromatolysis and plaque formation) and changes of neuroinflammatory markers in the hippocampus (increased TNF α, IL 1β, ROS and nitrite) along with changes of serum inflammatory markers (TNF α, IL 1β, ROS and nitrite) and alteration of systemic immune responses (higher phagocytic activity of blood WBC and splenic PMN, higher cytotoxicity and lower leukocyte adhesion inhibition index of splenic MNC) were measured in the intracerebroventricular colchicine injected rats (ICIR). Administration of aminoguanidine (p.o. 30/50mg/kg body weight) to ICIR resulted in recovery of neuroinflammation and partial prevention of neurodegeneration which could be corroborated with the partial recovery of memory impairments in this model. The recovery of serum inflammatory markers and the systemic immune responses in ICIR was also observed after administration of aminoguanidine. Therefore, the present study shows that aminoguanidine can protect the colchicine induced neurodegeneration, memory impairments, and changes of systemic immune systemic responses in ICIR by inhibiting the iNOS.

  6. Are cyclooxygenase-2 and nitric oxide involved in the dyskinesia of Parkinson's disease induced by l-DOPA?†

    PubMed Central

    Bortolanza, Mariza; Padovan-Neto, Fernando E.; Cavalcanti-Kiwiatkoski, Roberta; dos Santos-Pereira, Maurício; Mitkovski, Miso; Raisman-Vozari, Rita; Del-Bel, Elaine

    2015-01-01

    Inflammatory mechanisms are proposed to play a role in l-DOPA-induced dyskinesia. Cyclooxygenase-2 (COX2) contributes to inflammation pathways in the periphery and is constitutively expressed in the central nervous system. Considering that inhibition of nitric oxide (NO) formation attenuates l-DOPA-induced dyskinesia, this study aimed at investigating if a NO synthase (NOS) inhibitor would change COX2 brain expression in animals with l-DOPA-induced dyskinesia. To this aim, male Wistar rats received unilateral 6-hydroxydopamine microinjection into the medial forebrain bundle were treated daily with l-DOPA (21 days) combined with 7-nitroindazole or vehicle. All hemi-Parkinsonian rats receiving l-DOPA showed dyskinesia. They also presented increased neuronal COX2 immunoreactivity in the dopamine-depleted dorsal striatum that was directly correlated with dyskinesia severity. Striatal COX2 co-localized with choline-acetyltransferase, calbindin and DARPP-32 (dopamine-cAMP-regulated phosphoprotein-32), neuronal markers of GABAergic neurons. NOS inhibition prevented l-DOPA-induced dyskinesia and COX2 increased expression in the dorsal striatum. These results suggest that increased COX2 expression after l-DOPA long-term treatment in Parkinsonian-like rats could contribute to the development of dyskinesia. PMID:26009769

  7. Nitric oxide-activated calcium/calmodulin-dependent protein kinase regulates the abscisic acid-induced antioxidant defence in maize.

    PubMed

    Ma, Fangfang; Lu, Rui; Liu, Huiying; Shi, Ben; Zhang, Jianhua; Tan, Mingpu; Zhang, Aying; Jiang, Mingyi

    2012-08-01

    Nitric oxide (NO), hydrogen peroxide (H2O2), and calcium (Ca2+)/calmodulin (CaM) are all required for abscisic acid (ABA)-induced antioxidant defence. Ca2+/CaM-dependent protein kinase (CCaMK) is a strong candidate for the decoder of Ca2+ signals. However, whether CCaMK is involved in ABA-induced antioxidant defence is unknown. The results of the present study show that exogenous and endogenous ABA induced increases in the activity of ZmCCaMK and the expression of ZmCCaMK in leaves of maize. Subcellular localization analysis showed that ZmCCaMK is located in the nucleus, the cytoplasm, and the plasma membrane. The transient expression of ZmCCaMK and the RNA interference (RNAi) silencing of ZmCCaMK analysis in maize protoplasts revealed that ZmCCaMK is required for ABA-induced antioxidant defence. Moreover, treatment with the NO donor sodium nitroprusside (SNP) induced the activation of ZmCCaMK and the expression of ZmCCaMK. Pre-treatments with an NO scavenger and inhibitor blocked the ABA-induced increases in the activity and the transcript level of ZmCCaMK. Conversely, RNAi silencing of ZmCCaMK in maize protoplasts did not affect the ABA-induced NO production, which was further confirmed using a mutant of OsCCaMK, the homologous gene of ZmCCaMK in rice. Moreover, H2O2 was also required for the ABA activation of ZmCCaMK, and pre-treatments with an NO scavenger and inhibitor inhibited the H2O2-induced increase in the activity of ZmCCaMK. Taken together, the data clearly suggest that ZmCCaMK is required for ABA-induced antioxidant defence, and H2O2-dependent NO production plays an important role in the ABA-induced activation of ZmCCaMK.

  8. Nitric oxide-activated calcium/calmodulin-dependent protein kinase regulates the abscisic acid-induced antioxidant defence in maize

    PubMed Central

    Zhang, Aying; Jiang, Mingyi

    2012-01-01

    Nitric oxide (NO), hydrogen peroxide (H2O2), and calcium (Ca2+)/calmodulin (CaM) are all required for abscisic acid (ABA)-induced antioxidant defence. Ca2+/CaM-dependent protein kinase (CCaMK) is a strong candidate for the decoder of Ca2+ signals. However, whether CCaMK is involved in ABA-induced antioxidant defence is unknown. The results of the present study show that exogenous and endogenous ABA induced increases in the activity of ZmCCaMK and the expression of ZmCCaMK in leaves of maize. Subcellular localization analysis showed that ZmCCaMK is located in the nucleus, the cytoplasm, and the plasma membrane. The transient expression of ZmCCaMK and the RNA interference (RNAi) silencing of ZmCCaMK analysis in maize protoplasts revealed that ZmCCaMK is required for ABA-induced antioxidant defence. Moreover, treatment with the NO donor sodium nitroprusside (SNP) induced the activation of ZmCCaMK and the expression of ZmCCaMK. Pre-treatments with an NO scavenger and inhibitor blocked the ABA-induced increases in the activity and the transcript level of ZmCCaMK. Conversely, RNAi silencing of ZmCCaMK in maize protoplasts did not affect the ABA-induced NO production, which was further confirmed using a mutant of OsCCaMK, the homologous gene of ZmCCaMK in rice. Moreover, H2O2 was also required for the ABA activation of ZmCCaMK, and pre-treatments with an NO scavenger and inhibitor inhibited the H2O2-induced increase in the activity of ZmCCaMK. Taken together, the data clearly suggest that ZmCCaMK is required for ABA-induced antioxidant defence, and H2O2-dependent NO production plays an important role in the ABA-induced activation of ZmCCaMK. PMID:22865912

  9. Cinnamaldehyde and cinnamaldehyde-containing micelles induce relaxation of isolated porcine coronary arteries: role of nitric oxide and calcium

    PubMed Central

    Raffai, Gábor; Kim, Byungkuk; Park, Sanga; Khang, Gilson; Lee, Dongwon; Vanhoutte, Paul M

    2014-01-01

    Background and purpose Cinnamaldehyde, a major component of cinnamon, induces the generation of reactive oxygen species and exerts vasodilator and anticancer effects, but its short half-life limits its clinical use. The present experiments were designed to compare the acute relaxing properties of cinnamaldehyde with those of self-assembling polymer micelles either loaded with cinnamaldehyde or consisting of a polymeric prodrug [poly(cinnamaldehyde)] that incorporates the compound in its backbone. Methods Rings of porcine coronary arteries were contracted with the thromboxane A2 receptor agonist U46619 or 40 mM KCl, and changes in isometric tension were recorded. Results Cinnamaldehyde induced concentration-dependent but endothelium-independent, nitric oxide synthase (NOS)-independent, cyclooxygenase-independent, soluble guanylyl cyclase (sGC)-independent, calcium-activated potassium-independent, and TRPA1 channel-independent relaxations. Cinnamaldehyde also inhibited the contractions induced by 40 mM KCl Ca2+ reintroduction in 40 mM KCl Ca2+-free solution or by the Ca2+ channel opener Bay K8644. Cinnamaldehyde-loaded control micelles induced complete, partly endothelium-dependent relaxations sensitive to catalase and inhibitors of NOS or sGC, but not cyclooxygenase or TRPA1, channels. Cinnamaldehyde-loaded micelles also inhibited contractions induced by 40 mM KCl Ca2+ reintroduction or Bay K8644. Poly(cinnamaldehyde) micelles induced only partial, endothelium-dependent relaxations that were reduced by inhibitors of NOS or sGC and by catalase and the antioxidant tiron, but not by indomethacin or TRPA1 channel blockers. Conclusion The present findings demonstrate that cinnamaldehyde-loaded and poly(cinnamaldehyde) micelles possess vasodilator properties, but that the mechanism underlying the relaxation that they cause differs from that of cinnamaldehyde, and thus could be used both to relieve coronary vasospasm and for therapeutic drug delivery. PMID:24904214

  10. Abscisic acid (ABA) inhibits light-induced stomatal opening through calcium- and nitric oxide-mediated signaling pathways.

    PubMed

    Garcia-Mata, Carlos; Lamattina, Lorenzo

    2007-01-01

    Nitric oxide (NO) is an important signaling component of ABA-induced stomatal closure. However, only fragmentary data are available about NO effect on the inhibition of stomatal opening. Here, we present results supporting that, in Vicia faba guard cells, there is a critical Ca2+-dependent NO increase required for the ABA-mediated inhibition of stomatal opening. Light-induced stomatal opening was inhibited by exogenous NO in V. faba epidermal strips. Furthermore, ABA-mediated inhibition of stomatal opening was blocked by the specific NO scavenger cPTIO, supporting the involvement of endogenous NO in this process. Since the raise in Ca2+ concentration is a pre-requisite in ABA-mediated inhibition of stomatal opening, it was interesting to establish how does Ca2+, NO and ABA interact in the inhibition of light-induced stomatal opening. The permeable Ca2+ specific buffer BAPTA-AM blocked both ABA- and Ca2+- but not NO-mediated inhibition of stomatal opening. The NO synthase (NOS) specific inhibitor L-NAME prevented Ca2+-mediated inhibition of stomatal opening, indicating that a NOS-like activity was required for Ca2+ signaling. Furthermore, experiments using the NO specific fluorescent probe DAF-2DA indicated that Ca2+ induces an increase of endogenous NO. These results indicate that, in addition to the roles in ABA-triggered stomatal closure, both NO and Ca2+ are active components of signaling events acting in ABA inhibition of light-induced stomatal opening. Results also support that Ca2+ induces the NO production through the activation of a NOS-like activity.

  11. The Effect of Artemisia fragrans Willd: Essential Oil on Inducible Nitric Oxide Synthase Gene Expression and Nitric Oxide Production in Lipopolysaccharide-stimulated Murine Macrophage Cell Line.

    PubMed

    Farghadan, Maryam; Ghafoori, Hosein; Vakhshiteh, Faezeh; Shahzadeh Fazeli, Seyed Abolhassan; Farzaneh, Parvaneh; Kokhaei, Parviz

    2016-12-01

    The genus Artemisia is estimated to comprise over 800 species with anti-cancer, anti-fungal, anti-oxidant and anti-inflammatory properties. Artemisia fragrans (A. fragrans), a species that belongs to genus Artemisia, is rich in monoterpenes and sesquiterpenes derivatives. Due to anti-inflammatory properties of monoterpenes and sesquiterpenes, we aimed to investigate the effect of A. fragrans essential oil on mRNA expression of inducible nitric oxide synthase (iNOS) gene and nitric oxide (NO) production in Lipopolysaccharide (LPS) -stimulated RAW264.7 cell line. NO, which is synthesized by iNOS, is the main macrophage-derived inflammatory mediator. The oil obtained from the A. fragrans was prepared from aerial parts of the plant. Chemical composition of essential oil was analyzed by gas chromatography-mass spectrometry (GC/MS).The cytotoxicity of various concentrations of essential oil was evaluated by mitochondrial reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test assay. The effect of different doses (1.75-7 mg/mL) of A. fragrans oil on mRNA expression of iNOS gene and NO production in LPS-stimulated RAW 264.7 cells was assessed by real-time PCR method and Griess reagent, respectively. In GC/MS analyses of A. fragrans oil, 32 compounds were identified. The main components of the oil were camphor and 1, 8-cineole. The results demonstrated that the essential oil of A. fragrans (1.75- 7 mg/mL), in a dose-dependent manner, inhibits mRNA expression of iNOS induced by LPS in the RAW264.7 cells without cytotoxic effect even at higher doses. The results of iNOS were consistent with the results of NO production. Our preliminary results suggest the possible anti-inflammatory effect of A. fragrans. Further studies are needed to determine the full pharmacokinetics of A. fragrans activity in vivo.

  12. Nitric Oxide Mediates the Stress Response Induced by Diatom Aldehydes in the Sea Urchin Paracentrotus lividus

    PubMed Central

    Romano, Giovanna; Costantini, Maria; Buttino, Isabella; Ianora, Adrianna; Palumbo, Anna

    2011-01-01

    Diatoms are ubiquitous and abundant primary producers that have been traditionally considered as a beneficial food source for grazers and for the transfer of carbon through marine food webs. However, many diatom species produce polyunsaturated aldehydes that disrupt development in the offspring of grazers that feed on these unicellular algae. Here we provide evidence that production of the physiological messenger nitric oxide increases after treatment with the polyunsaturated aldehyde decadienal in embryos of the sea urchin Paracentrotus lividus. At high decadienal concentrations, nitric oxide mediates initial apoptotic events leading to loss of mitochondrial functionality through the generation of peroxynitrite. At low decadienal concentrations, nitric oxide contributes to the activation of hsp70 gene expression thereby protecting embryos against the toxic effects of this aldehyde. When nitric oxide levels were lowered by inhibiting nitric oxide synthase activity, the expression of hsp70 in swimming blastula decreased and the proportion of abnormal plutei increased. However, in later pluteus stages nitric oxide was no longer able to exert this protective function: hsp70 and nitric oxide synthase expression decreased with a consequent increase in the expression of caspase-8. Our findings that nitric oxide production increases rapidly in response to a toxic exogenous stimulus opens new perspectives on the possible role of this gas as an important messenger to environmental stress in sea urchins and for understanding the cellular mechanisms underlying toxicity during diatom blooms. PMID:22022485

  13. Nitric oxide mediates the stress response induced by diatom aldehydes in the sea urchin Paracentrotus lividus.

    PubMed

    Romano, Giovanna; Costantini, Maria; Buttino, Isabella; Ianora, Adrianna; Palumbo, Anna

    2011-01-01

    Diatoms are ubiquitous and abundant primary producers that have been traditionally considered as a beneficial food source for grazers and for the transfer of carbon through marine food webs. However, many diatom species produce polyunsaturated aldehydes that disrupt development in the offspring of grazers that feed on these unicellular algae. Here we provide evidence that production of the physiological messenger nitric oxide increases after treatment with the polyunsaturated aldehyde decadienal in embryos of the sea urchin Paracentrotus lividus. At high decadienal concentrations, nitric oxide mediates initial apoptotic events leading to loss of mitochondrial functionality through the generation of peroxynitrite. At low decadienal concentrations, nitric oxide contributes to the activation of hsp70 gene expression thereby protecting embryos against the toxic effects of this aldehyde. When nitric oxide levels were lowered by inhibiting nitric oxide synthase activity, the expression of hsp70 in swimming blastula decreased and the proportion of abnormal plutei increased. However, in later pluteus stages nitric oxide was no longer able to exert this protective function: hsp70 and nitric oxide synthase expression decreased with a consequent increase in the expression of caspase-8. Our findings that nitric oxide production increases rapidly in response to a toxic exogenous stimulus opens new perspectives on the possible role of this gas as an important messenger to environmental stress in sea urchins and for understanding the cellular mechanisms underlying toxicity during diatom blooms.

  14. Role of reactive nitrogen species generated via inducible nitric oxide synthase in vesicant-induced lung injury, inflammation and altered lung functioning

    SciTech Connect

    Sunil, Vasanthi R.; Shen, Jianliang; Patel-Vayas, Kinal; Gow, Andrew J.; Laskin, Jeffrey D.; Laskin, Debra L.

    2012-05-15

    Pulmonary toxicity induced by sulfur mustard and related vesicants is associated with oxidative stress. In the present studies we analyzed the role of reactive nitrogen species (RNS) generated via inducible nitric oxide synthase (iNOS) in lung injury and inflammation induced by vesicants using 2-chloroethyl ethyl sulfide (CEES) as a model. C57Bl/6 (WT) and iNOS −/− mice were sacrificed 3 days or 14 days following intratracheal administration of CEES (6 mg/kg) or control. CEES intoxication resulted in transient (3 days) increases in bronchoalveolar lavage (BAL) cell and protein content in WT, but not iNOS −/− mice. This correlated with expression of Ym1, a marker of oxidative stress in alveolar macrophages and epithelial cells. In contrast, in iNOS −/− mice, Ym1 was only observed 14 days post-exposure in enlarged alveolar macrophages, suggesting that they are alternatively activated. This is supported by findings that lung tumor necrosis factor and lipocalin Lcn2 expression, mediators involved in tissue repair were also upregulated at this time in iNOS −/− mice. Conversely, CEES-induced increases in the proinflammatory genes, monocyte chemotactic protein-1 and cyclooxygenase-2, were abrogated in iNOS −/− mice. In WT mice, CEES treatment also resulted in increases in total lung resistance and decreases in compliance in response to methacholine, effects blunted by loss of iNOS. These data demonstrate that RNS, generated via iNOS play a role in the pathogenic responses to CEES, augmenting oxidative stress and inflammation and suppressing tissue repair. Elucidating inflammatory mechanisms mediating vesicant-induced lung injury is key to the development of therapeutics to treat mustard poisoning. -- Highlights: ► Lung injury, inflammation and oxidative stress are induced by the model vesicant CEES ► RNS generated via iNOS are important in the CEES-induced pulmonary toxicity ► iNOS −/− mice are protected from CEES-induced lung toxicity and

  15. Taurocholate-induced nitric oxide signaling and the ensuing production of reactive oxygen species lead to an increase in epithelial permeability in cultivated mouse gastric epithelium.

    PubMed

    Mustonen, Harri; Kiviluoto, Tuula; Puolakkainen, Pauli; Paimela, Hannu; Mentula, Panu; Kemppainen, Esko; Kivilaakso, Eero

    2008-12-01

    We have here elucidated whether ulcerogenic agents affect the production of NO and reactive oxygen species (ROS). The ulcerogenic agents dose dependently induced NO and ROS production in mouse gastric epithelial cells. Taurocholate (TC, 5 mM) exposure did not affect cell viability, but it increased inducible nitric oxide synthase (iNOS) expression, NO production, ROS production, and epithelial permeability. Epithelial permeability was inhibited with NOS inhibitors or antioxidants. Oxidative stress induced by acetylsalicylic acid (ASA) and ethanol was not inhibited with NOS inhibitors. ASA induced ROS production even at low concentrations (1 mM), which did not affect cell viability. Ethanol-induced ROS production was linked to cell viability, suggesting direct oxidative stress caused by ethanol. Taurocholate-induced NO signaling and the ensuing production of ROS might contribute to initiation of defensive or adaptive cellular mechanisms. ASA-induced ROS signaling might have similar effects, whereas ethanol induced direct oxidative stress, having an influence on cell viability.

  16. The new nitric oxide donor cyclohexane nitrate induces vasorelaxation, hypotension, and antihypertensive effects via NO/cGMP/PKG pathway

    PubMed Central

    Mendes-Júnior, Leônidas das G.; Guimarães, Driele D.; Gadelha, Danilo D. A.; Diniz, Thiago F.; Brandão, Maria C. R.; Athayde-Filho, Petrônio F.; Lemos, Virginia S.; França-Silva, Maria do S.; Braga, Valdir A.

    2015-01-01

    We investigated the cardiovascular effects induced by the nitric oxide donor Cyclohexane Nitrate (HEX). Vasodilatation, NO release and the effects of acute or sub-chronic treatment with HEX on cardiovascular parameters were evaluated. HEX induced endothelium-independent vasodilatation (Maximum effect [efficacy, ME] = 100.4 ± 4.1%; potency [pD2] = 5.1 ± 0.1). Relaxation was attenuated by scavenging nitric oxide (ME = 44.9 ± 9.4% vs. 100.4 ± 4.1%) or by inhibiting the soluble guanylyl cyclase (ME = 38.5 ± 9.7% vs. 100.4 ± 4.1%). In addition, pD2 was decreased after non-selective blockade of K+ channels (pD2 = 3.6 ± 0.1 vs. 5.1 ± 0.1) or by inhibiting KATP channels (pD2 = 4.3 ± 0.1 vs. 5.1 ± 0.1). HEX increased NO levels in mesenteric arteries (33.2 ± 2.3 vs. 10.7 ± 0.2 au, p < 0.0001). Intravenous acute administration of HEX (1–20 mg/kg) induced hypotension and bradycardia in normotensive and hypertensive rats. Furthermore, starting at 6 weeks after the induction of 2K1C hypertension, oral treatment with the HEX (10 mg/Kg/day) for 7 days reduced blood pressure in hypertensive animals (134 ± 6 vs. 170 ± 4 mmHg, respectively). Our data demonstrate that HEX is a NO donor able to produce vasodilatation via NO/cGMP/PKG pathway and activation of the ATP-sensitive K+ channels. Furthermore, HEX acutely reduces blood pressure and heart rate as well as produces antihypertensive effect in renovascular hypertensive rats. PMID:26379557

  17. Preventive effect of gomisin J from Schisandra chinensis on angiotensin II-induced hypertension via an increased nitric oxide bioavailability.

    PubMed

    Ye, Byeong Hyeok; Lee, Seung Jin; Choi, Young Whan; Park, So Youn; Kim, Chi Dae

    2015-03-01

    Gomisin J (GJ) is a small molecular weight lignan found in Schisandra chinensis and has been demonstrated to have vasodilatory activity. In this study, the authors investigated the effect of GJ on blood pressure (BP) in angiotensin II (Ang II)-induced hypertensive mice. In addition, we determined the relative potencies of gomisin A (GA) and GJ with respect to vasodilatory activity and antihypertensive effects. C57/BL6 mice infused s.c. with Ang II (2 μg kg(-1) min(-1) for 2 weeks) showed an increase in BP and a decrease in plasma nitric oxide (NO) metabolites. In the thoracic aortas of Ang II-induced hypertensive mice, a decrease in vascular NO was accompanied by an increase in reactive oxygen species (ROS) production. Furthermore, these alterations in BP, plasma concentrations of NO metabolites and in the vascular productions of NO and ROS in Ang II-treated mice were reversed by the co-administration of GJ (1 and 3 μg kg(-1) min(-1)). In in vitro studies, Ang II decreased the cellular concentration of NO, which was accompanied by a reduction in phosphorylated endothelial nitric oxide synthase (eNOS) and an increase in ROS production. These eNOS phosphorylation and ROS production changes in Ang II-treated cells were also reversed by GJ pretreatment (0-3 μg ml(-1)). Interestingly, the vasodilatory and antihypertensive effects of GJ were more prominent than those of GA. Collectively, an increase in BP in mice treated with Ang II was markedly attenuated by GJ, which was attributed to the preservations of vascular NO bioavailability and eNOS function, and to the inhibition of ROS production in Ang II-induced hypertensive mice.

  18. Nitric Oxide Induces Cardiac Protection by Preventing Extracellular Matrix Degradation through the Complex Caveolin-3/EMMPRIN in Cardiac Myocytes.

    PubMed

    Cuadrado, Irene; Castejon, Borja; Martin, Ana M; Saura, Marta; Reventun-Torralba, Paula; Zamorano, Jose Luis; Zaragoza, Carlos

    2016-01-01

    Inhibition of Extracellular Matrix degradation by nitric oxide (NO) induces cardiac protection against coronary ischemia/reperfusion (IR). Glycosylation of Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) stimulates enzymatic activation of matrix metalloproteinases (MMPs) in the heart, although the mechanisms leading to EMMPRIN glycosylation are poorly understood. We sought to determine if NO may induce cardiac protection by preventing glycosylation of EMMPRIN in a mouse model of IR. Here we found that Caveolin-3 binds to low glycosylated EMMPRIN (LG-EMMPRIN) in cardiac cells and in the hearts of healthy mice, whereas IR disrupted the complex in nitric oxide synthase 2 (NOS2) knockout (KO) mice. By contrast, the binding was partially restored when mice were fed with an NO donor (DEA-NO) in the drinking water, showing a significant reduction on infarct size (NOS2KO: 34.6±5 vs NOS2KO+DEA-NO: 20.7±9), in expression of matrix metalloproteinases, and cardiac performance was improved (left ventricular ejection fraction (LVEF). NOS2KO: 31±4 vs NOS2KO+DEA-NO: 46±6). The role of Caveolin-3/EMMPRIN in NO-mediated cardiac protection was further assayed in Caveolin-3 KO mice, showing no significant improvement on infarct size (Caveolin-3 KO: 34.8±3 vs Caveolin-3 KO+DEA-NO:33.7±5), or in the expression of MMPs, suggesting that stabilization of the complex Caveolin-3/LG-EMMPRIN may play a significant role in the cardioprotective effect of NO against IR.

  19. Nitric Oxide Induces Cardiac Protection by Preventing Extracellular Matrix Degradation through the Complex Caveolin-3/EMMPRIN in Cardiac Myocytes

    PubMed Central

    Cuadrado, Irene; Castejon, Borja; Martin, Ana M.; Saura, Marta; Reventun-Torralba, Paula; Zamorano, Jose Luis

    2016-01-01

    Inhibition of Extracellular Matrix degradation by nitric oxide (NO) induces cardiac protection against coronary ischemia/reperfusion (IR). Glycosylation of Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) stimulates enzymatic activation of matrix metalloproteinases (MMPs) in the heart, although the mechanisms leading to EMMPRIN glycosylation are poorly understood. We sought to determine if NO may induce cardiac protection by preventing glycosylation of EMMPRIN in a mouse model of IR. Here we found that Caveolin-3 binds to low glycosylated EMMPRIN (LG-EMMPRIN) in cardiac cells and in the hearts of healthy mice, whereas IR disrupted the complex in nitric oxide synthase 2 (NOS2) knockout (KO) mice. By contrast, the binding was partially restored when mice were fed with an NO donor (DEA-NO) in the drinking water, showing a significant reduction on infarct size (NOS2KO: 34.6±5 vs NOS2KO+DEA-NO: 20.7±9), in expression of matrix metalloproteinases, and cardiac performance was improved (left ventricular ejection fraction (LVEF). NOS2KO: 31±4 vs NOS2KO+DEA-NO: 46±6). The role of Caveolin-3/EMMPRIN in NO-mediated cardiac protection was further assayed in Caveolin-3 KO mice, showing no significant improvement on infarct size (Caveolin-3 KO: 34.8±3 vs Caveolin-3 KO+DEA-NO:33.7±5), or in the expression of MMPs, suggesting that stabilization of the complex Caveolin-3/LG-EMMPRIN may play a significant role in the cardioprotective effect of NO against IR. PMID:27649573

  20. Folic Acid Promotes Recycling of Tetrahydrobiopterin and Protects Against Hypoxia-Induced Pulmonary Hypertension by Recoupling Endothelial Nitric Oxide Synthase

    PubMed Central

    Chalupsky, Karel; Kračun, Damir; Kanchev, Ivan; Bertram, Katharina

    2015-01-01

    Abstract Aims: Nitric oxide (NO) derived from endothelial NO synthase (eNOS) has been implicated in the adaptive response to hypoxia. An imbalance between 5,6,7,8-tetrahydrobiopterin (BH4) and 7,8-dihydrobiopterin (BH2) can result in eNOS uncoupling and the generation of superoxide instead of NO. Dihydrofolate reductase (DHFR) can recycle BH2 to BH4, leading to eNOS recoupling. However, the role of DHFR and eNOS recoupling in the response to hypoxia is not well understood. We hypothesized that increasing the capacity to recycle BH4 from BH2 would improve NO bioavailability as well as pulmonary vascular remodeling (PVR) and right ventricular hypertrophy (RVH) as indicators of pulmonary hypertension (PH) under hypoxic conditions. Results: In human pulmonary artery endothelial cells and murine pulmonary arteries exposed to hypoxia, eNOS was uncoupled as indicated by reduced superoxide production in the presence of the nitric oxide synthase inhibitor, L-(G)-nitro-L-arginine methyl ester (L-NAME). Concomitantly, NO levels, BH4 availability, and expression of DHFR were diminished under hypoxia. Application of folic acid (FA) restored DHFR levels, NO bioavailability, and BH4 levels under hypoxia. Importantly, FA prevented the development of hypoxia-induced PVR, right ventricular pressure increase, and RVH. Innovation: FA-induced upregulation of DHFR recouples eNOS under hypoxia by improving BH4 recycling, thus preventing hypoxia-induced PH. Conclusion: FA might serve as a novel therapeutic option combating PH. Antioxid. Redox Signal. 23, 1076–1091. PMID:26414244

  1. Secondary stimulation from Bacillus Calmette-Guérin induced macrophages induce nitric oxide independent cell-death in bladder cancer cells.

    PubMed

    Thiel, Tomas; Ryk, Charlotta; Chatzakos, Vicky; Hallén Grufman, Katarina; Bavand-Chobot, Nasrin; Flygare, Jenny; Wiklund, N Peter; de Verdier, Petra J

    2014-06-28

    The anti-tumour mechanisms following Bacillus Calmette-Guérin (BCG) treatment of bladder-cancer remain largely unknown. Previous studies have shown involvement of nitric-oxide (NO) formation in the BCG-mediated effect. We analyzed the effects of macrophage secreted factors (MSFs) from BCG-stimulated RAW264.7 cells on the bladder-cancer cell line MBT2. Direct treatment with BCG did not induce NO in MBT2-cells whereas supernatant from BCG-stimulated macrophages increased NOS2 mRNA and protein expression, NO concentrations and cell-death. Blocking NO-synthesis with the NOS-inhibitor L-NAME did not affect levels of cell-death suggesting cytotoxic pathways involving other signalling molecules than NO. Several such candidate genes were identified in a microarray.

  2. Reduced nitric oxide-mediated relaxation and endothelial nitric oxide synthase expression in the tail arteries of streptozotocin-induced diabetic rats.

    PubMed

    Mokhtar, Siti Safiah; Vanhoutte, Paul M; Leung, Susan Wai Sum; Suppian, Rapeah; Yusof, Mohd Imran; Rasool, Aida Hanum Ghulam

    2016-02-15

    Diabetes is associated with endothelial dysfunction, which is characterized by impaired endothelium-dependent relaxations. The present study aimed to examine the role of nitric oxide (NO), prostacyclin and endothelium-dependent hyperpolarization (EDH), in the relaxation of ventral tail arteries of rats under diabetic conditions. Relaxations of tail arteries of control and diabetic rats were studied in wire myograph. Western blotting and immunostaining were used to determine the presence of proteins. Acetylcholine-induced relaxations were significantly smaller in arteries of diabetic compared to control rats (Rmax; 70.81 ± 2.48% versus 85.05 ± 3.15%). Incubation with the combination of non-selective cyclooxygenase (COX) inhibitor, indomethacin and potassium channel blockers, TRAM 34 and UCL 1684, demonstrated that NO-mediated relaxation was attenuated significantly in diabetic compared to control rats (Rmax; 48.47 ± 5.84% versus 68.39 ± 6.34%). EDH-type (in the presence of indomethacin and NO synthase inhibitor, LNAME) and prostacyclin-mediated (in the presence of LNAME plus TRAM 34 and UCL 1684) relaxations were not significantly reduced in arteries of diabetic compared to control rats [Rmax: (EDH; 17.81 ± 6.74% versus 34.16 ± 4.59%) (prostacyclin; 15.85 ± 3.27% versus 17.23 ± 3.75%)]. Endothelium-independent relaxations to sodium nitroprusside, salbutamol and prostacyclin were comparable in the two types of preparations. Western blotting and immunostaining indicated that diabetes diminished the expression of endothelial NO synthase (eNOS), while increasing those of COX-1 and COX-2. Thus, since acetylcholine-induced NO-mediated relaxation was impaired in diabetes because of reduced eNOS protein expression, pharmacological intervention improving NO bioavailability could be useful in the management of diabetic endothelial dysfunction.

  3. In vivo footprinting of the mouse inducible nitric oxide synthase gene: inducible protein occupation of numerous sites including Oct and NF-IL6.

    PubMed Central

    Goldring, C E; Reveneau, S; Algarté, M; Jeannin, J F

    1996-01-01

    A wide variety of cells usefully but sometimes destructively produce nitric oxide via inducible nitric oxide synthase (iNOS). Data obtained by gel shift analysis and reporter assays have linked murine iNOS gene induction by cytokines and bacterial products with the binding of a number of proteins to a proximal promoter, as well as to a distal enhancer of the iNOS gene. Nevertheless, these techniques do not necessarily reflect protein occupation of sites in vivo. To address this, we have used dimethyl sulphate in vivo footprinting to determine binding events in the two murine iNOS transcription control regions, using a classical lipopolysaccharide induction of RAW 264.7 macrophages. Protein-DNA interactions are absent before activation. Exposure to lipopolysaccharide induces protection at a NF-kappaB site and hypersensitivity at a shared gamma-activated site/interferon-stimulated response element within the enhancer. Protections are seen at a NF-IL6, and an Oct site within the promoter. We also observe modulations in guanine methylation at two regions which do not correspond to any known putative binding elements. Furthermore, we confirm the probable involvement of interferon regulatory factor-1 (binding to its -901 to -913 site) and the binding of NF-kappaB to its proximal site. Our data demonstrate an abundance of hitherto-unrecognised protein-DNA binding events upon simple lipopolysaccharide activation of the iNOS gene and suggests a role for protein-protein interactions in its transcriptional induction. PMID:8649986

  4. Role of oxidative stress, inflammation, nitric oxide and transforming growth factor-beta in the protective effect of diosgenin in monocrotaline-induced pulmonary hypertension in rats.

    PubMed

    Ahmed, Lamiaa A; Obaid, Al Arqam Z; Zaki, Hala F; Agha, Azza M

    2014-10-05

    Pulmonary hypertension is a progressive disease of various origins that is associated with right ventricular dysfunction. In the present study, the protective effect of diosgenin was investigated in monocrotaline-induced pulmonary hypertension in rats. Pulmonary hypertension was induced by a single subcutaneous injection of monocrotaline (60 mg/kg). Diosgenin (100 mg/kg) was given by oral administration once daily for 3 weeks. At the end of the experiment, mean arterial blood pressure, electrocardiography and echocardiography were recorded. Rats were then sacrificed and serum was separated for determination of total nitrate/nitrite level. Right ventricles and lungs were isolated for estimation of oxidative stress markers, tumor necrosis factor-alpha, total nitrate/nitrite and transforming growth factor-beta contents. Myeloperoxidase and caspase-3 activities in addition to endothelial and inducible nitric oxide synthase protein expression were also determined. Moreover, histological analysis of pulmonary arteries and cardiomyocyte cross-sectional area was performed. Diosgenin treatment provided a significant improvement toward preserving hemodynamic changes and alleviating oxidative stress, inflammatory and apoptotic markers induced by monocrotaline in rats. Furthermore, diosgenin therapy prevented monocrotaline-induced changes in nitric oxide production, endothelial and inducible nitric oxide synthase protein expression as well as histological analysis. These findings support the beneficial effect of diosgenin in pulmonary hypertension induced by monocrotaline in rats.

  5. Binding of CAP70 to Inducible Nitric Oxide Synthase and Implications for the Vectorial Release of Nitric Oxide in Polarized Cells

    PubMed Central

    Navarro-Lérida, Inmaculada; Martínez-Moreno, Mónica; Ventoso, Iván; Álvarez-Barrientos, Alberto

    2007-01-01

    In this article we analyze the mechanisms by which the C-terminal four amino acids of inducible nitric oxide synthase (NOS2) interact with proteins that contain PDZ (PSD-95/DLG/ZO-1) domains resulting in the translocation of NOS2 to the cellular apical domain. It has been reported that human hepatic NOS2 associates to EBP50, a protein with two PDZ domains present in epithelial cells. We describe herein that NOS2 binds through its four carboxy-terminal residues to CAP70, a protein that contains four PDZ modules that is targeted to apical membranes. Interestingly, this interaction augments both the cytochrome c reductase and ·NO-synthase activities of NOS2. Binding of CAP70 to NOS2 also results in an increase in the population of active NOS2 dimers. In addition, CAP70 participates in the correct subcellular targeting of NOS2 in a process that is also dependent on the acylation state of the N-terminal end of NOS2. Hence, nonpalmitoylated NOS2 is unable to progress toward the apical side of the cell despite its interaction with either EBP50 or CAP70. Likewise, if we abrogate the interaction of NOS2 with either EBP50 or CAP70 by fusing the GFP reporter to the carboxy-terminal end of NOS2 palmitoylation is not sufficient to confer an apical targeting. PMID:17507652

  6. Renal Nitric Oxide Synthase and Antioxidant Preservation in Cyp1a1-Ren-2 Transgenic Rats With Inducible Malignant Hypertension

    PubMed Central

    2013-01-01

    BACKGROUND Dietary administration of 0.30% indole-3-carbinol (I3C) to Cyp1a1-Ren2 transgenic rats (TGRs) generates angiotensin II (ANG II)–dependent malignant hypertension (HTN) and increased renal vascular resistance. However, TGRs with HTN maintain a normal or slightly reduced glomerular filtration rate. We tested the hypothesis that maintenance of renal function in hypertensive Cyp1a1-Ren2 TGRs is due to preservation of the intrarenal nitric oxide (NO) and antioxidant systems. METHODS Kidney cortex, kidney medulla, aortic endothelial (e) and neuronal (n) nitric oxide synthase (NOS), superoxide dismutases (SODs), and p22phox (nicotinamide adenine dinucleotide phosphate-oxidase subunit) protein abundances were measured along with kidney cortex total antioxidant capacity (TAC) and NOx. TGRs were fed a normal diet that contained 0.3% I3C or 0.3% I3C + candesartan (AT1 receptor antagonist; 25mg/L in drinking water) (n = 5–6 per group) for 10 days. RESULTS Blood pressure increased and body weight decreased in I3C-induced TGRs, while candesartan blunted these responses. Abundances of NOS, SOD, and p22phox as well as TAC were maintained in the kidney cortex of I3C-induced TGRs with and without candesartan, while kidney cortex NOx production increased in both groups. Kidney medulla eNOS and extracellular (EC) SOD decreased and nNOS were unchanged in both groups of I3C-induced TGRs. In addition, a compensatory increase occurred in kidney medulla Mn SOD in I3C-induced TGRs + candesartan. Aortic eNOS and nNOS∝ fell and p22phox and Mn SOD increased in hypertensive I3C-induced TGRs; all changes were reversed with candesartan. CONCLUSIONS The preservation of renal cortical NO and antioxidant capacity is associated with preserved renal function in Cyp1a1-Ren2 TGRs with ANG II-dependent malignant HTN. PMID:23764378

  7. Nicotine improves ethanol-induced impairment of memory: possible involvement of nitric oxide in the dorsal hippocampus of mice.

    PubMed

    Raoufi, N; Piri, M; Moshfegh, A; Shahin, M-S

    2012-09-06

    In the present study, the possible involvement of nitric oxide (NO) systems in the dorsal hippocampus in nicotine's effect on ethanol-induced amnesia and ethanol state-dependent memory was investigated. Adult male mice were cannulated in the CA1 regions of the dorsal hippocampus and trained on a passive avoidance learning task for memory assessment. We found that pre-training intraperitoneal (i.p.) administration of ethanol (1 g/kg) decreased inhibitory avoidance memory when tested 24 h later. The response induced by pre-training ethanol was significantly reversed by pre-test administration of the drug. Similar to ethanol, pre-test administration of nicotine (0.4 and 0.8 μg/mouse, intra-CA1) alone and nicotine (0.2, 0.4 and 0.8 μg/mouse) plus an ineffective dose of ethanol also significantly reversed the amnesia induced by ethanol. Ethanol amnesia was also prevented by pre-test administration of L-arginine (1.2 μg/mouse, intra-CA1), a NO precursor. Interestingly, an ineffective dose of nicotine (0.2 μg/mouse) in combination with a low dose of L-arginine (0.8 μg/mouse) synergistically improved memory performance impaired by ethanol given before training. In contrast, pre-test intra-CA1 microinjection of L-NAME (NG-nitro-L-arginine methyl ester), a nitric oxide synthase (NOS) inhibitor (0.4 and 0.8 μg/mouse), which reduced memory retrieval in inhibitory avoidance task by itself, in combination with an effective dose of nicotine (0.4 μg/mouse) prevented the improving effect of nicotine on memory impaired by pre-training ethanol. Moreover, intra-CA1 microinjection of L-NAME reversed the L-arginine-induced potentiation of the nicotine response. The results suggest the importance of NO system(s) in the CA1 regions of the dorsal hippocampus for improving the effect of nicotine on the ethanol-induced amnesia.

  8. Effect of magnesium supplementation on blood pressure and vascular reactivity in nitric oxide synthase inhibition-induced hypertension model.

    PubMed

    Basralı, Filiz; Koçer, Günnur; Ülker Karadamar, Pınar; Nasırcılar Ülker, Seher; Satı, Leyla; Özen, Nur; Özyurt, Dilek; Şentürk, Ümit Kemal

    2015-01-01

    The aim of this study was to assess the effect of oral magnesium supplementation (Mg-supp) on blood pressure (BP) and possible mechanism in nitric oxide synthase (NOS) inhibition-induced hypertension model. Hypertension and/or Mg-supp were created by N-nitro-l-arginine methyl ester (25 mg/kg/day by drinking water) and magnesium-oxide (0.8% by diet) for 6 weeks. Systolic BP was measured weekly by tail-cuff method. The effects of hypertension and/or Mg-supp in thoracic aorta and third branch of mesenteric artery constriction and relaxation responses were evaluated. NOS-inhibition produced a gradually developing hypertension and the magnitude of the BP was significantly attenuated after five weeks of Mg-supp. The increased phenylephrine-induced contractile and decreased acetylcholine (ACh)-induced dilation responses were found in both artery segments of hypertensive groups. Mg-supp was restored ACh-relaxation response in both arterial segments and also Phe-constriction response in thoracic aorta but not in mesenteric arteries. The contributions of NO, prostaglandins and K(+) channels to the dilator response of ACh were similar in the aorta of all the groups. The contribution of the NO to the ACh-mediated relaxation response of mesenteric arteries was suppressed in hypertensive rats, whereas this was corrected by Mg-supp. The flow-mediated dilation response of mesenteric arteries in hypertensive rats failed and could not be corrected by Mg-supp. Whereas, vascular eNOS protein and magnesium levels were not changed and plasma nitrite levels were reduced in hypertensive rats. The results of this study showed that Mg-supp lowered the arterial BP in NOS-inhibition induced hypertension model by restoring the agonist-induced relaxation response of the arteries.

  9. Endothelial nitric oxide synthase is protective in the initiation of caerulein-induced acute pancreatitis in mice.

    PubMed

    DiMagno, Matthew J; Williams, John A; Hao, Yibai; Ernst, Stephen A; Owyang, Chung

    2004-07-01

    The effect of inhibiting nitric oxide (NO) synthase (NOS) or enhancing NO on the course of acute pancreatitis (AP) is controversial, in part because three NOS isoforms exist: neuronal (nNOS), endothelial (eNOS), and inducible (iNOS). We investigated whether inhibition or selective gene deletion of NOS isoforms modified the initiation phase of caerulein-induced AP in mice and explored whether this affected pancreatic microvascular blood flow (PMBF). We investigated the effects of nonspecific NOS inhibition with N(omega)-nitro-l-arginine (l-NNA; 10 mg/kg ip) or targeted deletion of eNOS, nNOS, or iNOS genes on the initiation phase of caerulein-induced AP in mice using in vivo and in vitro models. Western blot analysis was performed to assess eNOS phosphorylation status, an indicator of enzyme activity, and microsphere studies were used to measure PMBF. l-NNA and eNOS deletion, but not nNOS or iNOS deletion, increased pancreatic trypsin activity and serum lipase during the initiation phase of in vivo caerulein-induced AP. l-NNA and eNOS did not affect trypsin activity in caerulein-hyperstimulated isolated acini, suggesting that nonacinar events mediate the effect of NOS blockade in vivo. The initiation phase of AP in wild-type mice was associated with eNOS Thr(495) residue dephosphorylation, which accompanies eNOS activation, and a 178% increase in PMBF; these effects were absent in eNOS-deleted mice. Thus eNOS is the main isoform influencing the initiation of caerulein-induced AP. eNOS-derived NO exerts a protective effect through actions on nonacinar cell types, most likely endothelial cells, to produce greater PMBF.

  10. Cordycepin induces human lung cancer cell apoptosis by inhibiting nitric oxide mediated ERK/Slug signaling pathway

    PubMed Central

    Hwang, Jung Hoo; Park, Soo Jung; Ko, Won Gyu; Kang, Seong-Mun; Lee, Da Bin; Bang, Junho; Park, Byung-Joo; Wee, Chung-Beum; Kim, Dae Joon; Jang, Ik-Soon; Ko, Jae-Hong

    2017-01-01

    Nitric oxide (NO) is an important signaling molecule and a component of the inflammatory cascade. Besides, it is also involved in tumorigenesis. Aberrant upregulation and activation of the ERK cascade by NO often leads to tumor cell development. However, the role of ERK inactivation induced by the negative regulation of NO during apoptosis is not completely understood. In this study, treatment of A549 and PC9 human lung adenocarcinoma cell lines with cordycepin led to a reduction in their viability. Analysis of the effect of cordycepin treatment on ERK/Slug signaling activity in the A549 cell line revealed that LPS-induced inflammatory microenvironments could stimulate the expression of TNF-α, CCL5, IL-1β, IL-6, IL-8 and upregulate NO, phospho-ERK (p-ERK), and Slug expression. In addition, constitutive expression of NO was observed. Cordycepin inhibited LPS-induced stimulation of iNOS, NO, p-ERK, and Slug expression. L-NAME, an inhibitor of NOS, inhibited p-ERK and Slug expression. It was also found that cordycepin-mediated inhibition of ERK downregulated Slug, whereas overexpression of ERK led to an upregulation of Slug levels in the cordycepin-treated A549 cells. Inhibition of Slug by siRNA induced Bax and caspase-3, leading to cordycepin-induced apoptosis. Cordycepin-mediated inhibition of ERK led to a reduction in phospho-GSK3β (p-GSK3β) and Slug levels, whereas LiCl, an inhibitor of GSK3β, upregulated p-GSK3β and Slug. Overall, the results obtained indicate that cordycepin inhibits the ERK/Slug signaling pathway through the activation of GSK3β which, in turn, upregulates Bax, leading to apoptosis of the lung cancer cells.

  11. Nitric Oxide Deficiency Accelerates Chlorophyll Breakdown and Stability Loss of Thylakoid Membranes during Dark-Induced Leaf Senescence in Arabidopsis

    PubMed Central

    Liu, Fang; Guo, Fang-Qing

    2013-01-01

    Nitric oxide (NO) has been known to preserve the level of chlorophyll (Chl) during leaf senescence. However, the mechanism by which NO regulates Chl breakdown remains unknown. Here we report that NO negatively regulates the activities of Chl catabolic enzymes during dark-induced leaf senescence. The transcriptional levels of the major enzyme genes involving Chl breakdown pathway except for RED CHL CATABOLITE REDUCTASE (RCCR) were dramatically up-regulated during dark-induced Chl degradation in the leaves of Arabidopsis NO-deficient mutant nos1/noa1 that exhibited an early-senescence phenotype. The activity of pheide a oxygenase (PAO) was higher in the dark-induced senescent leaves of nos1/noa1 compared with wild type. Furthermore, the knockout of PAO in nos1/noa1 background led to pheide a accumulation in the double mutant pao1 nos1/noa1, which retained the level of Chl during dark-induced leaf senescence. The accumulated pheide a in darkened leaves of pao1 nos1/noa1 was likely to inhibit the senescence-activated transcriptional levels of Chl catabolic genes as a feed-back inhibitory effect. We also found that NO deficiency led to decrease in the stability of photosynthetic complexes in thylakoid membranes. Importantly, the accumulation of pheide a caused by PAO mutations in combination with NO deficiency had a synergistic effect on the stability loss of thylakoid membrane complexes in the double mutant pao1 nos1/noa1 during dark-induced leaf senescence. Taken together, our findings have demonstrated that NO is a novel negative regulator of Chl catabolic pathway and positively functions in maintaining the stability of thylakoid membranes during leaf senescence. PMID:23418559

  12. Nitric oxide-induced biphasic mechanism of vascular relaxation via dephosphorylation of CPI-17 and MYPT1.

    PubMed

    Kitazawa, Toshio; Semba, Shingo; Huh, Yang Hoon; Kitazawa, Kazuyo; Eto, Masumi

    2009-07-15

    Nitric oxide (NO) from endothelium is a major mediator of vasodilatation through cGMP/PKG signals that lead to a decrease in Ca(2+) concentration. In addition, NO-mediated signals trigger an increase in myosin light chain phosphatase (MLCP) activity. To evaluate the mechanism of NO-induced relaxation through MLCP deinhibition, we compared time-dependent changes in Ca(2+), myosin light chain (MLC) phosphorylation and contraction to changes in phosphorylation levels of CPI-17 at Thr38, RhoA at Ser188, and MYPT1 at Ser695, Thr696 and Thr853 in response to sodium nitroprusside (SNP)-induced relaxation in denuded rabbit femoral artery. During phenylephrine (PE)-induced contraction, SNP reduced CPI-17 phosphorylation to a minimal value within 15 s, in parallel with decreases in Ca(2+) and MLC phosphorylation, followed by a reduction of contractile force having a latency period of about 15 s. MYPT1 phosphorylation at Ser695, the PKG-target site, increased concurrently with relaxation. Phosphorylation of RhoA, MYPT1 Thr696 and Thr853 differed significantly at 5 min but not within 1 min of SNP exposure. Inhibition of Ca(2+) release delayed SNP-induced relaxation while inhibition of Ca(2+) channel, BK(Ca) channel or phosphodiesterase-5 did not. Pretreatment of resting artery with SNP suppressed an increase in Ca(2+), contractile force and phosphorylation of MLC, CPI-17, MYPT1 Thr696 and Thr853 at 10 s after PE stimulation, but had no effect on phorbol ester-induced CPI-17 phosphorylation. Together, these results suggest that NO production suppresses Ca(2+) release, which causes an inactivation of PKC and rapid CPI-17 dephosphorylation as well as MLCK inactivation, resulting in rapid MLC dephosphorylation and relaxation.

  13. Pharmacological inhibition of inducible nitric oxide synthase (iNOS) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, convalesce behavior and biochemistry of hypertension induced vascular dementia in rats.

    PubMed

    Sharma, Bhupesh; Singh, Nirmal

    2013-02-01

    Cognitive disorders are likely to increase over the coming years (5-10). Vascular dementia (VaD) has heterogeneous pathology and is a challenge for clinicians. Current Alzheimer's disease drugs have had limited clinical efficacy in treating VaD and none have been approved by major regulatory authorities specifically for this disease. Role of iNOS and NADPH-oxidase has been reported in various pathological conditions but there role in hypertension (Hypt) induced VaD is still unclear. This research work investigates the salutiferous effect of aminoguanidine (AG), an iNOS inhibitor and 4'-hydroxy-3'-methoxyacetophenone (HMAP), a NADPH oxidase inhibitor in Hypt induced VaD in rats. Deoxycorticosterone acetate-salt (DOCA-S) hypertension has been used for development of VaD in rats. Morris water-maze was used for testing learning and memory. Vascular system assessment was done by testing endothelial function. Mean arterial blood pressure (MABP), oxidative stress [aortic superoxide anion, serum and brain thiobarbituric acid reactive species (TBARS) and brain glutathione (GSH)], nitric oxide levels (serum nitrite/nitrate) and cholinergic activity (brain acetyl cholinesterase activity-AChE) were also measured. DOCA-S treated rats have shown increased MABP with impairment of endothelial function, learning and memory, reduction in serum nitrite/nitrate & brain GSH levels along with increase in serum & brain TBARS, and brain AChE activity. AG as well as HMAP significantly convalesce Hypt induced impairment of learning, memory, endothelial function, and alterations in various biochemical parameters. It may be concluded that AG, an iNOS inhibitor and HMAP, a NADPH-oxidase inhibitor may be considered as potential agents for the management of Hypt induced VaD.

  14. Diclofenac enhances proinflammatory cytokine-induced phagocytosis of cultured microglia via nitric oxide production

    SciTech Connect

    Kakita, Hiroki; Aoyama, Mineyoshi; Nagaya, Yoshiaki; Asai, Hayato; Hussein, Mohamed Hamed; Suzuki, Mieko; Kato, Shin; Saitoh, Shinji; Asai, Kiyofumi

    2013-04-15

    Influenza-associated encephalopathy (IAE) is a central nervous system complication with a high mortality rate, which is increased significantly by the non-steroidal anti-inflammatory drug diclofenac sodium (DCF). In the present study, we investigated the effects of DCF on brain immune cells (i.e. microglia) stimulated with three proinflammatory cytokines, namely tumor necrosis factor-α, interleukin-1β, and interferon-γ. Similar to previous findings in astrocytes, all three cytokines induced the expression of inducible NO synthase (iNOS), as well as NO production, in microglia. The addition of DCF to the culture system augmented iNOS expression and NO production. Immunocytochemical analysis and the phagocytosis assay revealed that cytokine treatment induced morphological changes to and phagocytosis by the microglia. The addition of DCF to the culture system enhanced microglial activation, as well as the phagocytic activity of cytokine-stimulated microglia. Inhibitors of nuclear factor (NF)-κB inhibited iNOS gene expression in cytokine-stimulated microglia with or without DCF, suggesting that the NF-κB pathway is one of the main signaling pathways involved. The iNOS inhibitor N{sup G}-monomethyl-L-arginine (L-NMMA) reduced both cytokine-induced phagocytosis and phagocytosis induced by the combination of cytokines plus DCF. Furthermore, the NO donor sodium nitroprusside induced phagocytosis, indicating that NO production is a key regulator of microglial phagocytosis. In conclusion, DCF acts synergistically with proinflammatory cytokines to increase the production of NO in microglia, leading to phagocytic activity of the activated microglia. These findings, together with previous observations regarding astrocytes, may explain the significant increase in mortality of IAE patients treated with DCF. - Highlights: ► Influenza-associated encephalopathy (IAE) is associated with a high mortality rate. ► Hyperimmunization in the brain is believed to be responsible for

  15. The vitamin D receptor and inducible nitric oxide synthase associated pathways in acquired resistance to Cooperia oncophora infection in cattle

    PubMed Central

    2011-01-01

    Cooperia oncophora is an economically important gastrointestinal nematode in ruminants. Acquired resistance to Cooperia oncophora infection in cattle develops rapidly as a result of prior infections. Naïve cattle, when given a primary infection of high-dose infective L3 larvae, develop a strong immunity to subsequent reinfection. Compared to primary infection, reinfection resulted in a marked reduction in worm establishment. In order to understand molecular mechanisms underlying the development of acquired resistance, we characterized the transcriptomic responses of the bovine small intestine to a primary infection and reinfection. A total of 23 pathways were significantly impacted during infection. The vitamin D receptor activation was strongly induced only during reinfection, suggesting that this pathway may play an important role in the development of acquired resistance via its potential roles in immune regulation and intestinal mucosal integrity maintenance. The expression of inducible nitric oxide synthase (NOS2) was strongly induced during reinfection but not during primary infection. As a result, several canonical pathways associated with NOS2 were impacted. The genes involved in eicosanoid synthesis, including prostaglandin synthase 2 (PTGS2 or COX2), remained largely unchanged during infection. The rapid development of acquired resistance may help explain the lack of relative pathogenicity by Cooperia oncophora infection in cattle. Our findings facilitate the understanding of molecular mechanisms underlying the development of acquired resistance, which could have an important implication in vaccine design. PMID:21414188

  16. Transcriptional basis for hyporesponsiveness of the human inducible nitric oxide synthase gene to lipopolysaccharide/interferon-gamma.

    PubMed

    Zhang, X; Laubach, V E; Alley, E W; Edwards, K A; Sherman, P A; Russell, S W; Murphy, W J

    1996-04-01

    The work reported here resolves, at the level of gene regulation, the controversy as to whether or not human monocytes/macrophages can produce nitric oxide (NO) when stimulated with lipopolysaccharide (LPS), with or without co-stimulation by interferon-gamma (IFN-gamma). Studies included structural comparison of the promoters for human and mouse inducible NO synthase (iNOS) genes, transfection and assay of human and mouse iNOS promoter regions in response to LPS +/- IFN-gamma, and electrophoretic mobility shift assays of kappa B response elements. Two explanations for hyporesponsiveness of the human iNOS promoter to LPS +/- IFN-gamma were found: (1) multiple inactivating nucleotide substitutions in the human counterpart of the enhancer element that has been shown to regulate LPS/IFN-gamma induced expression of the mouse iNOS gene; and (2) and absence of one or more nuclear factors in human macrophages (e.g., an LPS-inducible nuclear factor-kappa B/Rel complex), that is (are) required for maximal expression of the gene. The importance of resolution of this controversy is that future research in this area should be directed toward the understanding of alternative mechanisms that can result in the successful production of NO.

  17. Nitric oxide mediates effects of acute, not chronic, naltrexone on LPS-induced hepatic encephalopathy in cirrhotic rats.

    PubMed

    Ghiassy, Bentolhoda; Rahimi, Nastaran; Javadi-Paydar, Mehrak; Gharedaghi, Mohammad Hadi; Norouzi-Javidan, Abbas; Dehpour, Ahmad R

    2017-01-01

    Recent studies suggest endogenous opioids and nitric oxide (NO) are involved in the pathophysiology of hepatic encephalopathy (HE). In this study, the interaction between the opioid receptor antagonist and NO was investigated on lipopolysaccharide (LPS)-induced HE in cirrhotic rats. Male rats were divided in the sham- and bile duct ligation (BDL)-operated groups. Animals were treated with saline; naltrexone (10 mg/kg, i.p.); or L-NAME (3 mg/kg, i.p.), alone or in combination with naltrexone. To induce HE, LPS (1 mg/kg, i.p.) was injected 1 h after the final drug treatment. HE scoring, hepatic histology, and plasma NO metabolites levels and mortality rate were recorded. Deteriorated level of consciousness and mortality after LPS administration significantly ameliorated following both acute and chronic treatment with naltrexone in cirrhotic rats. However, acute and chronic administration of L-NAME did not change HE scores in cirrhotic rats. The effects of acute but not chronic treatment of naltrexone on HE parameters were reversed by L-NAME. Plasma NOx concentrations elevated in BDL rats, which were decreased after acute and chronic treatment by naltrexone or L-NAME, significantly. We suggest both acute and chronic treatment with naltrexone improved LPS-induced HE. But, only acute treatment with naltrexone may affect through NO pathway.

  18. Hydrogen-rich water alleviates aluminum-induced inhibition of root elongation in alfalfa via decreasing nitric oxide production.

    PubMed

    Chen, Meng; Cui, Weiti; Zhu, Kaikai; Xie, Yanjie; Zhang, Chunhua; Shen, Wenbiao

    2014-02-28

    One of the earliest and distinct symptoms of aluminum (Al) toxicity is the inhibition of root elongation. Although hydrogen gas (H2) is recently described as an important bio-regulator in plants, whether and how H2 regulates Al-induced inhibition of root elongation is largely unknown. To address these gaps, hydrogen-rich water (HRW) was used to investigate a physiological role of H2 and its possible molecular mechanism. Individual or simultaneous (in particular) exposure of alfalfa seedlings to Al, or a fresh but not old nitric oxide (NO)-releasing compound sodium nitroprusside (SNP), not only increased NO production, but also led to a significant inhibition of root elongation. Above responses were differentially alleviated by pretreatment with 50% saturation of HRW. The addition of HRW also alleviated the appearance of Al toxicity symptoms, including the improvement of seedling growth and less accumulation of Al. Subsequent results revealed that the removal of NO by the NO scavenger, similar to HRW, could decrease NO production and alleviate Al- or SNP-induced inhibition of root growth. Thus, we proposed that HRW alleviated Al-induced inhibition of alfalfa root elongation by decreasing NO production. Such findings may be applicable to enhance crop yield and improve stress tolerance.

  19. Nitric oxide and brassinosteroids mediated fungal endophyte-induced volatile oil production through protein phosphorylation pathways in Atractylodes lancea plantlets.

    PubMed

    Ren, Cheng-Gang; Dai, Chuan-Chao

    2013-11-01

    Fungal endophytes have been isolated from almost every plant, infecting their hosts without causing visible disease symptoms, and yet have still proved to be involved in plant secondary metabolites accumulation. To decipher the possible physiological mechanisms of the endophytic fungus-host interaction, the role of protein phosphorylation and the relationship between endophytic fungus-induced kinase activity and nitric oxide (NO) and brassinolide (BL) in endophyte-enhanced volatile oil accumulation in Atractylodes lancea plantlets were investigated using pharmacological and biochemical approaches. Inoculation with the endophytic fungus Gilmaniella sp. AL12 enhanced the activities of total protein phosphorylation, Ca²⁺-dependent protein kinase, and volatile oil accumulation in A. lancea plantlets. The upregulation of protein kinase activity could be blocked by the BL inhibitor brassinazole. Furthermore, pretreatments with the NO-specific scavenger cPTIO significantly reduced the increased activities of protein kinases in A. lancea plantlets inoculated with endophytic fungus. Pretreatments with different protein kinase inhibitors also reduced fungus-induced NO production and volatile oil accumulation, but had barely no effect on the BL level. These data suggest that protein phosphorylation is required for endophyte-induced volatile oil production in A. lancea plantlets, and that crosstalk between protein phosphorylation and the NO pathway may occur and act as a downstream signaling event of the BL pathway.

  20. In site bioimaging of hydrogen sulfide uncovers its pivotal role in regulating nitric oxide-induced lateral root formation.

    PubMed

    Li, Yan-Jun; Chen, Jian; Xian, Ming; Zhou, Li-Gang; Han, Fengxiang X; Gan, Li-Jun; Shi, Zhi-Qi

    2014-01-01

    Hydrogen sulfide (H2S) is an important gasotransmitter in mammals. Despite physiological changes induced by exogenous H2S donor NaHS to plants, whether and how H2S works as a true cellular signal in plants need to be examined. A self-developed specific fluorescent probe (WSP-1) was applied to track endogenous H2S in tomato (Solanum lycopersicum) roots in site. Bioimaging combined with pharmacological and biochemical approaches were used to investigate the cross-talk among H2S, nitric oxide (NO), and Ca(2+) in regulating lateral root formation. Endogenous H2S accumulation was clearly associated with primordium initiation and lateral root emergence. NO donor SNP stimulated the generation of endogenous H2S and the expression of the gene coding for the enzyme responsible for endogenous H2S synthesis. Scavenging H2S or inhibiting H2S synthesis partially blocked SNP-induced lateral root formation and the expression of lateral root-related genes. The stimulatory effect of SNP on Ca(2+) accumulation and CaM1 (calmodulin 1) expression could be abolished by inhibiting H2S synthesis. Ca(2+) chelator or Ca(2+) channel blocker attenuated NaHS-induced lateral root formation. Our study confirmed the role of H2S as a cellular signal in plants being a mediator between NO and Ca(2+) in regulating lateral root formation.

  1. Benzo[a]pyrene-induced nitric oxide production acts as a survival signal targeting mitochondrial membrane potential.

    PubMed

    Hardonnière, Kévin; Huc, Laurence; Podechard, Normand; Fernier, Morgane; Tekpli, Xavier; Gallais, Isabelle; Sergent, Odile; Lagadic-Gossmann, Dominique

    2015-10-01

    Benzo[a]pyrene (B[a]P), the prototype molecule of polycyclic aromatic hydrocarbons, exhibits genotoxic and carcinogenic effects, which has led the International Agency for Research on Cancer to recognize it as a human carcinogen. Besides the well-known apoptotic signals triggered by B[a]P, survival signals have also been suggested to occur, both signals likely involved in cancer promotion. Our previous work showed that B[a]P induced an hyperpolarization of mitochondrial membrane potential (ΔΨm) in rat hepatic epithelial F258 cells. Elevated ΔΨm plays a role in tumor development and progression, and nitric oxide (NO) has been suggested to be responsible for increases in ΔΨm. The present study therefore aimed at evaluating the impact of B[a]P on NO level in F258 cells, and at testing the putative role for NO as a survival signal, notably in link with ΔΨm. Our data demonstrated that B[a]P exposure resulted in an NO production which was dependent upon the activation of the inducible NO synthase. This enzyme activation involved AhR and possibly p53 activation. Preventing NO production not only increased B[a]P-induced cell death but also blocked mitochondrial hyperpolarization. This therefore points to a role for NO as a survival signal upon B[a]P exposure, possibly targeting ΔΨm.

  2. The Nitric oxide/CGMP/KATP pathway mediates systemic and central antinociception induced by resistance exercise in rats.

    PubMed

    Galdino, Giovane S; Xavier, Carlos H; Almeida, Renato; Silva, Grazielle; Fontes, Marcos A; Menezes, Gustavo; Duarte, Igor D; Perez, Andrea C

    2015-01-01

    Resistance exercise (RE) is characterized to increase strength, tone, mass, and/or muscular endurance and also for produces many beneficial effects, such as blood pressure and osteoporosis reduction, diabetes mellitus control, and analgesia. However, few studies have investigated endogenous mechanisms involved in the RE-induced analgesia. Thus, the aim of this study was evaluate the role of the NO/CGMP/KATP pathway in the antinociception induced by RE. Wistar rats were submitted to acute RE in a weight-lifting model. The nociceptive threshold was measured by mechanical nociceptive test (paw-withdrawal). To investigate the involvement of the NO/CGMP/KATP pathway the following nitric oxide synthase (NOS) non-specific and specific inhibitors were used: N-nitro-l-arginine (NOArg), Aminoguanidine, N5-(1-Iminoethyl)-l-ornithine dihydrocloride (l-NIO), Nω-Propyl-l-arginine (l-NPA); guanylyl cyclase inhibitor, 1H-[1,2,4]oxidiazolo[4,3-a]quinoxalin-1-one (ODQ); and KATP channel blocker, Glybenclamide; all administered subcutaneously, intrathecally and intracerebroventricularly. Plasma and cerebrospinal fluid (CSF) nitrite levels were determined by spectrophotometry. The RE protocol produced antinociception, which was significantly reversed by NOS specific and unspecific inhibitors, guanylyl cyclase inhibitor (ODQ) and KATP channel blocker (Glybenclamide). RE was also responsible for increasing nitrite levels in both plasma and CSF. These finding suggest that the NO/CGMP/KATP pathway participates in antinociception induced by RE.

  3. Effects of D-penicillamine on pentylenetetrazole-induced seizures in mice: involvement of nitric oxide/NMDA pathways.

    PubMed

    Rahimi, Nastaran; Sadeghzadeh, Mitra; Javadi-Paydar, Mehrak; Heidary, Mahmoud Reza; Jazaeri, Farahnaz; Dehpour, Ahmad R

    2014-10-01

    Besides the clinical applications of penicillamine, some reports show that use of D-penicillamine (D-pen) has been associated with adverse effects such as seizures. So, the purpose of this study was to evaluate the effects of D-pen on pentylenetetrazole (PTZ)-induced seizures in male NMRI mice. It also examined whether N-methyl-D-aspartate (NMDA) receptor/nitrergic system blockage was able to alter the probable effects of D-pen. Different doses of D-pen (0.1, 0.5, 1, 10, 100, 150, and 250 mg/kg) were administered intraperitoneally (i.p.) 90 min prior to induction of seizures. D-Penicillamine at a low dose (0.5 mg/kg, i.p.) had anticonvulsant effects, whereas at a high dose (250 mg/kg, i.p.), it was proconvulsant. Both anti- and proconvulsant effects of D-pen were blocked by a single dose of a nonspecific inhibitor of nitric oxide synthase (NOS), L-NAME (10 mg/kg, i.p.), and a single dose of a specific inhibitor of neuronal nitric oxide synthase (nNOS), 7-nitroindazole (30 mg/kg, i.p.). A selective inhibitor of iNOS, aminoguanidine (100 mg/kg, i.p.), had no effect on these activities. An NMDA receptor antagonist, MK-801 (0.05 mg/kg, i.p.), alters the anti- and proconvulsant effects of D-pen. The results of the present study showed that the nitric oxide system and NMDA receptors may contribute to the biphasic effects of D-pen, which remain to be clarified further.

  4. Deregulation of Hepatic Insulin Sensitivity Induced by Central Lipid Infusion in Rats Is Mediated by Nitric Oxide

    PubMed Central

    Marsollier, Nicolas; Kassis, Nadim; Mezghenna, Karima; Soty, Maud; Fioramonti, Xavier; Lacombe, Amélie; Joly, Aurélie; Pillot, Bruno; Zitoun, Carine; Vilar, José; Mithieux, Gilles; Gross, René; Lajoix, Anne-Dominique; Routh, Vanessa; Magnan, Christophe; Cruciani-Guglielmacci, Céline

    2009-01-01

    Background Deregulation of hypothalamic fatty acid sensing lead to hepatic insulin-resistance which may partly contribute to further impairment of glucose homeostasis. Methodology We investigated here whether hypothalamic nitric oxide (NO) could mediate deleterious peripheral effect of central lipid overload. Thus we infused rats for 24 hours into carotid artery towards brain, either with heparinized triglyceride emulsion (Intralipid, IL) or heparinized saline (control rats). Principal Findings Lipids infusion led to hepatic insulin-resistance partly related to a decreased parasympathetic activity in the liver assessed by an increased acetylcholinesterase activity. Hypothalamic nitric oxide synthases (NOS) activities were significantly increased in IL rats, as the catalytically active neuronal NOS (nNOS) dimers compared to controls. This was related to a decrease in expression of protein inhibitor of nNOS (PIN). Effect of IL infusion on deregulated hepatic insulin-sensitivity was reversed by carotid injection of non selective NOS inhibitor NG-monomethyl-L-arginine (L-NMMA) and also by a selective inhibitor of the nNOS isoform, 7-Nitro-Indazole (7-Ni). In addition, NO donor injection (L-arginine and SNP) within carotid in control rats mimicked lipid effects onto impaired hepatic insulin sensitivity. In parallel we showed that cultured VMH neurons produce NO in response to fatty acid (oleic acid). Conclusions/Significance We conclude that cerebral fatty acid overload induces an enhancement of nNOS activity within hypothalamus which is, at least in part, responsible fatty acid increased hepatic glucose production. PMID:19680547

  5. Inhibition of nerve stimulation-induced vasodilatation in corpora cavernosa of the pithed rat by blockade of nitric oxide synthase.

    PubMed Central

    Finberg, J. P.; Levy, S.; Vardi, Y.

    1993-01-01

    1. The effect of inhibition of nitric oxide synthase by NG-nitro-L-arginine methyl ester (L-NAME) on nerve stimulation-induced vasodilation in corpora cavernosa was studied in the pithed rat. Corporal vasodilation was estimated by the increase in ratio (corpora cavernosal pressure/systemic blood pressure; CP/BP) following electrical stimulation of the sacral part of the spinal cord. 2. L-NAME (2, 5, 10 and 25 mg kg-1) caused an increase in BP and a dose-dependent inhibition of the rise in the CP/BP ratio following stimulation. 3. The inhibitory effect of L-NAME (25 mg kg-1) on the corporal response to spinal cord stimulation, as well as the pressor response, was partially prevented by prior administration of L- but not D-arginine (400 mg kg-1, i.v.). 4. L-NAME (20 mg kg-1, i.v.) did not inhibit the rise in corporal pressure resulting from direct intracavernosal administration of papaverine (400 micrograms over 2 min). However, this response was inhibited by 5-hydroxytryptamine (20 micrograms kg-1, i.v.). 5. The results are indicative of a role of nitric oxide (NO) in the corporal vasodilator response to erectile stimulation. PMID:7683562

  6. The role of inducible nitric oxide synthase and haem oxygenase 1 in growth and development of dental tissue'.

    PubMed

    Speranza, Lorenza; Pesce, Mirko; Franceschelli, Sara; Mastrangelo, Filiberto; Patruno, Antonia; De Lutiis, Maria Anna; Tetè, Stefano; Felaco, Mario; Grilli, Alfredo

    2012-04-01

    In this study, the activity of the antioxidant enzyme network was assessed spectrophotometrically in samples of dental pulp and dental papilla taken from third-molar gem extracts. The production of nitric oxide by the conversion of l-(2,3,4,5)-[3H] arginine to l-(3H) citrulline, the activity of haem oxygenase 1 (HO-1) through bilirubin synthesis and the expression of inducible nitric oxide synthase (iNOS), HO-1 proteins and messenger RNA by Western blot and reverse-transcribed polymerase chain reaction were also tested. The objective of this study was to evaluate the role of two proteins, iNOS and HO-1, which are upregulated by a condition of oxidative stress present during dental tissue differentiation and development. This is fundamental for guaranteeing proper homeostasis favouring a physiological tissue growth. The results revealed an over-expression of iNOS and HO-1 in the papilla, compared with that in the pulp, mediated by the nuclear factor kappa B transcription factor activated by the reactive oxygen species that acts as scavengers for the superoxide radicals. HO-1, a metabolically active enzyme in the papilla, but not in the pulp, seems to inhibit the iNOS enzyme by a crosstalk between the two proteins. We suggest that the probable mechanism through which this happens is the interaction of HO-1 with haem, a cofactor dimer indispensible for iNOS, and the subsequent suppression of its metabolic activity.

  7. Toxoplasma gondii infection of activated J774-A1 macrophages causes inducible nitric oxide synthase degradation by the proteasome pathway.

    PubMed

    Padrão, Juliana da Cruz; Cabral, Gabriel Rabello de Abreu; da Silva, Maria de Fátima Sarro; Seabra, Sergio Henrique; DaMatta, Renato Augusto

    2014-10-01

    Classically activated macrophages produce nitric oxide (NO), which is a potent microbicidal agent. NO production is catalyzed by inducible nitric oxide synthase (iNOS), which uses arginine as substrate producing NO and citruline. However, it has been demonstrated that NO production is inhibited after macrophage infection of Toxoplasma gondii, the agent of toxoplasmosis, due to iNOS degradation. Three possible iNOS degradation pathways have been described in activated macrophages: proteasome, calpain and lysosomal. To identify the iNOS degradation pathway after T. gondii infection, J774-A1 macrophage cell line was activated with lipopolysaccharide and interferon-gamma for 24 h, treated with the following inhibitors, lactacystin (proteasome), calpeptin (calpain), or concanamycin A (lysosomal), and infected with the parasite. NO production and iNOS expression were evaluated after 2 and 6 h of infection. iNOS was degraded in J774-A1 macrophages infected with T. gondii. However, treatment with lactacystin maintained iNOS expression in J774-A1 macrophages infected for 2 h by T. gondii, and after 6 h iNOS was localized in aggresomes. iNOS was degraded after parasite infection of J774-A1 macrophages treated with calpeptin or concanamycin A. NO production confirmed iNOS expression profiles. These results indicate that T. gondii infection of J774-A1 macrophages caused iNOS degradation by the proteasome pathway.

  8. Suppressive effects of coumarins from Mammea siamensis on inducible nitric oxide synthase expression in RAW264.7 cells.

    PubMed

    Morikawa, Toshio; Sueyoshi, Mayumi; Chaipech, Saowanee; Matsuda, Hisashi; Nomura, Yukiko; Yabe, Mikuko; Matsumoto, Tomoko; Ninomiya, Kiyofumi; Yoshikawa, Masayuki; Pongpiriyadacha, Yutana; Hayakawa, Takao; Muraoka, Osamu

    2012-08-15

    A methanol extract of the flowers of Mammea siamensis (Calophyllaceae) was found to inhibit nitric oxide (NO) production in lipopolysaccharide-activated RAW264.7 cells. From the extract, two new geranylated coumarins, mammeasins A (1) and B (2), were isolated together with 17 known compounds including 15 coumarins. The structures of 1 and 2 were determined on the basis of their spectroscopic properties as well as of their chemical evidence. Among the isolates, 1 (IC(50) = 1.8 μM), 2 (6.4 μM), surangins B (3, 5.0 μM), C (4, 6.8 μM), and D (5, 6.2 μM), kayeassamins E (7, 6.1 μM), F (8, 6.0 μM), and G (9, 0.8 μM), mammea A/AD (11, 1.3 μM), and mammea E/BB (16, 7.9 μM) showed NO production inhibitory activity. Compounds 1, 9, and 11 were found to inhibit induction of inducible nitric oxide synthase (iNOS). With regard to mechanism of action of these active constituents (1, 9, and 11), suppression of STAT1 activation is suggested to be mainly involved in their suppression of iNOS induction.

  9. Melampolides from the leaves of Smallanthus sonchifolius and their inhibitory activity of lps-induced nitric oxide production.

    PubMed

    Hong, Seong Su; Lee, Seon A; Han, Xiang Hua; Lee, Min Hee; Hwang, Ji Sang; Park, Jeong Sook; Oh, Ki-Wan; Han, Kun; Lee, Myung Koo; Lee, Heesoon; Kim, Wook; Lee, Dongho; Hwang, Bang Yeon

    2008-02-01

    Two new melampolide-type sesquiterpene lactones, 8beta-epoxyangeloyloxy-9alpha-ethoxy-14-oxo-acanthospermolide (1) and 8beta-angeloyloxy-9alpha-ethoxy-14-oxo-acanthospermolide (2), were isolated from the leaves of yacon [Smallanthus sonchifolia (POEPP. et ENDL.) H. Robinson] along with eleven known melampolides, allo-schkuhriolide (3), enhydrin (4), polymatin A (5), fluctuanin (6), 8beta-angeloyloxy-9alpha-acetoxy-14-oxo-acanthospermolide (7), 8beta-angeloyloxy-14-oxo-acanthospermolide (8), 8beta-methacryloyloxymelampolid-14-oic acid methyl ester (9), uvedalin (10), polymatin B (11), 8beta-tigloyloxymelampolid-14-oic acid methyl ester (12), and sonchifolin (13). Their structures were established on the basis of spectroscopic evidence including 1D- and 2D-NMR experiments. All isolates were evaluated for inhibition of LPS-induced nitric oxide production in murine macrophage RAW 264.7 cells.

  10. Infection-responsive expansion of the hematopoietic stem and progenitor cell compartment in zebrafish is dependent upon inducible nitric oxide.

    PubMed

    Hall, Christopher J; Flores, Maria Vega; Oehlers, Stefan H; Sanderson, Leslie E; Lam, Enid Y; Crosier, Kathryn E; Crosier, Philip S

    2012-02-03

    Hematopoietic stem cells (HSCs) are rare multipotent cells that contribute to all blood lineages. During inflammatory stress, hematopoietic stem and progenitor cells (HSPCs) can be stimulated to proliferate and differentiate into the required immune cell lineages. Manipulating signaling pathways that alter HSPC capacity holds great promise in the treatment of hematological malignancies. To date, signaling pathways that influence HSPC capacity, in response to hematopoietic stress, remain largely unknown. Using a zebrafish model of demand-driven granulopoiesis to explore the HSPC response to infection, we present data supporting a model where the zebrafish ortholog of the cytokine-inducible form of nitric oxide synthase (iNOS/NOS2) Nos2a acts downstream of the transcription factor C/ebpβ to control expansion of HSPCs following infection. These results provide new insights into the reactive capacity of HSPCs and how the blood system is "fine-tuned" in response to inflammatory stress.

  11. Effect of N-nitrosodimethylamine on inducible nitric oxide synthase expression and production of nitric oxide by neutrophils and mononuclear cells: the role of JNK signalling pathway.

    PubMed

    Ratajczak-Wrona, Wioletta; Jablonska, Ewa; Garley, Marzena; Jablonski, Jakub; Radziwon, Piotr

    2011-07-01

    In neutrophils (PMN) and mononuclear cells (PBMC), one of the enzymes responsible for nitric oxide (NO) synthesis is inducible nitric oxide synthase (iNOS). Changes in iNOS expression result from various signalling pathways including the mitogen-activated protein kinase (MAPK) pathway activated by endogenic and exogenic factors. N-nitrosodimethylamine (NDMA) is a xenobiotic with widespread occurrence in human environment and has an effect on cells of the immune system. The aim of this study was to determine the effect of NDMA on iNOS expression and NO production by human PMN and PBMC cells in the light of superoxide anion production by PMN cells. Moreover, the role of JNK and p38 pathways in NO production with involvement of iNOS was studied. Additionally, the function of JNK pathway in generation of superoxide anion was determined. Moreover, nitrotyrosine expression was studied in PMN and PBMC cells in the presence of NDMA. This work shows that NDMA increases iNOS expression and NO production by PMN and PBMC cells. In addition, elevated expression of phospho-JNK and phospho-p38, which are markers of JNK and p38 MAPK pathways activation, were observed. Lower iNOS expression and NO production by neutrophils exposed to extended action of NDMA were observed after application of inhibitor of JNK and p38 pathways. Lower phospho-p38 expression in PMN stimulated by NDMA was found as a result of arresting JNK pathway, whereas, application of inhibitor of p38 pathway resulted in enhanced phospho-JNK expression in PMN and PBMC cells. Increased ability to release superoxide anion by NDMA-stimulated PMN cells was observed. This ability was reduced after the application of inhibitor of JNK pathway. In PMN and PBMC cells exposed to NDMA, an increased expression of nitrotyrosine, which is dependant on JNK and p38 pathways that are activated by this particular xenobiotic, was observed. Generally, increased induction of iNOS related to elevated production of NO by PMN and PBMC

  12. Effects of Intracerebroventricularly (ICV) Injected Ghrelin on Cardiac Inducible Nitric Oxide Synthase Activity/Expression in Obese Rats.

    PubMed

    Sudar Milovanovic, E; Jovanovic, A; Misirkic-Marjanovic, M; Vucicevic, Lj; Janjetovic, K; Isenovic, E R

    2015-11-01

    The aim of this study was to examine the effects of ghrelin on regulation of cardiac inducible nitric oxide synthase (iNOS) activity/expression in high fat (HF), obese rats.For this study, male Wistar rats fed with HF diet (30% fat) for 4 weeks were injected every 24 h for 5 days intracerebroventricularly (ICV) with ghrelin (0.3 nmol/5 µl) or with an equal volume of phosphate buffered saline (PBS). Control rats were ICV injected with an equal volume of PBS. Glucose, insulin and nitric oxide (NO) concentrations were measured in serum, while arginase activity and citrulline concentrations were measured in heart lysate. Protein iNOS and regulatory subunit of nuclear factor-κB (NFκB-p65), phosphorylation of enzymes protein kinase B (Akt) at Ser(473), and extracellular signal-regulated kinases 1/2 (ERK1/2) at Tyr(202)/Tyr(204) were determined in heart lysate by Western blot. For gene expression of iNOS qRT-PCR was used.Results show significantly (p<0.01) higher serum NO production in ghrelin treated HF rats compared with HF rats. Ghrelin significantly reduced citrulline concentration (p<0.05) and arginase activity (p<0.01) in HF rats. In ghrelin treated HF rats, gene and protein expression of iNOS and NFκB-p65 levels were significantly (p<0.05) increased compared with HF rats. Increased phosphorylation of Akt (p<0.01) and decreased (p<0.05) ERK1/2 phosphorylation were detected in HF ghrelin treated rats compared with HF rats hearts.Results from this study indicate that exogenous ghrelin induces expression and activity of cardiac iNOS via Akt phosphorylation followed by NFκB activation in HF rats.

  13. Contribution of inducible and neuronal nitric oxide synthases to mitochondrial damage and melatonin rescue in LPS-treated mice.

    PubMed

    García, José Antonio; Ortiz, Francisco; Miana, Javier; Doerrier, Carolina; Fernández-Ortiz, Marisol; Rusanova, Iryna; Escames, Germaine; García, José Joaquín; Acuña-Castroviejo, Darío

    2017-01-21

    NOS isoform activation is related to liver failure during sepsis, but the mechanisms driving mitochondrial impairment remain unclear. We induced sepsis by LPS administration to inducible nitric oxide synthase (iNOS(-/-)) and neuronal nitric oxide synthase (nNOS(-/-)) mice and their respective wild-type controls to examine the contribution of iNOS to mitochondrial failure in the absence of nNOS. To achieve this goal, the determination of messenger RNA (mRNA) expression and protein content of iNOS in cytosol and mitochondria, the mitochondrial respiratory complex content, and the levels of nitrosative and oxidative stress (by measuring 3-nitrotyrosine residues and carbonyl groups, respectively) were examined in the liver of control and septic mice. We detected strongly elevated iNOS mRNA expression and protein levels in liver cytosol and mitochondria of septic mice, which were related to enhanced oxidative and nitrosative stress, and with fewer changes in respiratory complexes. The absence of the iNOS, but not nNOS, gene absolutely prevented mitochondrial impairment during sepsis. Moreover, the nNOS gene did not modify the expression and the effects of iNOS here shown. Melatonin administration counteracted iNOS activation and mitochondrial damage and enhanced the expression of the respiratory complexes above the control values. These effects were unrelated to the presence or absence of nNOS. iNOS is a main target to prevent liver mitochondrial impairment during sepsis, and melatonin represents an efficient antagonist of these iNOS-dependent effects whereas it may boost mitochondrial respiration to enhance liver survival.

  14. The role of nitric oxide in the PKA inhibitor induced spatial memory deficits in rat: involvement of choline acetyltransferase.

    PubMed

    Najafi, Sheyda; Payandemehr, Borna; Tabrizian, Kaveh; Shariatpanahi, Marjan; Nassireslami, Ehsan; Azami, Kian; Mohammadi, Mojdeh; Asadi, Farideh; Roghani, Ali; Sharifzadeh, Mohammad

    2013-08-15

    Several lines of evidence show that cAMP-PKA signaling pathway plays critical role in memory functions and suggest nitric oxide as an important modulator in learning and memory. In this study, we assessed the effects of intra-hippocampal infusion of H-89, a selective PKAII inhibitor, and 1400 W, a selective inducible nitric oxide synthase (iNOS) inhibitor, on spatial memory in rats. By using the Morris water maze, spatial memory retention parameters were examined 48 h after the infusions through measuring escape latency, traveled distance, and swimming speed. The rats receiving intra-hippocampal infusions of 1400 W (100 µM/side) showed a significant reduction (*P<0.05) in escape latency and traveled distance in comparison with the control saline group. In contrast, a significant increase (**P<0.01) in escape latency and traveled distance was observed after infusion of 10 µM H-89. Moreover, among combination groups, co-administration of 1400 W (400 µM/side) with 10 µM/side of H-89 caused a significant reduction (*P<0.05) in escape latency and traveled distance in comparison with the H-89 group. Also, we evaluated the molecular effects of 1400 W on the expression of choline acetyltransferase (ChAT), a cholinergic marker, in the CA1 region of the hippocampus and medial septal area (MSA). Immunohistochemical analysis of post-training bilateral intra-hippocampal infusion of 1400 W revealed a significant increase in ChAT immunoreactivity levels in both the CA1 and the MSA regions. Overall, the results suggest that 1400 W has protective effect against H89-induced spatial memory impairment. Moreover, the observed memory improvements caused by 1400 W infusions, might be due to interaction of iNOS with the cholinergic system.

  15. Inducible nitric oxide synthase response and associated cytokine gene expression in the spleen of mice infected with Clonorchis sinensis.

    PubMed

    Shen, Ji-Qing; Yang, Qing-Li; Xue, Yan; Cheng, Xiao-Bing; Jiang, Zhi-Hua; Yang, Yi-Chao; Chen, Ying-Dan; Zhou, Xiao-Nong

    2015-05-01

    Clonorchis sinensis is a food-borne parasite that induces a permanent increase of nitrosation in the body upon infection. The spleen is an important secondary lymphoid organ for the regulation of immune responses locally and in the whole body. However, the functions and mechanisms of the spleen in nitric oxide (NO) responses after C. sinensis infection remain unknown. In this study, BALB/c mice were infected with 20, 40, and 80 C. sinensis metacercariae to simulate mild, moderate, and severe infections, respectively. We examined the expression of inducible nitric oxide synthase (iNOS) in the spleen and the relevant cytokine transcription in splenocytes from the mice infected with different amounts of metacercariae. The iNOS of the mice infected with 80 metacercariae was expressed in the spleen as early as 10 days post-infection (dpi) and gradually increased until 90 dpi. The iNOS expression in the mice infected with 40 metacercariae was detected only at 45 and 90 dpi, but not in the mice infected with 20 metacercariae. The level of interferon (IFN)-γ messenger RNA (mRNA) transcription in splenocytes significantly increased at 10 and 20 dpi (P < 0.05) in response to mild/moderate infection but gradually decreased to normal levels after 45 dpi. The level of IL-12p35 mRNA transcription did not change at 10 and 20 dpi but significantly decreased after 45 dpi under moderate/severe infection (P < 0.05/0.01/0.001). The level of IL-18 mRNA transcription significantly increased at 10 dpi (P < 0.05/0.01) but significantly decreased after 20 dpi (P < 0.05/0.01/0.001). These results suggest that spleen is an important organ for iNOS/NO responses, which correspond to the severity of C. sinensis infection, but cannot be attributed to the expression of the Th1 cytokines.

  16. Nitric oxide and protein S-nitrosylation are integral to hydrogen peroxide-induced leaf cell death in rice.

    PubMed

    Lin, Aihong; Wang, Yiqin; Tang, Jiuyou; Xue, Peng; Li, Chunlai; Liu, Linchuan; Hu, Bin; Yang, Fuquan; Loake, Gary J; Chu, Chengcai

    2012-01-01

    Nitric oxide (NO) is a key redox-active, small molecule involved in various aspects of plant growth and development. Here, we report the identification of an NO accumulation mutant, nitric oxide excess1 (noe1), in rice (Oryza sativa), the isolation of the corresponding gene, and the analysis of its role in NO-mediated leaf cell death. Map-based cloning revealed that NOE1 encoded a rice catalase, OsCATC. Furthermore, noe1 resulted in an increase of hydrogen peroxide (H(2)O(2)) in the leaves, which consequently promoted NO production via the activation of nitrate reductase. The removal of excess NO reduced cell death in both leaves and suspension cultures derived from noe1 plants, implicating NO as an important endogenous mediator of H(2)O(2)-induced leaf cell death. Reduction of intracellular S-nitrosothiol (SNO) levels, generated by overexpression of rice S-nitrosoglutathione reductase gene (GSNOR1), which regulates global levels of protein S-nitrosylation, alleviated leaf cell death in noe1 plants. Thus, S-nitrosylation was also involved in light-dependent leaf cell death in noe1. Utilizing the biotin-switch assay, nanoliquid chromatography, and tandem mass spectrometry, S-nitrosylated proteins were identified in both wild-type and noe1 plants. NO targets identified only in noe1 plants included glyceraldehyde 3-phosphate dehydrogenase and thioredoxin, which have been reported to be involved in S-nitrosylation-regulated cell death in animals. Collectively, our data suggest that both NO and SNOs are important mediators in the process of H(2)O(2)-induced leaf cell death in rice.

  17. Planar laser-induced fluorescence measurements of high-enthalpy free jet flow with nitric oxide

    NASA Technical Reports Server (NTRS)

    Palmer, Jennifer L.; Mcmillin, Brian K.; Hanson, Ronald K.

    1992-01-01

    Planar laser-induced fluorescence (PLIF) measurements of property fields in a high-enthalpy, supersonic, underexpanded free jet generated in a reflection-type shock tunnel are reported. PLIF images showing velocity and temperature sensitivity are presented. The inferred radial velocity and relative rotational temperature fields are found to be in agreement with those predicted by a numerical simulation of the flowfield using the method of characteristics.

  18. Respective role of lipoxygenase and nitric oxide-synthase pathways in plasma histamine-induced macromolecular leakage inconscious hamsters

    PubMed Central

    Gimeno, G; Carpentier, P H; Desquand-Billiald, S; Finet, M; Hanf, R

    1999-01-01

    phase of histamine-induced hypotension.Thus, plasma histamine can trigger both an immediate cysteinyl-leukotriene (Cys-LT)-dependent and a late nitric oxide (NO)-mediated inflammatory cascade. Although the cyclo-oxygenase (COX) pathway might account for histamine-induced venule dilatation, it would not influence histamine-induced extravasation. PMID:10372823

  19. Ethylene mediates brassinosteroid-induced stomatal closure via Gα protein-activated hydrogen peroxide and nitric oxide production in Arabidopsis.

    PubMed

    Shi, Chenyu; Qi, Cheng; Ren, Hongyan; Huang, Aixia; Hei, Shumei; She, Xiaoping

    2015-04-01

    Brassinosteroids (BRs) are essential for plant growth and development; however, whether and how they promote stomatal closure is not fully clear. In this study, we report that 24-epibrassinolide (EBR), a bioactive BR, induces stomatal closure in Arabidopsis (Arabidopsis thaliana) by triggering a signal transduction pathway including ethylene synthesis, the activation of Gα protein, and hydrogen peroxide (H(2)O(2)) and nitric oxide (NO) production. EBR initiated a marked rise in ethylene, H(2)O(2) and NO levels, necessary for stomatal closure in the wild type. These effects were abolished in mutant bri1-301, and EBR failed to close the stomata of gpa1 mutants. Next, we found that both ethylene and Gα mediate the inductive effects of EBR on H(2)O(2) and NO production. EBR-triggered H(2)O(2) and NO accumulation were canceled in the etr1 and gpa1 mutants, but were strengthened in the eto1-1 mutant and the cGα line (constitutively overexpressing the G protein α-subunit AtGPA1). Exogenously applied H(2)O(2) or sodium nitroprusside (SNP) rescued the defects of etr1-3 and gpa1 or etr1 and gpa1 mutants in EBR-induced stomatal closure, whereas the stomata of eto1-1/AtrbohF and cGα/AtrbohF or eto1-1/nia1-2 and cGα/nia1-2 constructs had an analogous response to H(2)O(2) or SNP as those of AtrbohF or Nia1-2 mutants. Moreover, we provided evidence that Gα plays an important role in the responses of guard cells to ethylene. Gα activator CTX largely restored the lesion of the etr1-3 mutant, but ethylene precursor ACC failed to rescue the defects of gpa1 mutants in EBR-induced stomatal closure. Lastly, we demonstrated that Gα-activated H(2)O(2) production is required for NO synthesis. EBR failed to induce NO synthesis in mutant AtrbohF, but it led to H(2)O(2) production in mutant Nia1-2. Exogenously applied SNP rescued the defect of AtrbohF in EBR-induced stomatal closure, but H(2)O(2) did not reverse the lesion of EBR-induced stomatal closure in Nia1-2. Together, our

  20. Nitric oxide induces thioredoxin-1 nuclear translocation: Possible association with the p21Ras survival pathway

    SciTech Connect

    Arai, Roberto J.; Yodoi, J.; Debbas, V.; Laurindo, Francisco R.; Stern, A.; Monteiro, Hugo P. . E-mail: hpmonte@uol.com.br

    2006-10-06

    One of the major redox-regulating molecules with thiol reducing activity is thioredoxin-1 (TRX-1). TRX-1 is a multifunctional protein that exists in the extracellular millieu, cytoplasm, and nucleus, and has a distinct role in each environment. It is well known that TRX-1 promptly migrates to the nuclear compartment in cells exposed to oxidants. However, the intracellular location of TRX-1 in cells exposed to nitrosothiols has not been investigated. Here, we demonstrated that the exposure of HeLa cells to increasing concentrations of the nitrosothiol S-nitroso-N-acetylpenicillamine (SNAP) promoted TRX-1 nuclear accumulation. The SNAP-induced TRX-1 translocation to the nucleus was inhibited by FPTIII, a selective inhibitor of p21Ras. Furthermore, TRX-1 migration was attenuated in cells stably transfected with NO insensitive p21Ras (p21{sup RasC118S}). Downstream to p21Ras, the MAP Kinases ERK1/2 were activated by SNAP under conditions that promote TRX-1 nuclear translocation. Inhibition of MEK prevented SNAP-stimulated ERK1/2 activation and TRX-1 nuclear migration. In addition, cells treated with p21Ras or MEK inhibitor showed increased susceptibility to cell death induced by SNAP. In conclusion, our observations suggest that the nuclear translocation of TRX-1 is induced by SNAP involving p21Ras survival pathway.

  1. Chemical constituents isolated from the Mongolian medicinal plant Sophora alopecuroides L. and their inhibitory effects on LPS-induced nitric oxide production in RAW 264.7 macrophages.

    PubMed

    Kwon, Jaeyoung; Basnet, Sunita; Lee, Jin Woo; Seo, Eun-Kyoung; Tsevegsuren, Nanzad; Hwang, Bang Yeon; Lee, Dongho

    2015-08-15

    Three new flavonostilbenes, alopecurones M-O (1-3), were isolated from the root bark of Sophora alopecuroides L. together with 21 known compounds. The structures of the isolated compounds were elucidated by using NMR, MS, and CD spectroscopic data. All isolates were evaluated for their potential to inhibit LPS-induced nitric oxide production in RAW 264.7 cells.

  2. Laser-induced fluorescence measurements of nitric oxide in laminar C2H6/O2/N2 flames at high pressure

    NASA Technical Reports Server (NTRS)

    Reisel, John R.; Carter, Campbell D.; Laurendeau, Normand M.

    1993-01-01

    Quantitative measurements of nitric oxide in C2H6/O2/N2 flames at 1-9 atm were successfully carried out using laser-induced fluorescence. The location of maximum NO concentration is found to shift towards leaner equivalence ratios with increasing pressure. Details of the experimental apparatus and measurement procedure are described.

  3. Iron(II) porphyrins induced conversion of nitrite into nitric oxide: A computational study.

    PubMed

    Zhang, Ting Ting; Liu, Yong Dong; Zhong, Ru Gang

    2015-09-01

    Nitrite reduction to nitric oxide by heme proteins was reported as a protective mechanism to hypoxic injury in mammalian physiology. In this study, the pathways of nitrite reduction to nitric oxide mediated by iron(II) porphyrin (P) complexes, which were generally recognized as models for heme proteins, were investigated by using density functional theory (DFT). In view of two type isomers of combination of nitrite and Fe(II)(P), N-nitro- and O-nitrito-Fe(II)-porphyrin complexes, and two binding sites of proton to the different O atoms of nitrite moiety, four main pathways for the conversion of nitrite into nitric oxide mediated by iron(II) porphyrins were proposed. The results indicate that the pathway of N-bound Fe(II)(P)(NO2) isomer into Fe(III)(P)(NO) and water is similar to that of O-bound isomer into nitric oxide and Fe(III)(P)(OH) in both thermodynamical and dynamical aspects. Based on the initial computational studies of five-coordinate nitrite complexes, the conversion of nitrite into NO mediated by Fe(II)(P)(L) complexes with 14 kinds of proximal ligands was also investigated. Generally, the same conclusion that the pathways of N-bound isomers are similar to those of O-bound isomer was obtained for iron(II) porphyrin with ligands. Different effects of ligands on the reduction reactions were also found. It is notable that the negative proximal ligands can improve reactive abilities of N-nitro-iron(II) porphyrins in the conversion of nitrite into nitric oxide compared to neutral ligands. The findings will be helpful to expand our understanding of the mechanism of nitrite reduction to nitric oxide by iron(II) porphyrins.

  4. Nitric oxide is the key mediator of death induced by fisetin in human acute monocytic leukemia cells.

    PubMed

    Ash, Dipankar; Subramanian, Manikandan; Surolia, Avadhesha; Shaha, Chandrima

    2015-01-01

    Nitric oxide (NO) has been shown to be effective in cancer chemoprevention and therefore drugs that help generate NO would be preferable for combination chemotherapy or solo use. This study shows a new evidence of NO as a mediator of acute leukemia cell death induced by fisetin, a promising chemotherapeutic agent. Fisetin was able to kill THP-1 cells in vivo resulting in tumor shrinkage in the mouse xenograft model. Death induction in vitro was mediated by an increase in NO resulting in double strand DNA breaks and the activation of both the extrinsic and the intrinsic apoptotic pathways. Double strand DNA breaks could be reduced if NO inhibitor was present during fisetin treatment. Fisetin also inhibited the downstream components of the mTORC1 pathway through downregulation of levels of p70 S6 kinase and inducing hypo-phosphorylation of S6 Ri P kinase, eIF4B and eEF2K. NO inhibition restored phosphorylation of downstream effectors of mTORC1 and rescued cells from death. Fisetin induced Ca(2+) entry through L-type Ca(2+) channels and abrogation of Ca(2+) influx reduced caspase activation and cell death. NO increase and increased Ca(2+) were independent phenomenon. It was inferred that apoptotic death of acute monocytic leukemia cells was induced by fisetin through increased generation of NO and elevated Ca(2+) entry activating the caspase dependent apoptotic pathways. Therefore, manipulation of NO production could be viewed as a potential strategy to increase efficacy of chemotherapy in acute monocytic leukemia.

  5. Nitric oxide as a mediator of fructose 1,6-bisphosphate protection in galactosamine-induced hepatotoxicity in rats.

    PubMed

    Alva, Norma; Cruz, David; Sanchez, Sergio; Valentín, Juana Ma; Bermudez, Jordi; Carbonell, Teresa

    2013-01-15

    Fructose 1,6-bisphosphate (F1,6BP) has been widely used as a therapeutic agent for different harmful conditions in a variety of tissues. The hypothesis of the present work was that the increase in nitric oxide production and the prevention of oxidative stress induced by exogenous F1,6BP mediate its protective effect against the hepatotoxic action of GalN. Experimental groups used were sham, F1,6BP (2g/kg bw i.p.), GalN (0.4g/kg bw i.p), l-NAME (10mg/kg bw i.v.), F1,6BP+GalN, l-NAME+GalN and l-NAME+F1,6BP+GalN. Animals were killed after 24h of bolus administration. F1,6BP induced an increase in NO and the redox ratio (GSH/GSSG) in liver. Western blot assays pointed to overexpression of liver eNOS in F1,6BP-treated rats. The hepatic injury induced by GalN increased transaminases in plasma and decreased the reduced/oxidized glutathione ratio in liver. The concomitant administration of F1,6BP reversed this damage, while the addition of l-NAME worsened the liver injury. We provided evidence that this F1,6BP-induced protection may be related to the increase in NO production through the positive modulation of eNOS, and the increase in intracellular reduced glutathione, thus providing a higher reducing capacity.

  6. Nitric oxide and histone deacetylases modulate cocaine-induced mu-opioid receptor levels in PC12 cells

    PubMed Central

    2012-01-01

    Background Cocaine exposure has been reported to alter central μ-opioid receptor (MOR) expression in vivo. The present study employed an in vitro cellular model to explore possible mechanisms that may be involved in this action of cocaine. Methods To assess the effects of cocaine on MOR levels, two treatment regimens were tested in PC12 cells: single continuous or multiple intermittent. MOR protein levels were assessed by western blot analysis and quantitative PCR was used to determine relative MOR mRNA expression levels. To evaluate the role of nitric oxide (NO) and histone acetylation in cocaine-induced MOR expression, cells were pre-treated with the NO synthase inhibitor Nω-nitro-L-arginine methylester (L-NAME) or the non-selective histone acetyltransferase inhibitor curcumin. Results Both cocaine treatment regimens significantly increased MOR protein levels and protein stability, but only multiple intermittent treatments increased MOR mRNA levels as well as c-fos mRNA levels and activator protein 1 binding activity. Both regimens increased NO production, and pre-treatment with L-NAME prevented cocaine-induced increases in MOR protein and mRNA levels. Single and multiple cocaine treatment regimens inhibited histone deacetylase activity, and pre-treatment with curcumin prevented cocaine-induced up-regulation of MOR protein expression. Conclusions In the PC12 cell model, both NO and histone deacetylase activity regulate cocaine-induced MOR expression at both the transcriptional and post-transcriptional levels. Based on these novel findings, it is hypothesized that epigenetic mechanisms are implicated in cocaine’s action on MOR expression in neurons. PMID:23079001

  7. Nitric oxide is the key mediator of death induced by fisetin in human acute monocytic leukemia cells

    PubMed Central

    Ash, Dipankar; Subramanian, Manikandan; Surolia, Avadhesha; Shaha, Chandrima

    2015-01-01

    Nitric oxide (NO) has been shown to be effective in cancer chemoprevention and therefore drugs that help generate NO would be preferable for combination chemotherapy or solo use. This study shows a new evidence of NO as a mediator of acute leukemia cell death induced by fisetin, a promising chemotherapeutic agent. Fisetin was able to kill THP-1 cells in vivo resulting in tumor shrinkage in the mouse xenograft model. Death induction in vitro was mediated by an increase in NO resulting in double strand DNA breaks and the activation of both the extrinsic and the intrinsic apoptotic pathways. Double strand DNA breaks could be reduced if NO inhibitor was present during fisetin treatment. Fisetin also inhibited the downstream components of the mTORC1 pathway through downregulation of levels of p70 S6 kinase and inducing hypo-phosphorylation of S6 Ri P kinase, eIF4B and eEF2K. NO inhibition restored phosphorylation of downstream effectors of mTORC1 and rescued cells from death. Fisetin induced Ca2+ entry through L-type Ca2+ channels and abrogation of Ca2+ influx reduced caspase activation and cell death. NO increase and increased Ca2+ were independent phenomenon. It was inferred that apoptotic death of acute monocytic leukemia cells was induced by fisetin through increased generation of NO and elevated Ca2+ entry activating the caspase dependent apoptotic pathways. Therefore, manipulation of NO production could be viewed as a potential strategy to increase efficacy of chemotherapy in acute monocytic leukemia. PMID:25973292

  8. AMP-activated protein kinase (AMPK) regulates the insulin-induced activation of the nitric oxide synthase in human platelets.

    PubMed

    Fleming, Ingrid; Schulz, Christian; Fichtlscherer, Birgit; Kemp, Bruce E; Fisslthaler, Beate; Busse, Rudi

    2003-11-01

    Little is known about the signaling cascades that eventually regulate the activity of the endothelial nitric oxide synthase (eNOS) in platelets. Here, we investigated the effects of insulin on the phosphorylation and activation of eNOS in washed human platelets and in endothelial cells. Insulin activated the protein kinase Akt in cultured endothelial cells and increased the phosphorylation of eNOS on Ser(1177) but failed to increase endothelial cyclic GMP levels or to elicit the relaxation of endothelium-intact porcine coronary arteries. In platelets, insulin also elicited the activation of Akt as well as the phosphorylation of eNOS and initiated NO production which was associated with increased cyclic GMP levels and the inhibition of thrombin-induced aggregation. The insulin-induced inhibition of aggregation was accompanied by a decreased Ca(2+) response to thrombin and was also prevented by N(omega) nitro-L-arginine. In platelets, but not in endothelial cells, insulin induced the activation of the AMP-activated protein kinase (AMPK), a metabolic stress-sensing kinase which was sensitive to the phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin and the AMPK inhibitor iodotubercidin. Moreover, the insulin-mediated inhibition of thrombin-induced aggregation was prevented by iodotubercidin. Insulin-independent activation of the AMPK using 5-aminoimidazole-4-carboxamide ribonucleoside, increased platelet eNOS phosphorylation, increased cyclic GMP levels and attenuated platelet aggregation. These results highlight the differences in the signal transduction cascade activated by insulin in endothelial cells and platelets, and demonstrate that insulin stimulates the formation of NO in human platelets, in the absence of an increase in Ca(2+), by acti-vating PI3-K and AMPK which phosphorylates eNOS on Ser(1177).

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

    PubMed

    McMurtry, Vanity; Saavedra, Joseph E; Nieves-Alicea, René; Simeone, Ann-Marie; Keefer, Larry K; Tari, Ana M

    2011-04-01

    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.

  10. Prostaglandins as negative regulators against lipopolysaccharide, lipoteichoic acid, and peptidoglycan-induced inducible nitric oxide synthase/nitric oxide production through reactive oxygen species-dependent heme oxygenase 1 expression in macrophages.

    PubMed

    Chien, Chih-Chiang; Shen, Shing-Chuan; Yang, Liang-Yo; Chen, Yen-Chou

    2012-11-01

    Although prostaglandins (PGs) were reported to exert proinflammatory and anti-inflammatory effects in macrophages, their action mechanisms remain unclear. The effects of PGs including PGJ2 (J2), Δ-PGJ2 (Δ), 15-deoxy-Δ PGJ2 (15d), PGE2 (E2), and PGF2α (F2α) on lipopolysaccharide (LPS)-, lipoteichoic acid (LTA)-, and peptidoglycan (PGN)-induced inducible nitric oxide (NO) synthase (iNOS)/NO production by RAW264.7 macrophages were investigated. First, we found that induction of cyclooxygenase 2 (COX-2) protein occurred at a time earlier than that of heme oxygenase 1 (HO-1) protein, and the addition of the COX-2 inhibitor NS398 reduced HO-1 protein expression in LPS-, LTA-, and PGN-treated RAW264.7 macrophages. Incubation of RAW264.7 macrophages with the indicated PGs showed that J2, Δ, and 15d significantly induced HO-1 protein expression; however, E2 and F2α did not. Heme oxygenase 1 protein induced by J2, Δ, and 15d was inhibited by the transcriptional inhibitor, actinomycin (Act) D; the translational inhibitor, cycloheximide; and the antioxidant, N-acetyl cysteine (NAC). Increases in intracellular peroxide levels by J2, Δ, and 15d were detected via a 2',7'™-dichlorofluorescein diacetate (DCFH-DA) analysis, and they were prevented by the addition of NAC. In addition, J2, Δ, and 15d produced significant inhibition of LPS-, LTA-, and PGN-induced iNOS protein and NO production by RAW264.7 cells, in accordance with increased HO-1 protein expression. Reductions of LPS-, LTA-, and PGN-induced phosphorylated c-Jun N-terminal kinase, c-Jun protein, and activator protein 1 luciferase activity by J2, Δ, and 15d were identified, and the addition of the HO-1 inhibitor, tin protoporphyrin, reversed the inhibitory effects of Δ and 15d on LPS- and LTA-induced iNOS/NO, phosphorylated c-Jun N-terminal kinase, and c-Jun protein expressions by macrophages. Knockdown of HO-1 protein expression by HO-1 small interfering RNA blocked Δ and 15d inhibition of LPS- and LTA-induced

  11. Ghrelin Protection against Lipopolysaccharide-Induced Gastric Mucosal Cell Apoptosis Involves Constitutive Nitric Oxide Synthase-Mediated Caspase-3 S-Nitrosylation

    PubMed Central

    Slomiany, Bronislaw L.; Slomiany, Amalia

    2010-01-01

    Ghrelin, a peptide hormone produced mainly in the stomach, has emerged as an important modulator of the inflammatory responses that are of significance to the maintenance of gastric mucosal integrity. Here, we report on the role of ghrelin in controlling the apoptotic processes induced in gastric mucosal cells by H. pylori lipopolysaccharide (LPS). The countering effect of ghrelin on the LPS-induced mucosal cell apoptosis was associated with the increase in constitutive nitric oxide synthase (cNOS) activity, and the reduction in caspase-3 and inducible nitric oxide synthase (NOS-2). The loss in countering effect of ghrelin on the LPS-induced changes in apoptosis and caspase-3 activity was attained with Src kinase inhibitor, PP2, as well as Akt inhibitor, SH-5, and cNOS inhibitor, L-NAME. Moreover, the effect of ghrelin on the LPS-induced changes in cNOS activity was reflected in the increased cNOS phosphorylation that was sensitive to SH-5. Furthermore, the ghrelin-induced up-regulation in cNOS activity was associated with the increase in caspase-3 S-nitrosylation that was susceptible to the blockage by L-NAME. Therefore, ghrelin protection of gastric mucosal cells against H. pylori LPS-induced apoptosis involves Src/Akt-mediated up-regulation in cNOS activation that leads to the apoptotic signal inhibition through the NO-induced caspase-3 S-nitrosylation. PMID:20369000

  12. Superoxide dismutase abolishes the platelet-derived growth factor-induced release of prostaglandin E2 by blocking induction of nitric oxide synthase: role of superoxide.

    PubMed

    Kelner, M J; Uglik, S F

    1995-09-10

    The ability of platelet-derived growth factor (PDGF) to induce prostaglandin E2 (PGE2) release in fibroblasts is abolished when copper-zinc superoxide dismutase activity is increased by transfection of an expression vector. The effect is specific to copper-zinc superoxide dismutase as glutathione peroxidase-overexpressing NIH3T3 cells, again produced by transfection of an expression vector, retain the ability to release PGE2 in response to growth factor stimulation. The defect in PDGF-induced PGE2 release occurs prior to action of prostaglandin H synthase/cyclooxygenase as release of arachadonic acid (in response to PDGF) does not occur in the superoxide dismutase-overexpressing clones. The defect in PDGF-induced release of PGE2 in superoxide dismutase-overexpressing clones differs from the defect found in pEJ-ras-transformed clones. The parent cells, the glutathione peroxidase-expressing cells, and the superoxide dismutase-overexpressing cells all release PGE2 in response to exogenous nitric oxide, whereas the pEJ-ras-transformed cells do not. The glutathione peroxidase-expressing cells also retained the ability to release nitrite in response to PDGF, whereas the superoxide dismutase-expressing clones do not. PDGF stimulates nitric oxide synthase activity in NIH3T3 cells, but not in the superoxide dismutase-expressing clones. These results indicate that superoxide dismutase overexpression blocks the PDGF-induced release of PGE2 by blocking induction of nitric oxide synthase. This indicates that the increase of nitric oxide synthase induced by PDGF is mediated in part by production of superoxide. These findings link cellular oxygen radical homeostasis to three different classes of messenger molecules (growth factors, nitric oxide, and prostaglandins).

  13. Anti-inflammatory effect of Mentha longifolia in lipopolysaccharide-stimulated macrophages: reduction of nitric oxide production through inhibition of inducible nitric oxide synthase.

    PubMed

    Karimian, Parastoo; Kavoosi, Gholamreza; Amirghofran, Zahra

    2013-01-01

    Mentha longifolia is an aromatic plant used in flavoring and preserving foods and as an anti-inflammatory folk medicine remedy. The present study assessed the effects of M. longifolia extracts, including essential oil and crude methanol extract and its fractions (ethyl acetate, butanol and hexane), on nitric oxide (NO) production and inducible NO synthase (iNOS) mRNA expression in lipopolysaccharide (LPS)-stimulated J774A.1 cells using real-time polymerase chain reaction (PCR). The cytotoxic effects of the extracts on the cells were examined and non-cytotoxic concentrations (<0.2 mg/ml) were used to examine their effects on NO production and iNOS mRNA expression. Only the hexane fraction that contained high levels of phenolic and flavonoid compounds at concentrations from 0.05-0.20 mg/ml significantly reduced NO production in LPS-stimulated cells (p < 0.001). Real-time PCR analysis indicated the ability of this fraction at the same concentrations to significantly decrease iNOS as well as TNFα mRNA expression in the cells (p < 0.001). All extracts were able to scavenge NO radicals in a concentration-dependent manner. At concentrations greater than 0.2 mg/ml, total radicals were 100% scavenged. In conclusion, M. longifolia possibly reduces NO secretion in macrophages by scavenging NO and inhibiting iNOS mRNA expression, and also decreases TNFα pro-inflammatory cytokine expression, thus showing its usefulness in the inflammatory disease process.

  14. Anticonvulsive Effects of Licofelone on Status Epilepticus Induced by Lithium-pilocarpine in Wistar Rats: a Role for Inducible Nitric Oxide Synthase

    PubMed Central

    Eslami, Seyyed Majid; Moradi, Mohammad Mobin; Ghasemi, Mehdi; Dehpour, Ahmad Reza

    2016-01-01

    Background and Purpose Status epilepticus (SE) is a neurological disorder with high prevalence and mortality rates, requiring immediate intervention. Licofelone is a cyclooxygenase (COX) and 5-lipoxygenase (5-LOX) inhibitor, which its effectiveness to treat osteoarthritis has been approved. Increasing evidence suggests an involvement of COX and LOX enzymes in epileptic disorders. Thus, in the present study we investigate possible effects of licofelone on prevention and termination of SE. We also evaluated whether the nitrergic system could participate in this effect of licofelone. Methods We have utilized lithium-pilocarpine model of SE in adult Wistar rats to assess the potential effect of licofelone on seizure susceptibility. Licofelone was administered 1 h before pilocarpine. To evaluate probable role of nitric oxide (NO) system, L-arginine (60 mg/kg, i.p.), as a NO precursor; L-NAME (15 mg/kg, i.p.), as a non-selective nitric oxide synthase (NOS) inhibitor; aminoguanidine (100 mg/kg, i.p.), as an inducible NOS (iNOS) inhibitor and 7-nitroindazole (60 mg/kg, i.p.), as a neuronal NOS inhibitor were injected 15 min before licofelone. Also, licofelone and diazepam 10 mg/kg were administered 30 minutes after onset of SE. Results Pre-treatment with licofelone at the dosage of 10 mg/kg, significantly prevented the onset of SE in all subjects (p < 0.001). L-arginine significantly inverted this anticonvulsant effect (p < 0.05). However, L-NAME and aminoguanidine, potentiated the anticonvulsant effect of licofelone (p < 0.05, p < 0.01). Licofelone could not terminate seizures after onset which was terminated by diazepam. Conclusions Our findings showed that anticonvulsive effects of licofelone on SE could be mediated by iNOS. Also, we suggest that COX/5-LOX activation is possibly required in the initial stage of onset but SE recruits extra excitatory pathways with prolongation. PMID:28101475

  15. Inhibition of lipopolysaccharide-induced cyclooxygenase-2 and inducible nitric oxide synthase expression by 4-[(2'-O-acetyl-α-L-rhamnosyloxy)benzyl]isothiocyanate from Moringa oleifera.

    PubMed

    Park, Eun-Jung; Cheenpracha, Sarot; Chang, Leng Chee; Kondratyuk, Tamara P; Pezzuto, John M

    2011-01-01

    Moringa oleifera Lamarck is commonly consumed for nutritional or medicinal properties. We recently reported the isolation and structure elucidation of novel bioactive phenolic glycosides, including 4-[(2'-O-acetyl-α-L-rhamnosyloxy)benzyl]isothiocyanate (RBITC), which was found to suppress inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production in lipopolysaccharide-stimulated RAW 264.7 mouse macrophage cells. Inhibitors of proteins such as cyclooxygenase-2 (COX-2) and iNOS are potential antiinflammatory and cancer chemopreventive agents. The inhibitory activity of RBITC on NO production (IC(50) = 0.96 ± 0.23 μM) was greater than that mediated by other well-known isothiocyanates such as sulforaphane (IC(50) = 2.86 ± 0.39 μM) and benzyl isothiocyanate (IC(50) = 2.08 ± 0.28 μM). RBITC inhibited expression of COX-2 and iNOS at both the protein and mRNA levels. Major upstream signaling pathways involved mitogen-activated protein kinases and nuclear factor-κB (NF-κB). RBITC inhibited phosphorylation of extracellular signal-regulated kinase and stress-activated protein kinase, as well as ubiquitin-dependent degradation of inhibitor κBα (IκBα). In accordance with IκBα degradation, nuclear accumulation of NF-κB and subsequent binding to NF-κB cis-acting element was attenuated by treatment with RBITC. These data suggest RBITC should be included in the dietary armamentarium of isothiocyanates potentially capable of mediating antiinflammatory or cancer chemopreventive activity.

  16. Selective Irreversible Inhibition of Neuronal and Inducible Nitric-oxide Synthase in the Combined Presence of Hydrogen Sulfide and Nitric Oxide*

    PubMed Central

    Heine, Christian L.; Schmidt, Renate; Geckl, Kerstin; Schrammel, Astrid; Gesslbauer, Bernd; Schmidt, Kurt; Mayer, Bernd; Gorren, Antonius C. F.

    2015-01-01

    Citrulline formation by both human neuronal nitric-oxide synthase (nNOS) and mouse macrophage inducible NOS was inhibited by the hydrogen sulfide (H2S) donor Na2S with IC50 values of ∼2.4·10−5 and ∼7.9·10−5 m, respectively, whereas human endothelial NOS was hardly affected at all. Inhibition of nNOS was not affected by the concentrations of l-arginine (Arg), NADPH, FAD, FMN, tetrahydrobiopterin (BH4), and calmodulin, indicating that H2S does not interfere with substrate or cofactor binding. The IC50 decreased to ∼1.5·10−5 m at pH 6.0 and increased to ∼8.3·10−5 m at pH 8.0. Preincubation of concentrated nNOS with H2S under turnover conditions decreased activity after dilution by ∼70%, suggesting irreversible inhibition. However, when calmodulin was omitted during preincubation, activity was not affected, suggesting that irreversible inhibition requires both H2S and NO. Likewise, NADPH oxidation was inhibited with an IC50 of ∼1.9·10−5 m in the presence of Arg and BH4 but exhibited much higher IC50 values (∼1.0–6.1·10−4 m) when Arg and/or BH4 was omitted. Moreover, the relatively weak inhibition of nNOS by Na2S in the absence of Arg and/or BH4 was markedly potentiated by the NO donor 1-(hydroxy-NNO-azoxy)-l-proline, disodium salt (IC50 ∼ 1.3–2.0·10−5 m). These results suggest that nNOS and inducible NOS but not endothelial NOS are irreversibly inhibited by H2S/NO at modest concentrations of H2S in a reaction that may allow feedback inhibition of NO production under conditions of excessive NO/H2S formation. PMID:26296888

  17. Nitric oxide and calcium ions in apoptotic esophageal carcinoma cells induced by arsenite

    PubMed Central

    Shen, Zhong-Ying; Shen, Wen-Ying; Chen, Ming-Hua; Shen, Jian; Cai, Wei-Jie; Yi, Zeng

    2002-01-01

    AIM: To Quantitatively analyze the nitri oxide (NO) and Ca2+ in apoptosis of esophageal carcinoma cells induced by arsenic trioxide (As2O3). METHODS: The cell line SHEEC1, a malignant esophageal epithelial cell induced by HPV in synergy with TPA in our laboratory, was cultured in a serum-free medium and treated with As2O3. Before and after administration of As2O3, NO production in cultured medium was detected quantitatively using the Griess Colorimetric method. Intracellular Ca2+ was labeled by using the fluorescent dye Fluo3-AM and detected under confocal laser scanning microscope (CLSM), which was able to acquire data in real-time enabling Ca2+ dynamics of individual cells in vitro. The apoptotic cells were examined under electron microscopy. RESULTS: Intracellular concentration of Ca2+ increased from 1.00 units to 1.09-1.38 units of fluorescent intensity at As2O3 treatment and NO products subsequently released from As2O3-treated cells increased from 0.98-1.00 × 10-2 μmol·L-1 up to 1.48-1.52 × 10-2 μmol·L-1 and maintained in a high level continuously. Finally apoptosis of cells occurred, chromatin being agglutinated, cells shrunk, nuclei became round and mitochondria swelled. CONCLUSION: Ca2+ and NO increased with cell damage and apoptosis in cells treated by As2O3. The Ca2+ is an initial messenger to the apoptotic pathway. To investigate Ca2+ and NO will be a new direction for studying the apoptotic signaling messenger of the esophageal carcinoma cells induced by As2O3. PMID:11833068

  18. Expression of inducible nitric oxide synthase causes delayed neurotoxicity in primary mixed neuronal-glial cortical cultures.

    PubMed

    Dawson, V L; Brahmbhatt, H P; Mong, J A; Dawson, T M

    1994-11-01

    Nitric oxide (NO) is a potent biological messenger molecule in the central nervous system (CNS). There are several potential sources of NO production in the CNS, including neurons and endothelial cells which express NO synthase (NOS) constitutively. Astrocytes and microglia can be induced by cytokines to express a NOS isoform similar to macrophage NOS (mNOS). Primary mixed glial cultures exposed to lipopolysaccharide (LPS) or a combination of LPS and gamma-interferon (INF-gamma) produce nitrite, a breakdown product of NO formation, in a dose-dependent manner. Nitrite production is detectable at 12 hr, peaks at 48 hr and is sustained for at least 96 hr. The NOS inhibitor, nitro-L-arginine (NArg), inhibits nitrite formation, but the immunosuppressant agent, FK506, does not. In mixed glial-neuronal cultures exposed to 50 ng LPS or 5 ng LPS and 1 microgram INF-gamma, neurons begin to die at 48 hr, approx. 24-36 hr after detectable nitrite production. Neurotoxicity is attenuated by 100 microM NArg. These data indicate that expression of inducible mNOS causes delayed neurotoxicity.

  19. A small molecule deubiquitinase inhibitor increases localization of inducible nitric oxide synthase to the macrophage phagosome and enhances bacterial killing.

    PubMed

    Burkholder, Kristin M; Perry, Jeffrey W; Wobus, Christiane E; Donato, Nicholas J; Showalter, Hollis D; Kapuria, Vaibhav; O'Riordan, Mary X D

    2011-12-01

    Macrophages are key mediators of antimicrobial defense and innate immunity. Innate intracellular defense mechanisms can be rapidly regulated at the posttranslational level by the coordinated addition and removal of ubiquitin by ubiquitin ligases and deubiquitinases (DUBs). While ubiquitin ligases have been extensively studied, the contribution of DUBs to macrophage innate immune function is incompletely defined. We therefore employed a small molecule DUB inhibitor, WP1130, to probe the role of DUBs in the macrophage response to bacterial infection. Treatment of activated bone marrow-derived macrophages (BMM) with WP1130 significantly augmented killing of the intracellular bacterial pathogen Listeria monocytogenes. WP1130 also induced killing of phagosome-restricted bacteria, implicating a bactericidal mechanism associated with the phagosome, such as the inducible nitric oxide synthase (iNOS). WP1130 had a minimal antimicrobial effect in macrophages lacking iNOS, indicating that iNOS is an effector mechanism for WP1130-mediated bacterial killing. Although overall iNOS levels were not notably different, we found that WP1130 significantly increased colocalization of iNOS with the Listeria-containing phagosome during infection. Taken together, our data indicate that the deubiquitinase inhibitor WP1130 increases bacterial killing in macrophages by enhancing iNOS localization to the phagosome and suggest a potential role for ubiquitin regulation in iNOS trafficking.

  20. Involvement of nitric oxide in the mechanism of biochemical alterations induced by simulated microgravity in Microcystis aeruginosa

    NASA Astrophysics Data System (ADS)

    Xiao, Yuan; Liu, Yongding; Wang, Gaohong

    2012-03-01

    Simulated microgravity (SMG) can inhibit proliferation and enhance microcystin production of Microcystis aeruginosa. We investigated the role of nitric oxide (NO) in regulating the SMG induced changes of proliferation, photochemical system II photochemical activity, pigment, soluble protein and microcystin production in M. aeruginosa. M. aeruginosa was exposed to 0.1 mM sodium nitroprusside (SNP, NO donor) or 0.02 mM 2-(4-carboxyphenyl)-4, 4, 5, 5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO, NO scavenger) alone or in combination with SMG for 48 h. SMG and SNP inhibited the growth of M. aeruginosa while c-PTIO had no effect on cell number. As to yield, the negative effect of SMG was augmented by SNP and suppressed by c-PTIO. The intracellular concentrations of chlorophyll a, carotenoid, phycocyanin, soluble protein and microcystin were increased by SMG after 48 h. The effects of SMG on these metabolic processes could be enhanced by SNP and be partly eliminated by c-PTIO. Moreover, SNP and c-PTIO only functioned in these biochemical processes under SMG, unlike in the regulation of cell proliferation and yield. These results showed that the effects of SMG could be enhanced by adding exogenous NO and be mitigated by scavenging endogenous NO, revealing the involvement of NO in the changes in biochemistry processes induced by SMG in M. aeruginosa.

  1. The Dynamics of the Defense Strategy of Pea Induced by Exogenous Nitric Oxide in Response to Aphid Infestation

    PubMed Central

    Woźniak, Agnieszka; Formela, Magda; Bilman, Piotr; Grześkiewicz, Katarzyna; Bednarski, Waldemar; Marczak, Łukasz; Narożna, Dorota; Dancewicz, Katarzyna; Mai, Van Chung; Borowiak-Sobkowiak, Beata; Floryszak-Wieczorek, Jolanta; Gabryś, Beata; Morkunas, Iwona

    2017-01-01

    The aim of this study was to investigate the effect of exogenous nitric oxide (NO), i.e., S-nitrosoglutathione (GSNO) and sodium nitroprusside (SNP), on the metabolic status of Pisum sativum L. cv. Cysterski leaves infested by Acyrthosiphon pisum Harris, population demographic parameters and A. pisum feeding activity. A reduction in the level of semiquinone radicals in pea seedling leaves pretreated with exogenous NO occurred 24 h after A. pisum infestation, which was earlier than in non-pretreated leaves. A decrease in the level of O2•− was observed in leaves pretreated with GSNO and infested by aphids at 48 and 72 h post-infestation (hpi). Directly after the pretreatment with GSNO, an increase in the level of metal ions was recorded. NO considerably induced the relative mRNA levels for phenylalanine ammonia-lyase in 24-h leaves pretreated with NO donors, both non-infested and infested. NO stimulated the accumulation of pisatin in leaves until 24 h. The Electrical Penetration Graph revealed a reduction in the feeding activity of the pea aphid on leaves pretreated with NO. The present study showed that foliar application of NO donors induced sequentially defense reactions of pea against A. pisum and had a deterrent effect on aphid feeding and limited the population growth rate. PMID:28165429

  2. Serum depletion induced cancer stem cell-like phenotype due to nitric oxide synthesis in oncogenic HRas transformed cells

    PubMed Central

    Monji, Keisuke; Uchiumi, Takeshi; Hoshizawa, Saki; Yagi, Mikako; Matsumoto, Takashi; Setoyama, Daiki; Matsushima, Yuichi; Gotoh, Kazuhito; Amamoto, Rie; Kang, Donchon

    2016-01-01

    Cancer cells rewire their metabolism and mitochondrial oxidative phosphorylation (OXPHOS) to promote proliferation and maintenance. Cancer cells use multiple adaptive mechanisms in response to a hypo-nutrient environment. However, little is known about how cancer mitochondria are involved in the ability of these cells to adapt to a hypo-nutrient environment. Oncogenic HRas leads to suppression of the mitochondrial oxygen consumption rate (OCR), but oxygen consumption is essential for tumorigenesis. We found that in oncogenic HRas transformed cells, serum depletion reversibly increased the OCR and membrane potential. Serum depletion promoted a cancer stem cell (CSC)-like phenotype, indicated by an increase in CSC markers expression and resistance to anticancer agents. We also found that nitric oxide (NO) synthesis was significantly induced after serum depletion and that NO donors modified the OCR. An NOS inhibitor, SEITU, inhibited the OCR and CSC gene expression. It also reduced anchorage-independent growth by promoting apoptosis. In summary, our data provide new molecular findings that serum depletion induces NO synthesis and promotes mitochondrial OXPHOS, leading to tumor progression and a CSC phenotype. These results suggest that mitochondrial OCR inhibitors can be used as therapy against CSC. PMID:27655692

  3. Resident microglia from adult mice are refractory to nitric oxide-inducing stimuli due to impaired NOS2 gene expression.

    PubMed

    Brannan, Courtney A; Roberts, Margo R

    2004-11-01

    Microglia are the immunoregulatory cells of the central nervous system (CNS) and share many characteristics with resident macrophages in extracerebral tissues. Nitric oxide (NO) is secreted by macrophages following induction of the NO synthase gene NOS2 by stimuli elicited during a T-cell response and/or by microbial products. NO regulates both innate and adaptive immune responses, such as killing intracellular pathogens and inhibiting T-cell proliferation. Regulation of NO production by microglia, however, is poorly understood. We find that microglia from healthy adult mice produce negligible amounts of NO compared with resident macrophages during restimulation of peptide-specific CD8 T cells, and therefore cannot block T-cell proliferation. The impaired NO response extends to exogenous NOS2-inducing stimuli, including cytokines, CD40 ligation, and lipopolysaccharide. In contrast, microglia produce proinflammatory cytokines in response to these same stimuli, and therefore possess a relatively selective block in NO production. We go on to show that resident microglia fail to produce detectable levels of either the NOS2 enzyme or NOS2 RNA in response to NO-inducing stimuli. We therefore propose that microglia in the healthy adult brain exist in an "NO-incompetent" state in which NO production is blocked at the level of NOS2 RNA. The inability of resident microglia in the healthy CNS to produce NO may allow these immunoregulatory cells to modulate immune processes temporally, and may serve to protect the CNS from irreparable damage at the onset of infection or injury.

  4. Is there a role of inducible nitric oxide synthase activation in the delayed antiarrhythmic effect of sodium nitrite?

    PubMed

    Demeter-Haludka, Vivien; Juhász, László; Kovác, Mária; Gardi, János; Végh, Ágnes

    2017-04-01

    This study aimed to examine whether inducible nitric oxide synthase (iNOS) plays a role in the delayed antiarrhythmic effect of sodium nitrite. Twenty-one dogs were infused intravenously with sodium nitrite (0.2 μmol·kg(-1)·min(-1)) for 20 min, either in the absence (n = 12) or in the presence of the iNOS inhibitor S-(2-aminoethyl)-isothiourea (AEST) (total dose 2.0 mg·kg(-1) i.v., n = 9). Control dogs (n = 12) were given saline. Twenty-four hours later, all of the dogs were subjected to a 25 min period occlusion of the left anterior descending coronary artery followed by rapid reperfusion. Dogs treated with AEST and nitrite received again AEST prior to the occlusion. Compared with the controls, sodium nitrite markedly reduced the number of ectopic beats, the number and incidence of ventricular tachycardia, and the incidence of ventricular fibrillation during occlusion and increased survival (0% versus 50%) from the combined ischaemia and reperfusion insult. Although AEST completely inhibited iNOS activity, the nitrite-induced increase in NO bioavailability during occlusion was not substantially modified. Furthermore, AEST attenuated but did not completely abolish the antiarrhythmic effect of nitrite. The marked delayed antiarrhythmic effect of sodium nitrite is not entirely due to the activation of iNOS; other mechanisms may certainly play a role.

  5. Serum depletion induced cancer stem cell-like phenotype due to nitric oxide synthesis in oncogenic HRas transformed cells.

    PubMed

    Monji, Keisuke; Uchiumi, Takeshi; Hoshizawa, Saki; Yagi, Mikako; Matsumoto, Takashi; Setoyama, Daiki; Matsushima, Yuichi; Gotoh, Kazuhito; Amamoto, Rie; Kang, Donchon

    2016-11-15

    Cancer cells rewire their metabolism and mitochondrial oxidative phosphorylation (OXPHOS) to promote proliferation and maintenance. Cancer cells use multiple adaptive mechanisms in response to a hypo-nutrient environment. However, little is known about how cancer mitochondria are involved in the ability of these cells to adapt to a hypo-nutrient environment. Oncogenic HRas leads to suppression of the mitochondrial oxygen consumption rate (OCR), but oxygen consumption is essential for tumorigenesis. We found that in oncogenic HRas transformed cells, serum depletion reversibly increased the OCR and membrane potential. Serum depletion promoted a cancer stem cell (CSC)-like phenotype, indicated by an increase in CSC markers expression and resistance to anticancer agents. We also found that nitric oxide (NO) synthesis was significantly induced after serum depletion and that NO donors modified the OCR. An NOS inhibitor, SEITU, inhibited the OCR and CSC gene expression. It also reduced anchorage-independent growth by promoting apoptosis. In summary, our data provide new molecular findings that serum depletion induces NO synthesis and promotes mitochondrial OXPHOS, leading to tumor progression and a CSC phenotype. These results suggest that mitochondrial OCR inhibitors can be used as therapy against CSC.

  6. The volatile oil of Nardostachyos Radix et Rhizoma induces endothelial nitric oxide synthase activity in HUVEC cells.

    PubMed

    Maiwulanjiang, Maitinuer; Bi, Cathy W C; Lee, Pinky S C; Xin, Guizhong; Miernisha, Abudureyimu; Lau, Kei M; Xiong, Aizhen; Li, Ning; Dong, Tina T X; Aisa, Haji A; Tsim, Karl W K

    2015-01-01

    Nardostahyos Radix et Rhizoma (NRR; the root and rhizome of Nardostachys jatamansi DC.) is a widely used medicinal herb. Historically, NRR is being used for the treatment of cardiovascular and neurological diseases. To search for active ingredients of NRR, we investigated the vascular benefit of NRR volatile oil in (i) the vasodilation in rat aorta ring, and (ii) the release of nitric oxide (NO) and the phosphorylation of endothelial NO synthase (eNOS) in cultured human umbilical vein endothelial cells (HUVECs). By measuring the fluorescence signal in cultures, application of NRR volatile oil resulted in a rapid activation of NO release as well as the phosphorylation of eNOS: both inductions were markedly reduced by L-NAME. In parallel, the phosphorylation level of Akt kinase was markedly increased by the oil treatment, which was partially attenuated by PI3K/Akt inhibitor LY294002. This inhibitor also blocked the NRR-induced NO production and eNOS phosphorylation. In HUVECs, application of NRR volatile oil elevated the intracellular Ca(2+) level, and BAPTA-AM, a Ca(2+) chelator, reduced the Ca(2+) surge: the blockage were also applied to NRR-induced eNOS phosphorylation and NO production. These findings suggested the volatile oil of NRR was the major ingredient in triggering the vascular dilatation, and which was mediated via the NO production.

  7. Benidipine, a dihydropyridine-calcium channel blocker, inhibits lysophosphatidylcholine-induced endothelial injury via stimulation of nitric oxide release.

    PubMed

    Matsubara, Masahiro; Yao, Kozo; Hasegawa, Kazuhide

    2006-01-01

    Benidipine hydrochloride (benidipine), which is a long-lasting dihydropyridine calcium channel blocker, exerts antihypertensive action via inhibition of Ca(2+) influx through L-type voltage-dependent calcium channels. In addition, benidipine is shown to restore endothelial function. However, the mechanisms whereby benidipine has protective effects on endothelium are poorly defined. Nitric oxide (NO), which is produced by endothelial NO synthase (eNOS), plays important roles in endothelial function. In this study, we examined effects of benidipine on NO production from human umbilical vein endothelial cells. Benidipine (0.3-10 microM) augmented eNOS expression and total eNOS enzymatic activities. Benidipine also promoted the production of NO and the accumulation of cGMP, a second messenger of NO. Lysophosphatidylcholine (lysoPC), a component of oxidized low-density lipoproteins, induced caspase-3 activation followed by apoptosis of endothelial cells. Benidipine (0.3-10 microM) prevented lysoPC-induced caspase-3 activation, which was canceled by Nomega-nitro-L-arginine-methyl ester (L-NAME) (250-2500 microM), an inhibitor of NOS. Moreover, diethylenetetraamine NONOate (30-100 microM), a NO donor, inhibited the caspase-3 activation. These results suggested that the increase in NO production by benidipine might be involved in the inhibition of caspase induction. The direct enhancement of endothelial NO release by benidipine may be in part responsible for amelioration of endothelial dysfunction.

  8. The Volatile Oil of Nardostachyos Radix et Rhizoma Induces Endothelial Nitric Oxide Synthase Activity in HUVEC Cells

    PubMed Central

    Maiwulanjiang, Maitinuer; Bi, Cathy W. C.; Lee, Pinky S. C.; Xin, Guizhong; Miernisha, Abudureyimu; Lau, Kei M.; Xiong, Aizhen; Li, Ning; Dong, Tina T. X.; Aisa, Haji A.; Tsim, Karl W. K.

    2015-01-01

    Nardostahyos Radix et Rhizoma (NRR; the root and rhizome of Nardostachys jatamansi DC.) is a widely used medicinal herb. Historically, NRR is being used for the treatment of cardiovascular and neurological diseases. To search for active ingredients of NRR, we investigated the vascular benefit of NRR volatile oil in (i) the vasodilation in rat aorta ring, and (ii) the release of nitric oxide (NO) and the phosphorylation of endothelial NO synthase (eNOS) in cultured human umbilical vein endothelial cells (HUVECs). By measuring the fluorescence signal in cultures, application of NRR volatile oil resulted in a rapid activation of NO release as well as the phosphorylation of eNOS: both inductions were markedly reduced by L-NAME. In parallel, the phosphorylation level of Akt kinase was markedly increased by the oil treatment, which was partially attenuated by PI3K/Akt inhibitor LY294002. This inhibitor also blocked the NRR-induced NO production and eNOS phosphorylation. In HUVECs, application of NRR volatile oil elevated the intracellular Ca2+ level, and BAPTA-AM, a Ca2+ chelator, reduced the Ca2+ surge: the blockage were also applied to NRR-induced eNOS phosphorylation and NO production. These findings suggested the volatile oil of NRR was the major ingredient in triggering the vascular dilatation, and which was mediated via the NO production. PMID:25643147

  9. Nitric oxide released from JS-K induces cell death by mitotic catastrophe as part of necrosis in glioblastoma multiforme.

    PubMed

    Günzle, Jessica; Osterberg, Nadja; Saavedra, Joseph E; Weyerbrock, Astrid

    2016-09-01

    The nitric oxide (NO) donor JS-K is specifically activated by glutathione S-transferases (GSTs) in GST-overexpressing cells. We have shown the induction of cell death in glioblastoma multiforme (GBM) cells at high JS-K doses but the mechanism remains unclear. The aim of this study was to determine whether NO-induced cell death is triggered by induction of apoptotic or necrotic pathways. For the first time, we demonstrate that NO induces cell death via mitotic catastrophe (MC) with non-apoptotic mechanisms in GBM cells. Moreover, the level of morphological changes indicating MC correlates with increased necrosis. Therefore, we conclude that MC is the main mechanism by which GBM cells undergo cell death after treatment with JS-K associated with necrosis rather than apoptosis. In addition, we show that PARP1 is not an exclusive marker for late apoptosis but is also involved in MC. Activating an alternative way of cell death can be useful for the multimodal cancer therapy of GBM known for its strong anti-apoptotic mechanisms and drug resistance.

  10. Lead-induced nitric oxide generation plays a critical role in lead uptake by Pogonatherum crinitum root cells.

    PubMed

    Yu, Qin; Sun, Lian; Jin, Haihong; Chen, Qian; Chen, Zunwei; Xu, Maojun

    2012-10-01

    The effects of lead (Pb) on endogenous nitric oxide (NO) generation, the role of NO in Pb uptake and the origin of Pb-induced NO production in Pogonatherum crinitum root cells were evaluated. Pb treatment induced rapid NO generation, showing that Pb exposure triggered endogenous NO signaling of the cells. Pre-treatment of the cells with the NO-specific scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline -1-oxyl-3-oxide (cPTIO) not only abolished the Pb-triggered NO burst but also reduced Pb contents of the cells. Moreover, Pb exposure enhanced nitrate reductase (NR) activity of the cells. The NR inhibitors tungstate and glutamine not only suppressed the Pb-enhanced NR activities but also reduced the Pb-triggered NO generation. Pre-treatment of the cells with tungstate and glutamine suppressed Pb accumulation and the suppression could be restored by application of exogenous NO via its donors sodium nitroprusside (SNP) and S-nitrosoglutathione (GSNO). Together, our results indicated that Pb exposure enhanced NR activity and triggered the NO burst of P. crinitum root cells. Furthermore, the data demonstrated that NR was responsible for the Pb-triggered NO burst and that NR-mediated NO generation played a critical role in Pb uptake by P. crinitum root cells. Thus, our results suggest a potential strategy for controlling Pb uptake by plants by targeting NR as a source of Pb-triggered NO production.

  11. Single-walled carbon nanotubes induce cell death and transcription of TNF-α in macrophages without affecting nitric oxide production.

    PubMed

    Kim, Kyong Hoon; Yeon, Seung-min; Kim, Hyun Gyung; Lee, Hwanbum; Kim, Sun Kyung; Han, Seung Hyun; Min, Kyung-Jin; Byun, Youngjoo; Lee, Eun Hee; Lee, Kenneth Sung; Yuk, Soon Hong; Ha, Un-Hwan; Jung, Yong Woo

    2014-02-01

    Single-walled carbon nanotubes (SWCNTs) are potent nanomaterials that have diverse shapes and features. The utilization of these molecules for drug delivery is being investigated; thus, it is important to determine whether they alter immune responses against pathogens. In this study, we show that macrophages treated with a mixture of lipopolysaccharide and SWCNTs produced normal levels of nitric oxide and inducible nitric oxide synthase mRNA. However, these treatments induced cell death, presumably via necrosis. In addition, treating cells with SWCNTs induced the expression of tumor necrosis factor-α mRNA, a potent pro-inflammatory cytokine. These results suggest that SWCNTs may influence immune responses, which could result in unexpected effects following their administration for the purpose of drug delivery.

  12. α-Terpineol attenuates morphine-induced physical dependence and tolerance in mice: role of nitric oxide

    PubMed Central

    Parvardeh, Siavash; Moghimi, Mahsa; Eslami, Pegah; Masoudi, Alireza

    2016-01-01

    Objective(s): Dependence and tolerance to opioid analgesics are major problems limiting their clinical application. α-Terpineol is a monoterpenoid alcohol with neuroprotective effects which is found in several medicinal plants such as Myrtus communis, Laurus nobilis, and Stachys byzantina. It has been shown that some of these medicinal plants such as S. byzantina attenuate dependence and tolerance to morphine. Since α-terpineol is one of the bioactive phytochemical constituent of these medicinal plants, the present study was conducted to investigate the effects of α-terpineol on morphine-induced dependence and tolerance in mice. Materials and Methods: The mice were rendered dependent or tolerant to morphine by a 3-day administration schedule. The hot-plate test and naloxone-induced withdrawal syndrome were used to evaluate tolerance and dependence on morphine, respectively. To investigate a possible role for nitric oxide (NO) in the protective effect of α-terpineol, the NO synthase inhibitor, L-N(G)-nitroarginine methyl ester (L-NAME) and NO precursor, L-arginine, were used. Results: Administration of α-terpineol (5, 10, and 20 mg/kg, IP) significantly decreased the number of jumps in morphine dependent animals. Moreover, α-terpineol (20 and 40 mg/kg, IP) attenuated tolerance to the analgesic effect of morphine. The inhibitory effects of α-terpineol on morphine-induced dependence and tolerance were enhanced by pretreatment with L-NAME (10 mg/kg, IP). However, L-arginine (300 mg/kg, IP) antagonized the protective effects of α-terpineol on dependence and tolerance to morphine. Conclusion: These findings indicate that α-terpineol prevents the development of dependence and tolerance to morphine probably through the influence on NO production. PMID:27081466

  13. Hypoxia and Reoxygenation Induce Endothelial Nitric Oxide Synthase Uncoupling in Endothelial Cells through Tetrahydrobiopterin Depletion and S-Glutathionylation

    PubMed Central

    2015-01-01

    Ischemia-reperfusion injury is accompanied by endothelial hypoxia and reoxygenation that trigger oxidative stress with enhanced superoxide generation and diminished nitric oxide (NO) production leading to endothelial dysfunction. Oxidative depletion of the endothelial NO synthase (eNOS) cofactor tetrahydrobiopterin can trigger eNOS uncoupling, in which the enzyme generates superoxide rather than NO. Recently, it has also been shown that oxidative stress can induce eNOS S-glutathionylation at critical cysteine residues of the reductase site that serves as a redox switch to control eNOS coupling. While superoxide can deplete tetrahydrobiopterin and induce eNOS S-glutathionylation, the extent of and interaction between these processes in the pathogenesis of eNOS dysfunction in endothelial cells following hypoxia and reoxygenation remain unknown. Therefore, studies were performed on endothelial cells subjected to hypoxia and reoxygenation to determine the severity of eNOS uncoupling and the role of cofactor depletion and S-glutathionylation in this process. Hypoxia and reoxygenation of aortic endothelial cells triggered xanthine oxidase-mediated superoxide generation, causing both tetrahydrobiopterin depletion and S-glutathionylation with resultant eNOS uncoupling. Replenishing cells with tetrahydrobiopterin along with increasing intracellular levels of glutathione greatly preserved eNOS activity after hypoxia and reoxygenation, while targeting either mechanism alone only partially ameliorated the decrease in NO. Endothelial oxidative stress, secondary to hypoxia and reoxygenation, uncoupled eNOS with an altered ratio of oxidized to reduced glutathione inducing eNOS S-glutathionylation. These mechanisms triggered by oxidative stress combine to cause eNOS dysfunction with shift of the enzyme from NO to superoxide production. Thus, in endothelial reoxygenation injury, normalization of both tetrahydrobiopterin levels and the glutathione pool are needed for maximal

  14. Exogenous Nitric Oxide Suppresses in Vivo X-ray-Induced Targeted and Non-Targeted Effects in Zebrafish Embryos

    PubMed Central

    Kong, E.Y.; Yeung, W.K.; Chan, T.K.Y.; Cheng, S.H.; Yu, K.N.

    2016-01-01

    The present paper studied the X-ray-induced targeted effect in irradiated zebrafish embryos (Danio rerio), as well as a non-targeted effect in bystander naïve embryos partnered with irradiated embryos, and examined the influence of exogenous nitric oxide (NO) on these targeted and non-targeted effects. The exogenous NO was generated using an NO donor, S-nitroso-N-acetylpenicillamine (SNAP). The targeted and non-targeted effects, as well as the toxicity of the SNAP, were assessed using the number of apoptotic events in the zebrafish embryos at 24 h post fertilization (hpf) revealed through acridine orange (AO) staining. SNAP with concentrations of 20 and 100 µM were first confirmed to have no significant toxicity on zebrafish embryos. The targeted effect was mitigated in zebrafish embryos if they were pretreated with 100 µM SNAP prior to irradiation with an X-ray dose of 75 mGy but was not alleviated in zebrafish embryos if they were pretreated with 20 µM SNAP. On the other hand, the non-targeted effect was eliminated in the bystander naïve zebrafish embryos if they were pretreated with 20 or 100 µM SNAP prior to partnering with zebrafish embryos having been subjected to irradiation with an X-ray dose of 75 mGy. These findings revealed the importance of NO in the protection against damages induced by ionizing radiations or by radiation-induced bystander signals, and could have important impacts on development of advanced cancer treatment strategies. PMID:27529238

  15. Increase of cellular recruitment, phagocytosis ability and nitric oxide production induced by hydroalcoholic extract from Chenopodium ambrosioides leaves.

    PubMed

    Cruz, Gustavo V B; Pereira, Paulo Vitor S; Patrício, Fernando J; Costa, Graciomar C; Sousa, Sanara M; Frazão, Josias B; Aragão-Filho, Walmir C; Maciel, Márcia C G; Silva, Lucilene A; Amaral, Flávia M M; Barroqueiro, Elizabeth S B; Guerra, Rosane N M; Nascimento, Flávia R F

    2007-04-20

    The leaves and the oil from the seeds of Chenopodium ambrosioides L. (Chenopodiaceae), a plant known in Brazil as 'mastruz', have been used by native people to treat parasitic diseases. Experimentally it was shown that Chenopodium ambrosioides inhibits the Ehrlich tumor growth, what could be due to an immunomodulatory effect of this product. The aim of this study was to investigate the effect of hydroalcoholic crude extract (HCE) from leaves of Chenopodium ambrosioides on macrophage activity and on lymphoid organs cellularity. C3H/HePas mice received the HCE (5mg/kg) by intraperitoneal via and were sacrificed 2 days later. HCE treatment did not alter the cell number in bone marrow, but it increased the cell number in peritoneal cavity, spleen and lymph node. The spreading and phagocytosis activity, the PMA-induced hydrogen peroxide (H(2)O(2)) release and the nitric oxide (NO) production were also increased when compared to control group. Similar results were obtained with concanavalin A (Con A), used as a positive control, with exception of the NO production that was only detected in HCE-derived macrophages. The in vitro treatment with HCE induced a dose-dependent NO production by resident macrophages, but did not enhance the NO production by HCE-derived macrophage, which however, was enhanced by Con A, suggesting that HCE and Con A induce NO production by different routes. In conclusion, HCE-treatment was able to increase the macrophages activity and also the cellular recruitment to secondary lymphoid organs, what could explain the previously related anti-tumor activity of Chenopodium ambrosioides.

  16. The comparative proteomics analysis revealed the modulation of inducible nitric oxide on the immune response of scallop Chlamys farreri.

    PubMed

    Sun, Zhibin; Jiang, Qiufen; Wang, Lingling; Zhou, Zhi; Wang, Mengqiang; Yi, Qilin; Song, Linsheng

    2014-10-01

    Nitric oxide (NO) is an important gasotransmitter which plays a key role on the modulation of immune response in all vertebrates and invertebrates. In the present study, the modulation of inducible NO on immune response of scallop Chlamys farreri was investigated via proteomic analysis. Total proteins from hepatopancreas of scallops treated with lipopolysaccharide (LPS) and/or the inhibitor of vertebrate inducible NO synthase (S-methylisothiourea sulfate, SMT) for 12 h were analyzed via 2-D PAGE and ImageMaster 2D Platinum. There were 890, 1189 and 1046 protein spots detected in the groups treated by phosphate buffered saline (PBS), LPS and LPS+SMT, respectively, and 26 differentially expressed protein spots were identified among them. These proteins were annotated with binding or catalytic activity, and most of them were involved in metabolic or cellular processes. Some immune-related or antioxidant-related molecules such as single Ig IL-1-related receptor, guanine nucleotide-binding protein subunit beta-like protein and peroxiredoxin were identified, and the changes of their expression levels in LPS group were intensified significantly after adding SMT. The decreased expression level of tyrosinase and increased level of glutathione S-transferase 4 in LPS group were diametrically reversed by appending SMT. Moreover, interferon stimulated exonuclease gene 20-like protein and copper chaperone for superoxide dismutase were only induced by LPS+SMT stimulation but not by LPS stimulation. These data indicated that NO could modulate many immunity processes in scallop, such as NF-κB transactivation, cytoskeleton reorganization and other pivotal processes, and it was also involved in the energy metabolism, posttranslational modification, detoxification and redox balance during the immune response.

  17. Nitric Oxide-Induced Autophagy in MC3T3-E1 Cells is Associated with Cytoprotection via AMPK Activation

    PubMed Central

    Yang, Jung Yoon; Park, Min Young; Park, Sam Young; Yoo, Hong Il; Kim, Min Seok; Kim, Jae Hyung

    2015-01-01

    Nitric oxide (NO) is important in the regulation of bone remodeling, whereas high concentration of NO promotes cell death of osteoblast. However, it is not clear yet whether NO-induced autophagy is implicated in cell death or survival of osteoblast. The present study is aimed to examine the role of NO-induced autophagy in the MC3T3-E1 cells and their underlying molecular mechanism. The effect of sodium nitroprusside (SNP), an NO donor, on the cytotoxicity of the MC3T3-E1 cells was determined by MTT assay and expression of apoptosis or autophagy associated molecules was evaluated by western blot analysis. The morphological observation of autophagy and apoptosis by acridine orange stain and TUNEL assay were performed, respectively. Treatment of SNP decreased the cell viability of the MC3T3-E1 cells in dose- and time-dependent manner. SNP increased expression levels of p62, ATG7, Beclin-1 and LC3-II, as typical autophagic markers and augmented acidic autophagolysosomal vacuoles, detected by acridine orange staining. However, pretreatment with 3-methyladenine (3MA), the specific inhibitor for autophagy, decreased cell viability, whereas increased the cleavage of PARP and caspase-3 in the SNP-treated MC3T3-E1 cells. AMP-activated protein kinase (AMPK), a major autophagy regulatory kinase, was activated in SNP-treated MC3T3-E1 cells. In addition, pretreatment with compound C, an inhibitor of AMPK, decreased cell viability, whereas increased the number of apoptotic cells, cleaved PARP and caspase-3 levels compared to those of SNP-treated MC3T3-E1 cells. Taken together, it is speculated that NO-induced autophagy functions as a survival mechanism via AMPK activation against apoptosis in the MC3T3-E1 cells. PMID:26557017

  18. Nitric oxide-induced murine hematopoietic stem cell fate involves multiple signaling proteins, gene expression, and redox modulation.

    PubMed

    Nogueira-Pedro, Amanda; Dias, Carolina C; Regina, Helena; Segreto, C; Addios, Priscilla C; Lungato, Lisandro; D'Almeida, Vania; Barros, Carlos C; Higa, Elisa M S; Buri, Marcus V; Ferreira, Alice T; Paredes-Gamero, Edgar Julian

    2014-11-01

    There are a growing number of reports showing the influence of redox modulation in cellular signaling. Although the regulation of hematopoiesis by reactive oxygen species (ROS) and reactive nitrogen species (RNS) has been described, their direct participation in the differentiation of hematopoietic stem cells (HSCs) remains unclear. In this work, the direct role of nitric oxide (NO(•)), a RNS, in the modulation of hematopoiesis was investigated using two sources of NO(•) , one produced by endothelial cells stimulated with carbachol in vitro and another using the NO(•)-donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP) in vivo. Two main NO(•) effects were observed: proliferation of HSCs-especially of the short-term HSCs-and its commitment and terminal differentiation to the myeloid lineage. NO(•)-induced proliferation was characterized by the increase in the number of cycling HSCs and hematopoietic progenitor cells positive to BrdU and Ki-67, upregulation of Notch-1, Cx43, PECAM-1, CaR, ERK1/2, Akt, p38, PKC, and c-Myc. NO(•)-induced HSCs differentiation was characterized by the increase in granulocytic-macrophage progenitors, granulocyte-macrophage colony forming units, mature myeloid cells, upregulation of PU.1, and C/EBPα genes concomitantly to the downregulation of GATA-3 and Ikz-3 genes, activation of Stat5 and downregulation of the other analyzed proteins mentioned above. Also, redox status modulation differed between proliferation and differentiation responses, which is likely associated with the transition of the proliferative to differentiation status. Our findings provide evidence of the role of NO(•) in inducing HSCs proliferation and myeloid differentiation involving multiple signaling.

  19. Nitric oxide modulates cadmium influx during cadmium-induced programmed cell death in tobacco BY-2 cells.

    PubMed

    Ma, Wenwen; Xu, Wenzhong; Xu, Hua; Chen, Yanshan; He, Zhenyan; Ma, Mi

    2010-07-01

    Nitric oxide (NO) is a bioactive gas and functions as a signaling molecule in plants exposed to diverse biotic and abiotic stresses including cadmium (Cd(2+)). Cd(2+) is a non-essential and toxic heavy metal, which has been reported to induce programmed cell death (PCD) in plants. Here, we investigated the role of NO in Cd(2+)-induced PCD in tobacco BY-2 cells (Nicotiana tabacum L. cv. Bright Yellow 2). In this work, BY-2 cells exposed to 150 microM CdCl(2) underwent PCD with TUNEL-positive nuclei, significant chromatin condensation and the increasing expression of a PCD-related gene Hsr203J. Accompanied with the occurring of PCD, the production of NO increased significantly. The supplement of NO by sodium nitroprusside (SNP) had accelerated the PCD, whereas the NO synthase inhibitor Nomega-nitro-L-arginine methyl ester hydrochloride (L-NAME) and NO-specific scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) alleviated this toxicity. To investigate the mechanism by which NO exerted its function, Cd(2+) concentration was measured subsequently. SNP led more Cd(2+) content than Cd(2+) treatment alone. By contrast, the prevention of NO by L-NAME decreased Cd(2+) accumulation. Using the scanning ion-selective electrode technique, we analyzed the pattern and rate of Cd(2+) fluxes. This analysis revealed the promotion of Cd(2+) influxes into cells by application of SNP, while L-NAME and cPTIO reduced the rate of Cd(2+) uptake or even resulted in net Cd(2+) efflux. Based on these founding, we concluded that NO played a positive role in CdCl(2)-induced PCD by modulating Cd(2+) uptake and thus promoting Cd(2+) accumulation in BY-2 cells.

  20. Overexpressed neuroglobin raises threshold for nitric oxide-induced impairment of mitochondrial respiratory activities and stress signaling in primary cortical neurons

    PubMed Central

    Singh, Shilpee; Zhuo, Ming; Gorgun, Murat; Englander, Ella W.

    2013-01-01

    Surges of nitric oxide compromise mitochondrial respiration primarily by competitive inhibition of oxygen binding to cytochrome c oxidase (complex IV) and are particularly injurious in neurons, which rely on oxidative phosphorylation for all their energy needs. Here, we show that transgenic overexpression of the neuronal globin protein, neuroglobin, helps diminish protein nitration, preserve mitochondrial function and sustain ATP content of primary cortical neurons challenged by extended nitric oxide exposure. Specifically, in transgenic neurons, elevated neuroglobin curtailed nitric oxide-induced alterations in mitochondrial oxygen consumption rates, including baseline oxygen consumption, consumption coupled with ATP synthesis, proton leak and spare respiratory capacity. Concomitantly, activation of genes involved in sensing and responding to oxidative/nitrosative stress, including the early-immediate c-Fos gene and the phase II antioxidant enzyme, heme oxygenase-1, was diminished in neuroglobin-overexpressing compared to wild-type neurons. Taken together, these differences reflect a lesser insult produced by similar concentrations of nitric oxide in neuroglobin-overexpressing compared to wild-type neurons, suggesting that abundant neuroglobin buffers nitric oxide and raises the threshold of nitric oxide-mediated injury in neurons. PMID:23587847

  1. Nitric oxide-releasing sulindac is a novel skin cancer chemopreventive agent for UVB-induced photocarcinogenesis

    SciTech Connect

    Chaudhary, Sandeep C.; Singh, Tripti; Kapur, Puneet; Weng, Zhiping; Arumugam, Aadithya; Elmets, Craig A.; Kopelovich, Levy; Athar, Mohammad

    2013-05-01

    Nitric oxide (NO)-releasing non-steroidal anti-inflammatory drugs (NO-NSAIDs) which have been synthesized to reduce gastro-intestinal and cardiovascular toxicities of NSAIDs, possess anti-proliferative, pro-apoptotic and anti-cancer activities. Here, we show that NO-sulindac inhibited UVB-induced skin tumorigenesis in SKH-1 hairless mice. Topical application of NO-sulindac reduced tumor incidence, number (p < 0.05) and volume (p < 0.005) as compared to UVB (alone)-irradiated vehicle-treated mice. An increase in TUNEL-positive cells in skin lesions was accompanied by the enhanced Bax:Bcl-2 ratio. The expression of pro-apoptotic Bax was increased whereas anti-apoptotic Bcl-2 reduced. However, proliferation was identified as the major target of NO-sulindac in this study. A reduced expression of PCNA and cyclin D1 associated with the dampening of cell cycle progression was observed. The mechanism of this inhibition was related to the reduction in UVB-induced Notch signaling pathway. UVB-induced inflammatory responses were diminished by NO-sulindac as observed by a remarkable reduction in the levels of phosphorylated MAP Kinases Erk1/2, p38 and JNK1/2. In this regard, NO-sulindac also inhibited NFκB by enhancing IκBα as evidenced by the reduced expression of iNOS and COX-2, the direct NFκB transcription target proteins. NO-sulindac significantly diminished the progression of benign lesions to invasive carcinomas by suppressing the tumor aggressiveness and retarding epithelial–mesenchymal transition. A marked decrease in the expression of mesenchymal markers such as Fibronectin, N-cadherin, SNAI, Slug and Twist and an increase in epithelial cell polarity marker E-cadherin were noted in NO-sulindac-treated tumors. Our data suggest that NO-sulindac is a potent inhibitor of UVB-induced skin carcinogenesis and acts by targeting proliferation-regulatory pathways. - Highlights: ► NO-sulindac is a potent chemopreventive agent for UVB-induced skin cancer. ► NO

  2. Effects of panax quinquefolium on streptozotocin-induced diabetic rats: role of C-peptide, nitric oxide and oxidative stress

    PubMed Central

    Amin, Kamal Adel; Awad, Ezzat Mohamed; Nagy, Mohammed Ahmad

    2011-01-01

    Background: Insulin-dependent diabetes mellitus are at high risk for vascular disorders as hypertension and nephropathy. Ginseng is one of the most widely used herbal medicines and is reported to have a wide range of therapeutic and pharmacological applications for antioxidant and vasorelaxation although the mechanism is not clear. This study, aimed to evaluate hypoglycemic, antioxidant and vasodilator effects of Panax quinquefolium aqueous ginseng extract (AGE) against streptozotocin (STZ)-induced diabetes in male rats. Furthermore explore the role of AGE in C-peptide and nitric oxide (NO) and their relation in STZ induced diabetic rats. Methods: Thirty White male Sprague daw-ley rats weighing 150-200 gm, about 4 month old were equally divided into the following: a control group (normal, nondiabetic), a diabetic group induced by intraperitoneal (I/P) injection of STZ (non-AGE-treated) and an AGE-treated diabetic group (STZ+AGE) (for 8 days). Serum level of urea, creatinine, glucose, insulin, C-peptide and NO were analyzed. Activities of hepatic glucose-6-phosphatase (G6Pase), hepatic glycogen phosphorylase and the renal antioxidant enzyme, catalase were analyzed. Also renal oxidative stress marker malondialdehyde (MDA) was measured. Results: Data showed that STZ treated rats produced a significant increased level of serum urea, creatinine, glucose, NO and renal MDA. Also, induced significantly higher activities of hepatic G6Pase and glycogen phosphorylase compared with controls, while give significant lowered serum insulin, C-peptide level and renal catalase activity. Whereas treatment with AGE led to a significant amelioration in the hyperglycemia (lower the activity of G6Pase and glycogen phosphorylase), hyperinsulinemia and oxidative stress markers. Besides declining the higher level of renal function test and NO. Conclusions: STZ induced-diabetes (DM) associated with renal function disturbances, hypoinsulinemia, defective antioxidant stability and increased

  3. Inhibition of neutral sphingomyelinase decreases elevated levels of inducible nitric oxide synthase and apoptotic cell death in ocular hypertensive rats

    SciTech Connect

    Aslan, Mutay; Basaranlar, Goksun; Unal, Mustafa; Ciftcioglu, Akif; Derin, Narin; Mutus, Bulent

    2014-11-01

    Endoplasmic reticulum (ER) stress and excessive nitric oxide production via induction of inducible nitric oxide synthase (NOS2) have been implicated in the pathogenesis of neuronal retinal cell death in ocular hypertension. Neutral sphingomyelinase (N-SMase)/ceramide pathway can regulate NOS2 expression, hence this study determined the role of selective neutral sphingomyelinase (N-SMase) inhibition on retinal NOS2 levels, ER stress, apoptosis and visual evoked potentials (VEPs) in a rat model of elevated intraocular pressure (EIOP). NOS2 expression and retinal protein nitration were significantly greater in EIOP and significantly decreased with N-SMase inhibition. A significant increase was observed in retinal ER stress markers pPERK, CHOP and GRP78 in EIOP, which were not significantly altered by N-SMase inhibition. Retinal TUNEL staining showed increased apoptosis in all EIOP groups; however N-SMase inhibition significantly decreased the percent of apoptotic cells in EIOP. Caspase-3, -8 and -9 activities were significantly increased in EIOP and returned to baseline levels following N-SMase inhibition. Latencies of all VEP components were significantly prolonged in EIOP and shortened following N-SMase inhibition. Data confirm the role of nitrative injury in EIOP and highlight the protective effect of N-SMase inhibition in EIOP via down-regulation of NOS2 levels and nitrative stress. - Highlights: • Inhibition of N-SMase decreases NOS2 levels in ocular hypertension. • Inhibition of N-SMase decreases protein nitration in ocular hypertension. • Inhibition of N-SMase decreases caspase activation in ocular hypertension. • Inhibition of N-SMase decreases apoptosis in ocular hypertension.

  4. Modulation of inducible nitric oxide synthase gene expression in RAW 264.7 murine macrophages by Pacific ciguatoxin.

    PubMed

    Kumar-Roiné, Shilpa; Matsui, Mariko; Chinain, Mireille; Laurent, Dominique; Pauillac, Serge

    2008-08-01

    To investigate the possible involvement of the nitric oxide radical (NO) in ciguatera fish poisoning (CFP), the in vitro effects of the main Pacific ciguatoxin (P-CTX-1B) and bacterial lipopolysaccharide (LPS) were comparatively studied on neuroblastoma Neuro-2a and on macrophage RAW 264.7 cell lines. NO accumulation was quantified by measuring nitrite levels in cellular supernatant using Griess reagent while the up-regulation of inducible nitric oxide synthase (iNOS) at the mRNA level was quantified via Real-Time Reverse-Transcription Polymerase Chain Reaction (RT-PCR). P-CTX-1B caused a concentration- and time-dependent induction of iNOS in RAW 264.7 cells but not in Neuro-2a cells. NO production was evidenced by increased nitrite levels in the 10 microM range after 48 h of RAW 264.7 cells exposure to LPS and P-CTX-1B (0.05 microg/ml and 6 nM, respectively). The expression of iNOS mRNA peaked at 8h for LPS then gradually decreased to low level at 48 h. In contrast, a sustained level was recorded with P-CTX-1B in the 8-48 h time interval. The addition of N(omega)-nitro-L-arginine methyl ester (L-NAME), a stereoselective NOS inhibitor, strongly diminished NO formation but had no effect on iNOS mRNA synthesis. The implication of NO in CFP paves the way for new therapies for both western and traditional medicines.

  5. Aloe vera toxic effects: expression of inducible nitric oxide synthase (iNOS) in testis of Wistar rat

    PubMed Central

    Asgharzade, Samira; Rafieian-kopaei, Mahmoud; Mirzaeian, Amin; Reiisi, Somaye; Salimzadeh, Loghman

    2015-01-01

    Objective(s): Nitric oxide (NO), a product of inducible nitric oxide synthase (iNOS), contributes in germ cell apoptosis. This study was aimed to evaluate the effects of Aloe vera gel (AVG) on male Wistar rat reproductive organ, serum NO level, and expression of iNOS gene in leydig cells. Materials and Methods: Adult male Wistar rats (n=36) were used for experiments in three groups. The experimental groups were orally administered with the AVG extract solution once-daily as follow: 150 mg.kg-1; group A, 300 mg.kg-1; group B, and only normal saline; group C (control group). They were mated with untreated females and the reproductive and chemical parameters were assessed for each group, including semen quality, serum testosterone, sperm fertility, gonad and body weight, serum NO concentration (by the Griess method), and iNOS gene expression (using RT-PCR). Results: The testes weight, serum testosterone, as well as sperm count and fertility of the AVG treated groups were significantly reduced when compared to the control (P<0.001). Concentration of serum NO was significantly increased (37.1±4.63 µM) in the administrated group with higher AVG concentration, compared to the control group (P<0.001; 10.19±0.87 µM); however, iNOS mRNA expression was increased in the treated animals (P<0.001). Conclusion: iNOS may play a functional role in spermatogenesis via apoptosis, reducing sperm count, but further studies are needed to illustrate the mechanisms by which AVG exerts its negative effects on spermatogenesis and sperm quality. PMID:26730330

  6. Recombinant arginine deiminase reduces inducible nitric oxide synthase iNOS-mediated neurotoxicity in a coculture of neurons and microglia.

    PubMed

    Yu, Hao-Hsin; Wu, Fe-Lin Lin; Lin, Shan-Erh; Shen, Li-Jiuan

    2008-10-01

    Modulation of nitric oxide (NO) production is considered a promising approach to therapy of diseases involving excessive inducible nitric oxide synthase (iNOS) expression, such as certain neuronal diseases. Recombinant arginine deiminase (rADI, EC3.5.3.6) catalyzes the conversion of L-arginine (L-arg), the sole substrate of NOS for NO production, to L-citrulline (L-cit) and ammonia. To understand the effect of the depletion of L-arg by rADI on NO concentration and neuroprotection, a direct coculture of neuron SHSY5Y cells and microglia BV2 cells treated with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) was used as a model of iNOS induction. The results showed that rADI preserved cell viability (4-fold higher compared with the cells treated with LPS/IFN-gamma only) by the MTT assay, corresponding with the results of neuronal viability by neuron-specific immunostaining assay. NO production (mean +/- SD) decreased from 67.0 +/- 1.3 to 19.5 +/- 5.5 microM after a 2-day treatment of rADI by the Griess assay; meanwhile, induction of iNOS protein expression by rADI was observed. In addition, rADI substantially preserved the neuronal function of dopamine uptake in the coculture. The replenishment of L-arg in the coculture eliminated the neuroprotective and NO-suppressive effects of rADI in the coculture, indicating that L-arg played a crucial role in the effects of rADI. These results highlight the important role of L-arg in the neuron-microglia coculture in excessive induction of iNOS. Regulation of L-arg by ADI demonstrated that rADI has a potentially therapeutic role in iNOS-related neuronal diseases.

  7. Inducible nitric oxide synthase activity contributes to the regulation of peripheral vascular tone in patients with cirrhosis and ascites

    PubMed Central

    Ferguson, J W; Dover, A R; Chia, S; Cruden, N L M; Hayes, P C; Newby, D E

    2006-01-01

    Background Overexpression of inducible nitric oxide synthase (iNOS) and increased nitric oxide generation may be associated with the hyperdynamic circulation of patients with cirrhosis. We have, for the first time, used the highly selective iNOS inhibitor, 1400W, to determine whether iNOS activity contributes to the regulation of vascular tone in patients with cirrhosis and ascites. Methods Bilateral forearm blood flow was measured using strain gauge plethysmography in eight patients with cirrhosis and ascites, and eight matched healthy volunteers during intrabrachial infusion of 1400W (0.1–1 μmol/min), NG‐monomethyl‐L‐arginine (L‐NMMA, a non‐selective NOS inhibitor; 2–8 μmol), and norepinephrine (a control vasoconstrictor; 60–480 pmol/min). Results In patients with cirrhosis, 1400W, L‐NMMA, and norepinephrine caused dose dependent reductions in forearm blood flow: peak reductions of 11 (5)%, 37 (4)%, and 48 (5)%, respectively (p<0.05 for all). In contrast, 1400W had no effect on blood flow (+4 (8)%; NS) in healthy controls despite similar reductions in blood flow with L‐NMMA and norepinephrine (39 (5)% and 49 (5)%, respectively; p<0.05 for both). Conclusions We have, for the first time, demonstrated that 1400W causes peripheral vasoconstriction in patients with cirrhosis but not healthy matched controls. This suggests that iNOS contributes to the regulation of peripheral vascular tone in patients with cirrhosis and ascites, and may contribute towards the hyperdynamic circulation associated with this condition. PMID:16299035

  8. Hypoxia-inducible factor-1 modulates the expression of vascular endothelial growth factor and endothelial nitric oxide synthase induced by eccentric exercise.

    PubMed

    Rodriguez-Miguelez, Paula; Lima-Cabello, Elena; Martínez-Flórez, Susana; Almar, Mar; Cuevas, María J; González-Gallego, Javier

    2015-04-15

    The present study investigated the effects of acute and chronic eccentric exercise on the hypoxia-inducible factor (HIF)-1α activation response and the concomitant modulation of vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) expression in rat skeletal muscle. Twenty-four male Wistar rats were randomly assigned to three experimental groups: rested control group, acutely exercised group after an intermittent downhill protocol for 90 min, and acutely exercise group with a previous eccentric training of 8 wk. HIF-1α activation, VEGF and eNOS gene expression, protein content, and promoter activation were assessed in vastus lateralis muscle biopsies. Acute eccentric exercise induced a marked activation of HIF-1α and resulted in increased VEGF and eNOS mRNA level and protein concentration. The binding of HIF-1α to the VEGF and eNOS promoters, measured by a chromatin immunoprecipitation assay, was undetectable in rested rats, whereas it was evident in acutely exercised animals. Acute exercise also increased myeloperoxidase, toll-like receptor-4, tumor necrosis factor-α, and interleukin-1β protein content, suggesting a contribution of proinflammatory stimuli to HIF-1α activation and VEGF overexpression. All of these effects were partially abolished by training. Moreover, training resulted in an increased capillary density. In summary, our findings indicate that eccentric exercise prompts an HIF-1α response in untrained skeletal muscle that contributes to the upregulation of VEGF and eNOS gene expression and is attenuated after an eccentric training program.

  9. A possible role for nitric oxide in glutamate (MSG)-induced Chinese restaurant syndrome, glutamate-induced asthma, 'hot-dog headache', pugilistic Alzheimer's disease, and other disorders.

    PubMed

    Scher, W; Scher, B M

    1992-07-01

    Endogenous glutamate is thought to be a major neurotransmitter. After binding to a cell membrane receptor there can be a stimulation of what can be called the nitric oxide (NO)-mediated neurotransmission pathway (NO-MNP). The activity of the enzyme that produces NO from arginine, NO synthase, and the level of NO become elevated. NO has little activity within the cell in which it is produced, but it rapidly leaks out of that cell and produces effects in neighboring cells. The NO-MNP can be activated to release NO in endothelial cells which in turn acts on neighboring vascular smooth muscle cells to induce vasodilation. Therefore, we suggest that exogenous, ingested glutamate, like endogenous glutamate, can lead to the same stimulation of the NO-MNP in sensitive individuals which would then cause the symptoms of the Chinese restaurant syndrome and/or glutamate-induced asthma. Further, since ingested nitrite and related compounds can be metabolized to NO, NO may more directly cause the symptoms of 'hot dog headache'. In addition, it has been suggested that NO production can also be controlled in endothelial cells by fluid forces that stimulate pressure receptors. Therefore, elevations of NO and stimulation of the NO-MNP may occur due to sudden, local, alterations of blood pressure during pugilistic activities and play a role in the symptoms of pugilistic Alzheimer's disease. If these ideas are correct, then inhibitors of the NO-MNP and/or temporary reduction of the plasma level of arginine may be useful in preventing at least some of the symptoms of these disorders.

  10. Nitric oxide content is associated with tolerance to bicarbonate-induced chlorosis in micropropagated Prunus explants.

    PubMed

    Cellini, Antonio; Corpas, Francisco Javier; Barroso, Juan Bautista; Masia, Andrea

    2011-09-01

    Iron (Fe) chlorosis is a common nutritional deficiency in fruit trees grown in calcareous soils. Grafting on tolerant rootstocks is the most efficient practice to cope with it. In the present work, three Prunus hybrid genotypes, commonly used as peach rootstocks, and one peach cultivar were cultivated with bicarbonate in the growth medium. Parameters describing oxidative stress and the metabolism of reactive nitrogen species were studied. Lower contents of nitric oxide and a decreased nitrosoglutathione reductase activity were found in the most sensitive genotypes, characterized by higher oxidative stress and reduced antioxidant defense. In the peach cultivar, which behaved as a tolerant genotype, a specifically nitrated polypeptide was found.

  11. Central blockade of nitric oxide transmission impairs exercise-induced neuronal activation in the PVN and reduces physical performance.

    PubMed

    Lima, Paulo M A; Santiago, Henrique P; Szawka, Raphael E; Coimbra, Cândido C

    2014-09-01

    The blockade of central nitric oxide (NO) signaling modifies the thermoregulatory and metabolic adjustments that occur during exercise, thereby impairing physical performance. However, the brain areas involved in this response remain unknown. Nitrergic neurons are present in the hypothalamic areas that are activated during exercise and participate in autonomic and neuroendocrine responses, such as, the hypothalamic paraventricular nucleus (PVN) and the supraoptic nucleus (SON). To investigate whether brain NO signaling affects thermoregulation during exercise through the activation of hypothalamic neurons, rats underwent acute submaximal treadmill exercise (18 mmin(-1), 5% inclination) until fatigue received an intracerebroventricular injection of 1.43 μmol Nω-nitro-l-arginine metil ester (L-NAME), a nitric oxide synthase inhibitor, or saline (SAL). Skin tail temperature (Tsk) and internal body temperature (Ti) were continuously recorded and c-Fos expression was determined in the PVN and the SON. L-NAME treatment reduced physical performance by 48%, which was positively correlated with tail vasodilation capacity, which was reduced by 28%, and negatively correlated with heat storage rate (HSR), which was increased by 38%. Physical exercise until fatigue increased the number of c-Fos-immunoreactive (ir) neurons in the PVN and the SON. L-NAME-treatment significantly reduced the exercise-induced c-Fos expression in the PVN, whereas it had no effect in the SON. Interestingly, the number of c-Fos-ir neurons in the PVN was closely correlated with physical performance and inversely associated with HSR. Thus, the inhibition of central NO attenuates neuronal activation induced by exercise in the PVN, impairs the autonomic regulation of heat dissipation, and anticipates the fatigue. Brain NO seems to play a role in exercise performance through the regulation of neuronal activation in the PVN, but not in the SON, although the SON neurons are also activated by running

  12. R59949, a diacylglycerol kinase inhibitor, inhibits inducible nitric oxide production through decreasing transplasmalemmal L-arginine uptake in vascular smooth muscle cells.

    PubMed

    Shimomura, Tomoko; Nakano, Tomoyuki; Goto, Kaoru; Wakabayashi, Ichiro

    2017-02-01

    Although diacylglycerol kinase (DGK) is known to be expressed in vascular smooth muscle cell, its functional significance remains to be clarified. We hypothesized that DGK is involved in the pathway of cytokine-induced nitric oxide (NO) production in vascular smooth muscle cells. The purpose of this study was to investigate the effects of R59949, a diacylglycerol kinase inhibitor, on inducible nitric oxide production in vascular smooth muscle cell. Cultured rat aortic smooth muscle cells (RASMCs) were used to elucidate the effects of R59949 on basal and interleukin-1β (IL-1β)-induced NO production. The effects of R59949 on protein and mRNA expression of induced nitric oxide synthase (iNOS) and on transplasmalemmal L-arginine uptake were also evaluated using RASMCs. Treatment of RASMCs with R59949 (10 μM) inhibited IL-1β (10 ng/ml)-induced NO production but not basal NO production. Neither protein nor mRNA expression level of iNOS after stimulation with IL-1β was significantly affected by R59949. Estimated enzymatic activities of iNOS in RASMCs were comparable in the absence and presence of R59949. Stimulation of RASMCs with IL-1β caused a marked increase in transplasmalemmal L-arginine uptake into RASMCs. L-Arginine uptake in the presence of IL-1β was markedly inhibited by R59949, while basal L-arginine uptake was not significantly affected by R59949. Both IL-1β-induced NO production and L-arginine uptake were abolished in the presence of cycloheximide (1 μM). The results indicate that R59949 inhibits inducible NO production through decreasing transplasmalemmal L-arginine uptake. DGK is suggested to be involved in cytokine-stimulated L-arginine transport and regulate its intracellular concentration in vascular smooth muscle cell.

  13. Pimaradienoic Acid Inhibits Carrageenan-Induced Inflammatory Leukocyte Recruitment and Edema in Mice: Inhibition of Oxidative Stress, Nitric Oxide and Cytokine Production

    PubMed Central

    Casagrande, Rubia; Verri, Waldiceu A.

    2016-01-01

    Pimaradienoic acid (PA; ent-pimara-8(14),15-dien-19-oic acid) is a pimarane diterpene found in plants such as Vigueira arenaria Baker (Asteraceae) in the Brazilian savannas. Although there is evidence on the analgesic and in vitro inhibition of inflammatory signaling pathways, and paw edema by PA, its anti-inflammatory effect deserves further investigation. Thus, the objective of present study was to investigate the anti-inflammatory effect of PA in carrageenan-induced peritoneal and paw inflammation in mice. Firstly, we assessed the effect of PA in carrageenan-induced leukocyte recruitment in the peritoneal cavity and paw edema and myeloperoxidase activity. Next, we investigated the mechanisms involved in the anti-inflammatory effect of PA. The effect of PA on carrageenan-induced oxidative stress in the paw skin and peritoneal cavity was assessed. We also tested the effect of PA on nitric oxide, superoxide anion, and inflammatory cytokine production in the peritoneal cavity. PA inhibited carrageenan-induced recruitment of total leukocytes and neutrophils to the peritoneal cavity in a dose-dependent manner. PA also inhibited carrageenan-induced paw edema and myeloperoxidase activity in the paw skin. The anti-inflammatory mechanism of PA depended on maintaining paw skin antioxidant activity as observed by the levels of reduced glutathione, ability to scavenge the ABTS cation and reduce iron as well as by the inhibition of superoxide anion and nitric oxide production in the peritoneal cavity. Furthermore, PA inhibited carrageenan-induced peritoneal production of inflammatory cytokines TNF-α and IL-1β. PA presents prominent anti-inflammatory effect in carrageenan-induced inflammation by reducing oxidative stress, nitric oxide, and cytokine production. Therefore, it seems to be a promising anti-inflammatory molecule that merits further investigation. PMID:26895409

  14. Unravelling the cardiovascular effects induced by alpha-terpineol: a role for the nitric oxide-cGMP pathway.

    PubMed

    Ribeiro, Thaís P; Porto, Dayanne L; Menezes, Camilla P; Antunes, Alessandra A; Silva, Darízy F; De Sousa, Damião P; Nakao, Lia S; Braga, Valdir A; Medeiros, Isac A

    2010-08-01

    1. Alpha-terpineol is a monoterpene found in the essential oils of several aromatic plant species. In the present study, we investigated the mechanisms underlying the cardiovascular changes induced by alpha-terpineol in rats. 2. In normotensive rats, administration of alpha-terpineol (1, 5, 10, 20 and 30 mg/kg, i.v.) produced a dose-dependent hypotension (-10 +/- 3, -20 +/- 8, -39 +/- 16, -52 +/- 21 and -57 +/- 23 mmHg, respectively; n = 5) followed by tachycardia. The hypotensive responses to 1, 5, 10, 20 and 30 mg/kg, i.v., alpha-terpineol were significantly attenuated following the administration of N(G)-nitro-L-arginine methyl ester (L-NAME; 20 mg/kg, i.v.; -2 +/- 1, -5 +/- 2, -7 +/- 3, -22 +/- 9 and -22 +/- 10 mmHg, respectively; P < 0.05; n = 5). 3. In 10 micromol/L phenylephrine (PE)-precontracted mesenteric artery rings, alpha-terpineol (10(-12) to 10(-5) mol/L) caused a concentration-dependent relaxation (maximum relaxation 61 +/- 6%; n = 7). After removal of the endothelium, the vasorelaxation elicited by alpha-terpineol was attenuated (maximum relaxation 20 +/- 1%; P < 0.05; n = 7). In addition, vasorelaxation induced by alpha-terpineol in rings pretreated with 100 or 300 micromol/L l-NAME, 30 micromol/L hydroxocobalamin or 10 micromol/L 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one was attenuated (maximum relaxation 18 +/- 3, 23 +/- 3, 24 +/- 7 and 21 +/- 1%, respectively; n = 6; P < 0.05). 4. Furthermore, in a rabbit aortic endothelial cell line, 10(-6), 10(-5) and 10(-4) mol/L alpha-terpineol induced concentration-dependent increases in nitric oxide (NO) levels (12 +/- 6, 18 +/- 9 and 34 +/- 12%Delta fluorescence, respectively; n = 3). 5. In conclusion, using combined functional and biochemical approaches in the present study, we were able to demonstrate that alpha-terpineol-induced hypotension and vasorelaxation are mediated, at least in part, by the endothelium, most likely via NO release and activation of the NO-cGMP pathway.

  15. Nitric Oxide-releasing Sulindac is a Novel Skin Cancer Chemopreventive Agent for UVB-induced Photocarcinogenesis

    PubMed Central

    Chaudhary, Sandeep C.; Singh, Tripti; Kapur, Puneet; Weng, Zhiping; Arumugam, Aadithya; Elmets, Craig A.; Kopelovich, Levy; Athar, Mohammad

    2013-01-01

    Nitric oxide (NO)-releasing non-steroidal anti-inflammatory drugs (NO-NSAIDs) which have been synthesized to reduce gastro-intestinal and cardiovascular toxicities of NSAIDs, possess anti-proliferative, pro-apoptotic and anti-cancer activities. Here, we show that NO-sulindac inhibited UVB-induced skin tumorigenesis in SKH-1 hairless mice. Topical application of NO-sulindac reduced tumor incidence, number (p<0.05) and volume (p<0.005) as compared to UVB (alone)-irradiated vehicle-treated mice. An increase in TUNEL-positive cells in skin lesions was accompanied by the enhanced Bax:Bcl-2 ratio. The expression of pro-apoptotic Bax was increased whereas anti-apoptotic Bcl-2 reduced. However, proliferation was identified as the major target of NO-sulindac in this study. A reduced expression of PCNA and cyclin D1 associated with the dampening of cell cycle progression was observed. The mechanism of this inhibition was related to the reduction in UVB-induced Notch signaling pathway. UVB-induced inflammatory responses were diminished by NO-sulindac as observed by a remarkable reduction in the levels of phosphorylated MAP Kinases Erk1/2, p38 and JNK1/2. In this regard, NO-sulindac also inhibited NFκB by enhancing IκBα as evidenced by the reduced expression of iNOS and COX-2, the direct NFκB transcription target proteins. NO-sulindac significantly diminished the progression of benign lesions to invasive carcinomas by suppressing the tumor aggressiveness and retarding epithelial-mesenchymal transition. A marked decrease in the expression of mesenchymal markers such as Fibronectin, N-cadherin, SNAI, Slug and Twist and an increase in epithelial cell polarity marker E-cadherin were noted in NO-sulindac-treated tumors. Our data suggest that NO-sulindac is a potent inhibitor of UVB-induced skin carcinogenesis and acts by targeting proliferation-regulatory pathways. PMID:23274568

  16. Paeonia japonica, Houttuynia cordata, and Aster scaber water extracts induce nitric oxide and cytokine production by lipopolysaccharide-activated macrophages.

    PubMed

    Kim, Jin; Park, Chang-Shin; Lim, Yunsook; Kim, Hyun-Sook

    2009-04-01

    Natural products are increasingly recognized as potential targets for drug discovery and development. We previously reported that Paeonia japonica, Houttuynia cordata, and Aster scaber enhanced macrophage activation both in vitro and in vivo. In the present study we investigated the immunomodulating effects of these plants on lipopolysacharide (LPS)-stimulated macrophages. An aqueous extract of each plant was administered to female BALB/c mice every other day for 4 weeks. Peritoneal macrophages were then collected and incubated to examine the immunoreactivity of macrophages against LPS at different time points. The expression levels of inducible nitric oxide (NO) synthetase (iNOS), cyclooxygenase (COX)-2, and inhibitory factor kappaB alpha (IkappaBalpha) proteins and the production of NO metabolite (nitrite), prostaglandin (PG) E(2), and the pro-inflammatory cytokines interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha were determined in the activated macrophages treated with extracts from each plant individually or combined. High levels of pro-inflammatory cytokines were produced by A. scaber-, P. japonica-, and H. cordata-treated macrophages following 24 hours of LPS stimulation. P. japonica, H. cordata, and A. scaber treatment also induced the production of nitrate by LPS-treated macrophages. Induction of iNOS mRNA and protein was also different in each group. PGE(2) secretion was up-regulated by all extract-treated macrophages at early time points; however, no significant differences were observed between the groups by 8 hours post-LPS stimulation. Treatment with A. scaber extract resulted in the highest levels of IkappaBalpha degradation. Our findings illustrate that the natural plant products P. japonica, H. cordata, and A. scaber may enhance immune function by modulating ex vivo pro-inflammatory cytokine and NO production as well as the expression of iNOS and COX-2.

  17. Neuropeptide Y modulation of interleukin-1{beta} (IL-1{beta})-induced nitric oxide production in microglia.

    PubMed

    Ferreira, Raquel; Xapelli, Sara; Santos, Tiago; Silva, Ana Paula; Cristóvão, Armando; Cortes, Luísa; Malva, João O

    2010-12-31

    Given the modulatory role of neuropeptide Y (NPY) in the immune system, we investigated the effect of NPY on the production of NO and IL-1β in microglia. Upon LPS stimulation, NPY treatment inhibited NO production as well as the expression of inducible nitric-oxide synthase (iNOS). Pharmacological studies with a selective Y(1) receptor agonist and selective antagonists for Y(1), Y(2), and Y(5) receptors demonstrated that inhibition of NO production and iNOS expression was mediated exclusively through Y(1) receptor activation. Microglial cells stimulated with LPS and ATP responded with a massive release of IL-1β, as measured by ELISA. NPY inhibited this effect, suggesting that it can strongly impair the release of IL-1β. Furthermore, we observed that IL-1β stimulation induced NO production and that the use of a selective IL-1 receptor antagonist prevented NO production upon LPS stimulation. Moreover, NPY acting through Y(1) receptor inhibited LPS-stimulated release of IL-1β, inhibiting NO synthesis. IL-1β activation of NF-κB was inhibited by NPY treatment, as observed by confocal microscopy and Western blotting analysis of nuclear translocation of NF-κB p65 subunit, leading to the decrease of NO synthesis. Our results showed that upon LPS challenge, microglial cells release IL-1β, promoting the production of NO through a NF-κB-dependent pathway. Also, NPY was able to strongly inhibit NO synthesis through Y(1) receptor activation, which prevents IL-1β release and thus inhibits nuclear translocation of NF-κB. The role of NPY in key inflammatory events may contribute to unravel novel gateways to modulate inflammation associated with brain pathology.

  18. The Influence of Vitamin D Treatment on the Inducible Nitric Oxide Synthase (INOS) Expression in Primary Hippocampal Neurons

    PubMed Central

    DURSUN, Erdinç; GEZEN-AK, Duygu; YILMAZER, Selma

    2014-01-01

    Introduction Neurodegeneration is a process that is characterized by the loss of neuronal structure and function and eventually ends with neuronal death. An elevated level of inducible nitric oxide synthase (iNOS) is suggested to accompany this process by inducing oxidative and nitrosative damage. Vitamin D is reported to protect glial cells against neurotoxicity via suppressing iNOS synthesis. Though there was no data about whether iNOS is regulated by vitamin D in hippocampal neurons. In this study our aim was to determine any alteration in iNOS expression of hippocampal neurons in response to vitamin D treatment. Method Twenty four and 48 hours of vitamin D treatments were performed on primary hippocampal neuron cultures that were prepared from Sprague dawley rat embryos (E18). The alterations in the iNOS mRNA expression were determined with quantative real time polymerase chain reaction (qRT-PCR). The cytotoxicity levels of each group were investigated by the measurement of lactate dehydrogenase (LDH) that is released to culture medium. Results No difference was observed between groups in 24 hours of treatment regarding the iNOS expression. Though the iNOS mRNA level of vitamin D treated group was significantly lower than that of control group on the 48th hours of treatment (p<.001). Vitamin D treatment also attenuated the LDH release which is an indicator of cytotoxicity (p<.001). Conclusion Our results indicated that vitamin D has the potential to prevent oxidative damage by suppressing iNOS expression.

  19. Inhaled nitric oxide alleviates hyperoxia suppressed phosphatidylcholine synthesis in endotoxin-induced injury in mature rat lungs

    PubMed Central

    Gong, Xiaohui; Guo, Chunbao; Huang, Shibing; Sun, Bo

    2006-01-01

    Background We investigated efficacy of inhaled nitric oxide (NO) in modulation of metabolism of phosphatidylcholine (PC) of pulmonary surfactant and in anti-inflammatory mechanism of mature lungs with inflammatory injury. Methods Healthy adult rats were divided into a group of lung inflammation induced by i.v. lipopolysaccharides (LPS) or a normal control (C) for 24 h, and then exposed to: room air (Air), 95% oxygen (O), NO (20 parts per million, NO), both O and NO (ONO) as subgroups, whereas [3H]-choline was injected i.v. for incorporation into PC of the lungs which were processed subsequently at 10 min, 4, 8, 12 and 24 h, respectively, for measurement of PC synthesis and proinflammatory cytokine production. Results LPS-NO subgroup had the lowest level of labeled PC in total phospholipids and disaturated PC in bronchoalveolar lavage fluid and lung tissue (decreased by 46–59%), along with the lowest activity of cytidine triphosphate: phosphocholine cytidylyltransferase (-14–18%) in the lungs, compared to all other subgroups at 4 h (p < 0.01), but not at 8 and 12 h. After 24-h, all LPS-subgroups had lower labeled PC than the corresponding C-subgroups (p < 0.05). LPS-ONO had higher labeled PC in total phospholipids and disaturated PC, activity of cytidylyltransferase, and lower activity of nuclear transcription factor-κB and expression of proinflammatory cytokine mRNA, than that in the LPS-O subgroup (p < 0.05). Conclusion In LPS-induced lung inflammation in association with hyperoxia, depressed PC synthesis and enhanced proinflammatory cytokine production may be alleviated by iNO. NO alone only transiently suppressed the PC synthesis as a result of lower activity of cytidylyltransferase. PMID:16403237

  20. The nitric oxide-sensitive p21Ras-ERK pathway mediates S-nitrosoglutathione-induced apoptosis

    SciTech Connect

    Tsujita, Maristela; Batista, Wagner L.; Ogata, Fernando T.; Monteiro, Hugo P. Arai, Roberto J.

    2008-05-16

    p21Ras protein plays a critical role in cellular signaling that induces either cell cycle progression or apoptosis. Nitric oxide (NO) has been consistently reported to activate p21Ras through the redox sensitive cysteine residue (118). In this study, we demonstrated that the p21Ras-ERK pathway regulates THP-1 monocyte/macrophage apoptosis induced by S-nitrosoglutathione (SNOG). This was apparent from studies in THP-1 cells expressing NO-insensitive p21Ras (p21Ras{sup C118S}) where the pro-apoptotic action of SNOG was almost abrogated. Three major MAP kinase pathways (ERK, JNK, and p38) that are downstream to p21Ras were investigated. It was observed that only the activation of ERK1/2 MAP kinases by SNOG in THP-1 cells was attributable to p21Ras. The inhibition of the ERK pathway by PD98059 markedly attenuated apoptosis in SNOG-treated THP-1 cells, but had a marginal effect on SNOG-treated THP-1 cells expressing NO-insensitive p21Ras. The inhibition of the JNK and p38 pathways by selective inhibitors had no marked effects on the percentage of apoptosis. The induction of p21Waf1 expression by SNOG was observed in THP-1 cells harboring mutant and wild-type p21Ras, however in cells expressing mutant Ras, the expression of p21Waf1 was significantly attenuated. The treatment of THP-1 cells expressing wild-type p21Ras with PD98059 resulted in significant attenuation of p21Waf1 expression. These results indicate that the redox sensitive p21Ras-ERK pathway plays a critical role in sensing and delivering the pro-apoptotic signaling mediated by SNOG.

  1. The nitric oxide-sensitive p21Ras-ERK pathway mediates S-nitrosoglutathione-induced apoptosis.

    PubMed

    Tsujita, Maristela; Batista, Wagner L; Ogata, Fernando T; Stern, Arnold; Monteiro, Hugo P; Arai, Roberto J

    2008-05-16

    p21Ras protein plays a critical role in cellular signaling that induces either cell cycle progression or apoptosis. Nitric oxide (NO) has been consistently reported to activate p21Ras through the redox sensitive cysteine residue (118). In this study, we demonstrated that the p21Ras-ERK pathway regulates THP-1 monocyte/macrophage apoptosis induced by S-nitrosoglutathione (SNOG). This was apparent from studies in THP-1 cells expressing NO-insensitive p21Ras (p21Ras(C118S)) where the pro-apoptotic action of SNOG was almost abrogated. Three major MAP kinase pathways (ERK, JNK, and p38) that are downstream to p21Ras were investigated. It was observed that only the activation of ERK1/2 MAP kinases by SNOG in THP-1 cells was attributable to p21Ras. The inhibition of the ERK pathway by PD98059 markedly attenuated apoptosis in SNOG-treated THP-1 cells, but had a marginal effect on SNOG-treated THP-1 cells expressing NO-insensitive p21Ras. The inhibition of the JNK and p38 pathways by selective inhibitors had no marked effects on the percentage of apoptosis. The induction of p21Waf1 expression by SNOG was observed in THP-1 cells harboring mutant and wild-type p21Ras, however in cells expressing mutant Ras, the expression of p21Waf1 was significantly attenuated. The treatment of THP-1 cells expressing wild-type p21Ras with PD98059 resulted in significant attenuation of p21Waf1 expression. These results indicate that the redox sensitive p21Ras-ERK pathway plays a critical role in sensing and delivering the pro-apoptotic signaling mediated by SNOG.

  2. Evidence against a role of inducible nitric oxide synthase in the endothelial protective effects of delayed preconditioning

    PubMed Central

    Laude, Karine; Richard, Vincent; Henry, Jean-Paul; Lallemand, Françoise; Thuillez, Christian

    2000-01-01

    Preconditioning the heart with brief periods of ischaemia induces delayed endothelial protection against reperfusion injury, but the precise mechanisms involved in this endogenous protein are still unclear. Induction of the type II-nitric oxide synthase (iNOS) acts as a mediator of the preconditioning against myocardial infarction and stunning. The present study was designed to assess whether iNOS also contributes to the delayed endothelial protective effects of preconditioning. Rats were subjected to 20 min ischaemia followed by 60 min reperfusion 24 h after sham surgery or preconditioning (one cycle or 2 min ischaemia/5 min reperfusion and two cycles of 5 min ischaemia/5 min reperfusion). At the end of the reperfusion, coronary segments were removed distal to the site of occlusion and mounted in wire myographs. Ischaemia-reperfusion (I/R) decreased the endothelium-dependent relaxations to acetylcholine (maximal relaxations: sham, 66±5%; I/R, 40±1%; P<0.05) and this impairment was prevented by preconditioning (maximal relaxation: 61±6%). Administration of N-(3-aminomethyl)benzyl)acetaminide (1400W), a highly selective inhibitor for iNOS, 10 min before prolonged ischaemia did not modify the beneficial effect of preconditioning (maximal relaxation: 66±5%). These data show that preconditioning induces delayed protection against reperfusion-injury. However, in contrast to the myocytes, these endothelial protective effects of delayed preconditioning do not involve iNOS. PMID:10928956

  3. Quinone Induced Activation of Keap1/Nrf2 Signaling by Aspirin Prodrugs Masquerading as Nitric Oxide

    PubMed Central

    Dunlap, Tareisha; Piyankarage, Sujeewa C.; Wijewickrama, Gihani T.; Abdul-Hay, Samer; Vanni, Michael; Litosh, Vladislav; Luo, Jia; Thatcher, Gregory R. J.

    2013-01-01

    The promising therapeutic potential of the NO-donating hybrid aspirin prodrugs (NO-ASA), includes induction of chemopreventive mechanisms, and has been reported in almost 100 publications. One example, NCX-4040 (pNO-ASA), is bioactivated by esterase to a quinone methide (QM) electrophile. In cell cultures, pNO-ASA and QM-donating X-ASA prodrugs that cannot release NO rapidly depleted intracellular GSH and caused DNA damage; however, induction of Nrf2 signaling elicited cellular defense mechanisms including upregulation of NAD(P)H:quinone oxidoreductase-1 (NQO1) and glutamate-cysteine ligase (GCL). In HepG2 cells, the “NO-specific” 4,5-diaminofluorescein reporter, DAF-DA, responded to NO-ASA and X-ASA, with QM-induced oxidative stress masquerading as NO. LC-MS/MS analysis demonstrated efficient alkylation of Cys residues of proteins including glutathione-S-transferase-P1 (GST-P1) and Kelch-like ECH-associated protein 1 (Keap1). Evidence was obtained for alkylation of Keap1 Cys residues associated with Nrf2 translocation to the nucleus, nuclear translocation of Nrf2, activation of antioxidant response element (ARE), and upregulation of cytoprotective target genes. At least in cell culture, pNO-ASA acts as a QM-donor, bioactivated by cellular esterase activity to release salicylates, NO3−, and an electrophilic QM. Finally, two novel aspirin prodrugs were synthesized, both potent activators of ARE, designed to release only the QM and salicylates on bioactivation. Current interest in electrophilic drugs acting via Nrf2 signaling suggests that QM-donating hybrid drugs can be designed as informative chemical probes in drug discovery. PMID:23035985

  4. Aerobic nitric oxide-induced thiol nitrosation in the presence and absence of magnesium cations.

    PubMed

    Kolesnik, Bernd; Heine, Christian L; Schmidt, Renate; Schmidt, Kurt; Mayer, Bernd; Gorren, Antonius C F

    2014-11-01

    Although different routes for the S-nitrosation of cysteinyl residues have been proposed, the main in vivo pathway is unknown. We recently demonstrated that direct (as opposed to autoxidation-mediated) aerobic nitrosation of glutathione is surprisingly efficient, especially in the presence of Mg(2+). In the present study we investigated this reaction in greater detail. From the rates of NO decay and the yields of nitrosoglutathione (GSNO) we estimated values for the apparent rate constants of 8.9 ± 0.4 and 0.55 ± 0.06 M(-1)s(-1) in the presence and absence of Mg(2+). The maximum yield of GSNO was close to 100% in the presence of Mg(2+) but only about half as high in its absence. From this observation we conclude that, in the absence of Mg(2+), nitrosation starts by formation of a complex between NO and O2, which then reacts with the thiol. Omission of superoxide dismutase (SOD) reduced by half the GSNO yield in the absence of Mg(2+), demonstrating O2(-) formation. The reaction in the presence of Mg(2+) seems to involve formation of a Mg(2+)•glutathione (GSH) complex. SOD did not affect Mg(2+)-stimulated nitrosation, suggesting that no O2(-) is formed in that reaction. Replacing GSH with other thiols revealed that reaction rates increased with the pKa of the thiol, suggesting that the nucleophilicity of the thiol is crucial for the reaction, but that the thiol need not be deprotonated. We propose that in cells Mg(2+)-stimulated NO/O2-induced nitrosothiol formation may be a physiologically relevant reaction.

  5. Regulation of inducible nitric oxide synthase expression in beta cells by environmental factors: heavy metals.

    PubMed Central

    Eckhardt, W; Bellmann, K; Kolb, H

    1999-01-01

    The expression of inducible NO synthase (iNOS) in pancreatic islet beta cells modulates endocrine cell functions and, at very high levels of NO production causes beta-cell death. We tested the hypothesis that environmental factors such as heavy-metal salts modulate iNOS expression in beta cells. A rat beta-cell line (insulinoma RINm5F) was cultured in the presence of low-dose interleukin (IL)-1beta for suboptimal induction of iNOS. PbCl2 (0. 1-10 microM) dose-dependently increased NO (measured as nitrite) formation (P<0.001). In contrast, HgCl2 suppressed nitrite production (0.1-10 microM, P<0.05). Measurements of iNOS activity by determining citrulline levels confirmed the potentiating effect of PbCl2 (P<0.05). There was a narrow time window of heavy-metal actions, ranging from -24 h (Hg2+) or -3 h (Pb2+) to +2 h, relative to the addition of IL-1beta. By semi-quantitative reverse transcriptase-PCR, enhanced levels of iNOS mRNA were found in the presence of Pb2+ (P<0.05) and decreased levels in the presence of Hg2+. The amount of iNOS protein as determined by Western blotting was increased in the presence of Pb2+. We conclude that Pb2+ upregulates and Hg2+ suppresses iNOS gene expression at the level of transcription, probably by acting on the signalling pathway. These observations may have important implications for understanding pathological effects of environmental factors on endocrine organ functions. PMID:10051441

  6. Hunterian Lecture. Regulation of hypoxia-inducible factor 1 (HIF-1) by nitric oxide in oral squamous cell carcinoma.

    PubMed Central

    Brennan, P. A.; Quintero, M.

    2005-01-01

    INTRODUCTION: Hypoxia is a common feature of many cancers, contributing to tumour progression as well as potentially compromising radiotherapy and chemotherapy. Hypoxia-inducible factor 1 (HIF-1) is an essential component in changing the transcriptional response of tumours under hypoxia and it targets the transcription of many genes involved in cancer biology. Over-expression of HIF-1 has been associated with increased patient mortality in several cancer types. Regulation of HIF-1 by the signalling molecule nitric oxide (NO) is becoming increasingly recognised. METHOD: Three oral cancer cell lines were used to investigate the effects of NO synthase enzymes (NOS) on HIF-1alpha expression under both normal oxygen and hypoxic conditions. The effect of NOS inhibition was evaluated with the drug L-NMMA. Protein expression was determined with Western blotting. RESULTS & CONCLUSIONS: HIF-1alpha expression occurred following exposure of cells to 3% oxygen for 8 h in all three cell lines, and its expression was found to be dependent on NOS activity, being reduced or inhibited by L-NMMA. Although the mechanism remains to be established, NO appears to play a role in the expression of HIF-1alpha in oral cancer. The possible clinical implications of targeting HIF-1 in cancer are discussed. PMID:15901372

  7. Effects of inducible nitric oxide synthase blockade within the periaqueductal gray on cardiovascular responses during mechanical, heat, and cold nociception.

    PubMed

    Chaitoff, Kevin A; Toner, Francis; Tedesco, Anthony; Maher, Timothy J; Ally, Ahmmed

    2012-02-01

    We have examined the role of inducible nitric oxide synthase (iNOS) within the dorsolateral periaqueductal gray mater (dlPAG) on cardiovascular responses during mechanical, thermal, and cold nociception in anesthetized rats. Mechanical stimulus was applied by a unilateral hindpaw pinch for 10 s that increased mean arterial pressure (MAP) and heart rate (HR). Bilateral microdialysis of a selective iNOS inhibitor, aminoguanidine (AGN; 10 μM), into the dlPAG for 30 min augmented MAP and HR responses during a mechanical stimulation. The cardiovascular responses recovered following discontinuation of the drug. Heat stimulus was generated by immersing one hindpaw metatarsus in a water bath at 52°C for 10 s, and this increased MAP and HR. Administration of AGN into the PAG potentiated these cardiovascular responses. Cardiovascular responses recovered following discontinuation of the drug. In contrast, application of a cold stimulus by immersing one hindpaw at 10°C for 10 s resulted in depressor and bradycardic responses. A second cold stimulus resulted in a response that was not significantly different from that prior to or after recovery from the AGN infusion. These results demonstrate that iNOS within the dlPAG plays a differential role in modulating cardiovascular responses during mechanical-, heat-, and cold-mediated nociception.

  8. Nitric Oxide Is Involved in Cadmium-Induced Programmed Cell Death in Arabidopsis Suspension Cultures1[C][W

    PubMed Central

    De Michele, Roberto; Vurro, Emanuela; Rigo, Chiara; Costa, Alex; Elviri, Lisa; Di Valentin, Marilena; Careri, Maria; Zottini, Michela; Sanità di Toppi, Luigi; Lo Schiavo, Fiorella

    2009-01-01

    Exposure to cadmium (Cd2+) can result in cell death, but the molecular mechanisms of Cd2+ cytotoxicity in plants are not fully understood. Here, we show that Arabidopsis (Arabidopsis thaliana) cell suspension cultures underwent a process of programmed cell death when exposed to 100 and 150 μm CdCl2 and that this process resembled an accelerated senescence, as suggested by the expression of the marker senescence-associated gene12 (SAG12). CdCl2 treatment was accompanied by a rapid increase in nitric oxide (NO) and phytochelatin synthesis, which continued to be high as long as cells remained viable. Hydrogen peroxide production was a later event and preceded the rise of cell death by about 24 h. Inhibition of NO synthesis by NG-monomethyl-arginine monoacetate resulted in partial prevention of hydrogen peroxide increase, SAG12 expression, and mortality, indicating that NO is actually required for Cd2+-induced cell death. NO also modulated the extent of phytochelatin content, and possibly their function, by S-nitrosylation. These results shed light on the signaling events controlling Cd2+ cytotoxicity in plants. PMID:19261736

  9. Nitric oxide attenuates hydrogen peroxide-induced barrier disruption and protein tyrosine phosphorylation in monolayers of intestinal epithelial cell.

    PubMed

    Katsube, Takanori; Tsuji, Hideo; Onoda, Makoto

    2007-06-01

    The intestinal epithelium provides a barrier to the transport of harmful luminal molecules into the systemic circulation. A dysfunctional epithelial barrier is closely associated with the pathogenesis of a variety of intestinal and systemic disorders. We investigated here the effects of nitric oxide (NO) and hydrogen peroxide (H(2)O(2)) on the barrier function of a human intestinal epithelial cell line, Caco-2. When treated with H(2)O(2), Caco-2 cell monolayers grown on permeable supports exhibited several remarkable features of barrier dysfunction as follows: a decrease in transepithelial electrical resistance, an increase in paracellular permeability to dextran, and a disruption of the intercellular junctional localization of the scaffolding protein ZO-1. In addition, an induction of tyrosine phosphorylation of numerous cellular proteins including ZO-1, E-cadherin, and beta-catenin, components of tight and adherens junctions, was observed. On the other hand, combined treatment of Caco-2 monolayers with H(2)O(2) and an NO donor (NOC5 or NOC12) relieved the damage to the barrier function and suppressed the protein tyrosine phosphorylation induced by H(2)O(2) alone. These results suggest that NO protects the barrier function of intestinal epithelia from oxidative stress by modulating some intracellular signaling pathways of protein tyrosine phosphorylation in epithelial cells.

  10. Gene therapy via inducible nitric oxide synthase: a tool for the treatment of a diverse range of pathological conditions.

    PubMed

    McCarthy, Helen O; Coulter, Jonathan A; Robson, Tracy; Hirst, David G

    2008-08-01

    Nitric oxide (NO(.)) is a reactive nitrogen radical produced by the NO synthase (NOS) enzymes; it affects a plethora of downstream physiological and pathological processes. The past two decades have seen an explosion in the understanding of the role of NO(.) biology, highlighting various protective and damaging modes of action. Much of the controversy surrounding the role of NO(.) relates to the differing concentrations generated by the three isoforms of NOS. Both calcium-dependent isoforms of the enzyme (endothelial and neuronal NOS) generate low-nanomolar/picomolar concentrations of NO(.). By contrast, the calcium-independent isoform (inducible NOS (iNOS)) generates high concentrations of NO(.), 2-3 orders of magnitude greater. This review summarizes the current literature in relation to iNOS gene therapy for the therapeutic benefit of various pathological conditions, including various states of vascular disease, wound healing, erectile dysfunction, renal dysfunction and oncology. The available data provide convincing evidence that manipulation of endogenous NO(.) using iNOS gene therapy can provide the basis for future clinical trials.

  11. Evidence for sodium azide as an artifact mediating the modulation of inducible nitric oxide synthase by C-reactive protein.

    PubMed

    Lafuente, Nuria; Azcutia, Verónica; Matesanz, Nuria; Cercas, Elena; Rodríguez-Mañas, Leocadio; Sánchez-Ferrer, Carlos F; Peiró, Concepción

    2005-03-01

    C-reactive protein (CRP) is an acute-phase protein identified as a cardiovascular risk marker. In recent years, an increasing number of studies have investigated the possible direct effects of CRP on the vasculature, using mainly commercial CRP. In the present work, a potential role for CRP as a modulator of inducible nitric oxide synthase (iNOS) induction was explored. Cultured human aortic vascular smooth muscle cells (HASMC) were stimulated for 18 hours with 10 ng/mL interleukin-1beta (IL-1beta), resulting in a marked increase of iNOS levels and NO production, as determined by Western blotting and nitrite measurement, respectively. Commercial CRP (1 to 100 microg/mL) concentration-dependently inhibited the effects elicited by IL-1beta. Unexpectedly, similar results were observed when the commercial CRP solution was replaced by the corresponding vehicle medium containing growing concentrations of sodium azide. The inhibitory effects of commercial CRP or vehicle medium were lost on sodium azide removal by dialysis. In conclusion, sodium azide from the commercial CRP solution, but not CRP itself, mainly accounts for the inhibitory effect on IL-1beta-evoked iNOS induction and NO release. Care should be taken before attributing any biologic role to commercial CRP containing sodium azide.

  12. Role of nitric oxide synthase, superoxide dismutase, and glutathione peroxidase in radiation-induced decrease in norepinephrine release

    SciTech Connect

    Kandasamy, S.B.

    1994-11-17

    Although the central nervous system (CNS) is considered to be relatively resistant to the direct effects of ionizing radiation, the dose and the time elapsed after radiation exposure can have a complex effect on the CNS. The hippocampus is important in critical functions such as learning, memory, and motor performance, and these functions are impaired after exposure to ionizing radiation. Noradrenergic systems are important in mediating arousal, food intake, and to some extent motor functions. Histofluorescence and immunohistochemical techniques have shown noradrenergic pathways in the hippocampus. Several factors can contribute to acute nervous system damage in vivo: (1) reduced systemic blood pressure following exposure to 25-100 Gy of gamma radiation, (2) decreased cerebral blood flow in various regions of the brain, including the hippocampus, (3) ischemia produced by the decreased blood flow, which is likely to affect neuronal activity; (4) free radical generation with resulting oxygen radicals implicated in cell damage following ischemia; (5) brain ischemia-induced release of an excessive amount of glutamate in the hippocampus, which acts on nitric oxide (NO) synthase to form NO through N-methvl-D-aspartate (NMDA) receptors, causing toxic effects.

  13. Inhibition of inducible nitric oxide synthase prevents graft injury after transplantation of livers from rats after cardiac death.

    PubMed

    Shi, Yanjun; Rehman, Hasibur; Wright, Gary L; Zhong, Zhi

    2010-11-01

    This study investigated the roles of inducible nitric oxide synthase (iNOS) in the failure of rat liver grafts from cardiac death donors (GCDD). Livers were explanted after 30-minute aorta clamping and implanted after 4-hour storage in University of Wisconsin solution. The iNOS expression increased slightly in grafts from non-cardiac death donors (GNCDD) but markedly in GCDD. Serum nitrite and nitrate and hepatic 3-nitrotyrosine adducts, indicators of NO and peroxynitrite production, respectively, were substantially higher after transplantation of GCDD than GNCDD. Production of reactive nitrogen species (RNS) was largely blocked by 1400W (N-[1-naphthyl]ethylenediamine dihydrochloride; 5 μM), a specific iNOS inhibitor. Alanine aminotransferase release, bilirubin, necrosis, and apoptosis were 6.4-fold, 6.5-fold, 2.3-fold, and 2.7-fold higher, respectively, after transplantation of GCDD than GNCDD. The inhibitor 1400W effectively blocked these alterations and also increased survival of GCDD to 80% from 33%. Increased RNS production and failure of GCDD were associated with activation of c-Jun-N-terminal kinase (JNK), an effect that was blocked by inhibition of iNOS. Inhibition of JNK also improved the outcome after transplantation of GCDD. Together, the data indicate that iNOS increases substantially in GCDD, leading to RNS overproduction, JNK activation, and more severe graft injury. Inhibitors of iNOS are suggested as effective therapies to improve the outcome after transplantation of GCDD.

  14. Epigenetic regulation of nitric oxide synthase 2, inducible (Nos2) by NLRC4 inflammasomes involves PARP1 cleavage.

    PubMed

    Buzzo, Carina de Lima; Medina, Tiago; Branco, Laura M; Lage, Silvia L; Ferreira, Luís Carlos de Souza; Amarante-Mendes, Gustavo P; Hottiger, Michael O; De Carvalho, Daniel D; Bortoluci, Karina R

    2017-02-02

    Nitric oxide synthase 2, inducible (Nos2) expression is necessary for the microbicidal activity of macrophages. However, NOS2 over-activation causes multiple inflammatory disorders, suggesting a tight gene regulation is necessary. Using cytosolic flagellin as a model for inflammasome-dependent NOS2 activation, we discovered a surprising new role for NLRC4/caspase-1 axis in regulating chromatin accessibility of the Nos2 promoter. We found that activation of two independent mechanisms is necessary for NOS2 expression by cytosolic flagellin: caspase-1 and NF-κB activation. NF-κB activation was necessary, but not sufficient, for NOS2 expression. Conversely, caspase-1 was necessary for NOS2 expression, but dispensable for NF-κB activation, indicating that this protease acts downstream NF-κB activation. We demonstrated that epigenetic regulation of Nos2 by caspase-1 involves cleavage of the chromatin regulator PARP1 (also known as ARTD1) and chromatin accessibility of the NF-κB binding sites located at the Nos2 promoter. Remarkably, caspase-1-mediated Nos2 transcription and NO production contribute to the resistance of macrophages to Salmonella typhimurium infection. Our results uncover the molecular mechanism behind the constricted regulation of Nos2 expression and open new therapeutic opportunities based on epigenetic activities of caspase-1 against infectious and inflammatory diseases.

  15. Simultaneous Laser-induced Fluorescence of Nitric Oxide and Atomic Oxygen in the Hypersonic Materials Environment Test System Arcjet Facility

    NASA Technical Reports Server (NTRS)

    Johansen, Craig; Lincoln, Daniel; Bathel, Brett; Inman, Jennifer; Danehy, Paul

    2014-01-01

    Simultaneous nitric oxide (NO) and atomic oxygen (O) laser induced fluorescence (LIF) experiments were performed in the Hypersonic Materials Environmental Test System (HYMETS) facility at the NASA Langley Research Center. The data serves as an experimental database for validation for chemical and thermal nonequilibrium models used in hypersonic flows. Measurements were taken over a wide range of stagnation enthalpies (6.7 - 18.5 MJ/kg) using an Earth atmosphere simulant with a composition of 75% N2, 20% O2, and 5% Ar (by volume). These are the first simultaneous measurements of NO and O LIF to be reported in literature for the HYMETS facility. The maximum O LIF mean signal intensity was observed at a stagnation enthalpy of approximately 12 MJ/kg while the maximum NO LIF mean signal intensity was observed at a stagnation enthalpy of 6.7 MJ/kg. Experimental results were compared to simple fluorescence model that assumes equilibrium conditions in the plenum and frozen chemistry in the isentropic nozzle expansion (Mach 5). The equilibrium calculations were performed using CANTERA v2.1.1 with 16 species. The fluorescence model captured the correlation in mean O and NO LIF signal intensities over the entire range of stagnation enthalpies tested. Very weak correlations between single-shot O and NO LIF intensities were observed in the experiments at all of the stagnation enthalpy conditions.

  16. Immunohistochemical study of constitutive neuronal and inducible nitric oxide synthase in the central nervous system of goat with natural listeriosis.

    PubMed

    Shin, T; Weinstock, D; Castro, M D; Acland, H; Walter, M; Kim, H Y; Purchase, H G

    2000-12-01

    The expression of both constitutive and inducible forms of nitric oxide synthase (NOS) was investigated by immunohistochemical staining of formalin-fixed paraffin-embedded sections in normal and Listeria monocytogenes-infected brains of goats. In normal control goats, a small number of neurons showed immunoreactivity of both iNOS and nNOS, and the number of iNOS-positive neurons was higher than the number of nNOS-positive neurons. In natural listeriosis, listeria antigens were easily immunostained in the inflammatory cells of microabscesses. In this lesion, the immunoreactivity of iNOS in neurons was more intense than the control, but nNOS was not. In microabscesses, nNOS was weakly visualized in macrophages and neutrophils, while iNOS was expressed in macrophages, but not in neutrophils. These findings suggest that normal caprine brain cells, including neurons, constitutively express iNOS and nNOS, and the expressions of these molecules is increased in Listeria monocytogenes infections. Furthermore, inflammatory cells, including macrophages, expressing both nNOS and iNOS may play important roles in the pathogenesis of bacterial meningoencephalitis in goat.

  17. Endogenous nitric oxide mediates He-Ne laser-induced adaptive responses in salt stressed-tall fescue leaves.

    PubMed

    Li, Yongfeng; Gao, Limei; Han, Rong

    2016-10-01

    The aim of this study was to investigate the role of endogenous nitric oxide in protective effects of He-Ne laser on salt stressed-tall fescue leaves. Salt stress resulted in significant increases of membrane injury, reactive oxygen species (ROS) production, polyamine accumulation, and activities of SOD, POD, and APX, while pronounced decreases of antioxidant contents, CAT activity and intracellular Ca(2+) concentration in seedlings leaves. He-Ne laser illumination caused a distinct alleviation of cellular injury that was reflected by the lower MDA amounts, polyamine accumulation and ROS levels at the stress period. In contrast, the laser treatment displayed a higher Ca(2+) concentration, antioxidant amounts, NO release, antioxidant enzyme, and NOS activities. These responses could be blocked due to the inhibition of NO biosynthesis by PTIO (NO scavenger) or LNNA (NOS inhibitor). The presented results demonstrated that endogenous NO might be involved in the progress of He-Ne laser-induced plant antioxidant system activation and ROS degradation in order to enhance adaptive responses of tall fescue to prolonged saline conditions.

  18. Effect of myrrh and thyme on Trichinella spiralis enteral and parenteral phases with inducible nitric oxide expression in mice.

    PubMed

    Attia, Rasha A H; Mahmoud, Abeer E; Farrag, Haiam Mohammed Mahmoud; Makboul, Rania; Mohamed, Mona Embarek; Ibraheim, Zedan

    2015-12-01

    Trichinellosis is a serious disease with no satisfactory treatment. We aimed to assess the effect of myrrh (Commiphora molmol) and, for the first time, thyme (Thymus vulgaris L.) against enteral and encysted (parenteral) phases of Trichinella spiralis in mice compared with albendazole, and detect their effect on inducible nitric oxide synthase (iNOS) expression. Oral administration of 500 mg/kg of myrrh and thyme led to adult reduction (90.9%, 79.4%), while 1,000 mg/kg led to larvae reduction (79.6%, 71.3%), respectively. Administration of 50 mg/kg of albendazole resulted in adult and larvae reduction (94.2%, 90.9%). Positive immunostaining of inflammatory cells infiltrating intestinal mucosa and submucosa of all treated groups was detected. Myrrh-treated mice showed the highest iNOS expression followed by albendazole, then thyme. On the other hand, both myrrh and thyme-treated groups showed stronger iNOS expression of inflammatory cells infiltrating and surrounding encapsulated T. spiralis larvae than albendazole treated group. In conclusion, myrrh and thyme extracts are highly effective against both phases of T. spiralis and showed strong iNOS expressions, especially myrrh which could be a promising alternative drug. This experiment provides a basis for further exploration of this plant by isolation and retesting the active principles of both extracts against different stages of T. spiralis.

  19. Effect of myrrh and thyme on Trichinella spiralis enteral and parenteral phases with inducible nitric oxide expression in mice

    PubMed Central

    Attia, Rasha AH; Mahmoud, Abeer E; Farrag, Haiam Mohammed Mahmoud; Makboul, Rania; Mohamed, Mona Embarek; Ibraheim, Zedan

    2015-01-01

    Trichinellosis is a serious disease with