Induction Of Inducible Nitric Oxide Synthase Research Articles

Augmentation of urea-synthetic capacity by inhibition of nitric oxide synthesis in butyrate-induced differentiated human hepatocytes

We have recently developed an in vitro differentiation model of immortalized non-transformed human hepatocytes using butyrate, and observed the induction of inducible NO synthase (iNOS). In this study, we analyzed the effect of NO on the urea-synthetic capacity of these cells. The inhibition of iNOS during butyrate treatment significantly increased the urea-synthetic capacity as compared to that of butyrate treatment alone, possibly through the further induction of ornithine transcarbamylase expression. Therefore, the inhibition of NO production might be useful for obtaining more differentiated hepatocytes in the process of in vitro induction of hepatocyte-specific differentiation.

Acid-dependent dismutation of nitrogen oxides may be a critical source of nitric oxide in human macrophages

The cytotoxic activity of the macrophage relies greatly on the secretion of a number of reactant intermediates, including superoxide (O2–), hydroxyl radical (OH–) and nitric oxide (NO). The latter, synthesized via cytokine-mediated induction of inducible NO-synthase (iNOS), is readily observed in murine macrophages. However, a poorly reproducible or minimal response to cytokine-stimulation in the human macrophage has questioned the presence or significance of this important pathway in man. Nevertheless, iNOS is present in other human phagocytic cells, e.g. neutrophils, while the NO metabolites, nitrite (NO2–) and nitrate (NO3–), are raised in human serum during infection. Low phagolysosomal pH is critical for the macrophage to destroy the engulfed pathogen. This acidic environment may allow synthesis of NO independently of iNOS via dismutation of NO2–to NO. Should this mechanism be active, assay for iNOS and NO by determination of NO2–could be misleading. In human macrophages, acid-induced conversion of imported nitrogen oxides (NOx) may take precedence over iNOS-mediated NO synthesis and should be investigated as a source of NO in these cells.

Cryptococcus neoformans neutralizes macrophage and astrocyte derived nitric oxide without interfering with inducible nitric oxide synthase induction or catalytic activity - possible involvement of nitric oxide consumption.

The effect of Cryptococcus neoformans on the accumulation of nitrite, an indicator of nitric oxide (NO) synthesis, was investigated in cytokine (interferon-gamma [IFN-gamma] and interleukin [IL]-1)-stimulated cultures of rat peritoneal macrophages and C6 astrocytoma cells. Cytokine-induced nitrite generation in cultures of both cell types was inhibited in a dose-dependent manner by live C. neoformans, but not by heat-killed cryptococcal cells or conditioned medium from yeast cultures. C. neoformans-mediated reduction of nitrite formation coincided with impairment of NO-dependent macrophage tumoricidal activity. Cytokine-triggered induction of inducible NO synthase (iNOS) was unaffected in C6 cells, and only marginally reduced in macrophages. When cells were pretreated with cytokines for 24 h to induce iNOS, and any further induction was prevented by inhibition of protein synthesis, C. neoformans was still able to reduce nitrite accumulation in cultures of both cell types. Finally, live C. neoformans, but not heat-killed yeast cells or yeast culture supernatant, significantly reduced nitrite production in a culture solution of NO-releasing compound S-nitrosoglutathione (GSNO). Thus, it appears that cryptococcal reduction of nitrite formation in macrophage and C6 cultures was caused by the consumption of NO by some yeast molecule, rather than by the inhibition of cellular NO synthesis.

Inhibitory effects of sesquiterpenes from bay leaf on nitric oxide production in lipopolysaccharide-activated macrophages: Structure requirement and role of heat shock protein induction

The methanolic extract from the leaves of Laurus nobilis (bay leaf, laurel) was found to inhibit nitric oxide (NO) production in lipopolysaccharide (LPS)-activated mouse peritoneal macrophages. Through bioassay-guided separation, fourteen known sesquiterpenes were isolated from the active fraction and were examined for ability to inhibit the NO production. Seven sesquiterpene lactones (costunolide, dehydrocostus lactone, eremanthine, zaluzanin C, magnolialide, santamarine and spirafolide) potently inhibited LPS-induced NO production (IC 50 = 1.2~3.8 μM). Other sesquiterpene constituents also showed the inhibitory activity (IC 50 >= 21 μM), but their inhibitory activities were less than those of sesquiterpene lactones. α-Methylene-γ-butyrolactone also showed inhibitory activity (IC 50 = 9.6 μM), while mokko lactone and watsonol A etc., reductants of the α-methylene-γ-butyrolactone moiety by NaBH 4 or DIBAL and a 2-mercaptoethanol adduct of dehydrocostus lactone showed little activity (IC 50 >= 18 μM). These results indicated that the α-methylene-γ-butyrolactone moiety is important for the activity. Furthermore, costunolide and dehydrocostus lactone inhibited inducible nitric oxide synthase (iNOS) induction in accordance with induction of heat shock protein 72 (HSP 72). These results suggested that, as one of their mechanisms of action, sesquiterpene lactones induce HSP 72 thereby preventing nuclear factor-κB activation followed by iNOS induction.

Extracellular Signal-Related Kinase (ERK) and p38 Mitogen-Activated Protein (MAP) Kinases Differentially Regulate the Lipopolysaccharide-Mediated Induction of Inducible Nitric Oxide Synthase and IL-12 in Macrophages:LeishmaniaPhosphoglycans Subvert Macrophage IL-12 Production by Targeting ERK MAP Kinase

AbstractMacrophage activation by cytokines or microbial products such as LPS results in the induction and release of several key immune effector molecules including NO and IL-12. These have been shown to play crucial roles in the development of immunity to intracellular pathogens such as Leishmania. The molecular mechanisms underlying the induction of these effector molecules are not fully understood. We now show that the extracellular signal-related kinase (ERK) and p38 mitogen-activated protein (MAP) kinases play differential roles in the regulation of LPS-stimulated inducible NO synthase and IL-12 gene expression. In macrophages, LPS stimulates the simultaneous activation of all three classes of MAP kinases, ERK, c-jun N-terminal kinase, and p38, albeit with differential activation kinetics. However, studies using inhibitors selective for ERK (PD98059) and p38 (SB203580) show that while p38 plays an essential role in the induction of inducible NO synthase, ERK MAP kinases play only a minor role in promoting NO generation. In contrast, while p38 promotes induction of IL-12 (p40) mRNA, ERK activation suppresses LPS-mediated IL-12 transcription. The biological relevance of these regulatory signals is demonstrated by our finding that Leishmania lipophosphoglycans, which promote parasite survival, act by stimulating ERK MAP kinase to inhibit macrophage IL-12 production. Thus, as ERK and p38 MAP kinases differentially regulate the induction of the macrophage effector molecules, inducible NO synthase and IL-12, these kinases are potential targets not only for the development of novel strategies to combat intracellular pathogens but also for therapeutic immunomodulation.

Modulation of inducible nitric oxide synthase induction by prostaglandin E 2 in macrophages: distinct susceptibility in murine J774 and RAW 264.7 macrophages☆

Prostaglandin E 2 (PGE 2) is the major cyclooxygenase metabolite in macrophages with complex proinflammatory and immunoregulatory properties. In the present study, we have compared the modulatory role of PGE 2/cAMP-dependent signaling on induced nitric oxide (NO) production in two murine macrophages, J774 and RAW 264.7. With no effect on NO release by itself, PGE 2 co-addition with lipopolysaccharide (LPS) resulted in a concentration-dependent enhancement in NO release and inducible NO synthase induction in J774, but not in RAW 264.7, macrophages. The potentiation effect of PGE 2 in J774 cells was still seen when applied within 9 h after LPS treatment. Whereas RAW 264.7 macrophages release PGE 2 with greater extent than J774 macrophages in response to LPS, indomethacin and NS-398, upon abolishing LPS-induced PGE 2 release, caused a more obvious inhibition of NO release from J774 than RAW 264.7 cells. Thus, we suggest a higher positive modulatory role of PGE 2—either endogenous or exogenous—on NO formation in J774 cells. Supporting these findings, exogenous PGE 2 triggers cAMP formation in J774 cells with higher potency and efficacy. Of interest, dBcAMP also elicits higher sensitivity in potentiating NO release in J774 cells. We conclude that the opposite effect of PGE 2/cAMP signaling on macrophage NO induction depends on its signaling efficacy and might be associated with the difference in endogenous PGE 2 levels.

Both the Neuronal and Inducible Isoforms Contribute to Upregulation of Retinal Nitric Oxide Synthase Activity by Brain-Derived Neurotrophic Factor

Although neurotrophins are best known for their trophic functions, growing evidence suggests that neurotrophins can also be neurotoxic, for instance by enhancing excitotoxic insults. We have shown recently that brain-derived neurotrophic factor (BDNF) limits its neuroprotective action on axotomized rat retinal ganglion cells (RGCs) by upregulating nitric oxide synthase (NOS) activity (Klöcker et al., 1998). The aim of the present study was to investigate this interaction of BDNF and NOS in the lesioned adult rat retina in more detail. We used NOS immunohistochemistry and NADPH-diaphorase (NADPH-d) reaction to characterize morphologically retinal NOS expression and activity. Using reverse transcription-PCR and Western blot analysis, we were able to identify the NOS isoforms being regulated. Six days after optic nerve lesion, we observed an increase in neuronal NOS (NOS-I) mRNA and protein expression in the inner retina. This did not lead to a marked increase in overall retinal NOS activity. Only RGC axons displayed strong de novo NADPH-d reactivity. In contrast, intraocular injection of BDNF resulted in a marked upregulation of NOS activity in NOS-I-immunoreactive structures, leaving the level of NOS-I expression unchanged. In addition, an induction of inducible NOS (NOS-II) was found after BDNF treatment. We identified microglial cells increasing in number and being activated by BDNF, which could serve as the cellular source of NOS-II. In summary, our data suggest that BDNF upregulates retinal NOS activity by both a post-translational regulation of NOS-I activity and an induction of NOS-II. These findings might be useful for developing pharmacological strategies to improve BDNF-mediated neuroprotection.

Inhibition of Experimental Melanin Protein-induced Uveitis (EMIU) by Targeting Nitric Oxide via Phosphatidylcholine-specific Phospholipase C

Experimental melanin protein-induced uveitis (EMIU) is an autoimmune uveitis induced by immunization with uveal melanin protein. Fas and FasL enhancement is reported in rats with EMIU. Tricyclodecan-9-yl-xanthogenate (D609), a specific inhibitor of phosphatidylcholine-specific phospholipase C, inhibits inducible nitric oxide synthase (iNOS) induction. In two independent experiments, 35 Lewis rats with EMIU received either D609 or PBS daily. The eyes and draining lymph nodes were collected for histology, analyses of nitrite, peroxide, and superoxide dismutase, Fas and FasL immunochemistry, in situ hybridization for iNOS mRNA and in situ apoptosis detection at the peak of the disease. Both experiments showed significant inhibition of EMIU by D609. Decreases in nitrite and peroxide, increase of superoxide dismutase and lower expressions of iNOS mRNA were found in D609-treated, as compared to PBS-treated eyes. There was mild enhancement of Fas and FasL in the eyes and lymph nodes of D609-injected animals. DNA fragmentation was increased in the lymph nodes of D609-treated rats. We conclude that iNOS activation is responsible for NO production in eyes with EMIU. The suppressive effect of D609 on EMIU may result from scavenging NO and activating apoptosis previously inhibited by NO along with other anti-inflammatory effects.

Inhibitory effects of vesnarinone in the progression of myocardial damage in experimental autoimmune myocarditis in rats.

Vesnarinone, a positive inotropic and immunomodulatory agent, diminishes nitric oxide (NO) levels by suppressing the induction of inducible NO synthase (iNOS) expressed in cytokine-stimulated macrophages and cardiomyocytes. We examined whether vesnarinone exerts inhibitory effects on the progression of myocardial damage in experimental autoimmune myocarditis in rats through suppression of iNOS. Myocarditis was induced in 30 Lewis rats by injection of porcine cardiac myosin and vesnarinone was orally administered to 20 of the 30 rats. On day 21 after immunization (the climax of inflammation), the hemodynamics were examined and the severity of myocarditis was evaluated by determining the area ratio (%) [affected/entire area] of myocardial lesions in histological sections. Levels of serum CK-MB, NOx (NO2(-)+NO3-), TNF-alpha and IL-1 beta, and cyclic GMP, iNOS mRNA, TNF-alpha and IL-1 beta in heart tissues were determined. Expression of iNOS and TNF-alpha protein were examined by immunohistochemical methods. Histopathological examination revealed extensive myocardial destruction and massive infiltration of inflammatory cells in the vesnarinone-untreated rats. The area ratio of the lesions in the treated rats was significantly lower than that in the untreated ones. Levels of CK-MB, NOx, cyclic GMP, cytokines and iNOS mRNA were significantly lower in the vesnarinone-treated rats. Infiltrating macrophages and cardiomyocytes in the untreated rats showed much higher levels of expression of iNOS and TNF-alpha than those in the vesnarinone-treated rats. Vesnarinone may prove to be useful in the treatment of myocarditis by attenuating NO production through suppression of iNOS induced by cytokines.

Involvement of p38 mitogen-activated protein kinase in lipopolysaccharide-induced iNOS and COX-2 expression in J774 macrophages.

Both the nitrite and prostaglandin E2 (PGE2) release caused by lipopolysaccharide (LPS) in J774 macrophages are inhibited by SB 203580, a specific p38 mitogen-activated protein kinase (MAPK) inhibitor, in a concentration-dependent manner. The 50% inhibitory concentration (IC50) for nitrite and PGE2 responses was 1 microm and 0.5 microm, respectively. Inhibition was marked following simultaneous treatment with SB 203580 and LPS, and was much reduced when SB 203580 was added 6 hr after LPS treatment. In parallel, LPS induction of inducible NO synthase (iNOS) and cyclo-oxygenase-2 (COX-2) proteins and their steady-state levels of mRNA were reduced by SB 203580. LPS activation of nuclear factor-kappa B (NF-kappaB), activator protein-1 (AP-1) and p38 MAPK was also inhibited by SB 203580. These results suggest a crucial role of p38 MAPK in regulation of the transcriptional level of endotoxin LPS-induced iNOS and COX-2 protein expression.

Pentoxifylline improves circulatory failure and survival in murine models of endotoxaemia

Pentoxifylline, a methylxanthine derivative, has been widely used to improve erythrocyte deformability and capillary blood circulation in patients with claudication and cerebrovascular disorders as well as in animals with sepsis. Here, we investigate the effects of pentoxifylline on the hypotension, vascular hyporeactivity to noradrenaline, release of tumour necrosis factor-α (TNF-α) and nitric oxide (NO), and inducible NO synthase protein expression in a rat model of circulatory shock induced by bacterial endotoxin ( Escherichia coli lipopolysaccharide). In addition, we have evaluated the effect of pentoxifylline on the 36-h survival rate in a murine model of endotoxaemia. Male Wistar–Kyoto rats were anaesthetised and instrumented for the measurement of mean arterial pressure and heart rate. Injection of lipopolysaccharide (10 mg/kg, i.v.) resulted in a significant fall in mean arterial pressure and an increase of heart rate. In contrast, animals pretreated with pentoxifylline (3 mg/kg, i.v., at 30 min prior to lipopolysaccharide) maintained a significantly higher mean arterial pressure but showed no effect on the tachycardia when compared to rats given only lipopolysaccharide (lipopolysaccharide-rats). The pressor effect of noradrenaline (1 μg/kg, i.v.) was also significantly reduced after the treatment of rats with lipopolysaccharide. Similarly, rings of thoracic aorta obtained from lipopolysaccharide-rats showed a significant reduction in the contractile responses elicited by noradrenaline (1 μM). Pretreatment of lipopolysaccharide-rats with pentoxifylline partially, but significantly, prevented this lipopolysaccharide-induced hyporeactivity to noradrenaline in vivo and ex vivo. The injection of lipopolysaccharide resulted in bell-shape changes in plasma TNF-α level which reached a peak at 60 min, whereas the effect of lipopolysaccharide on the plasma level of nitrate (an indicator of NO formation) was increased in a time-dependent manner. This increase of both TNF-α and nitrate levels induced by lipopolysaccharide was significantly reduced in lipopolysaccharide-rats pretreated with pentoxifylline. Endotoxaemia for 240 min caused a significantly increased protein expression of inducible NO synthase in the lung. In lipopolysaccharide-rats pretreated with pentoxifylline, inducible NO synthase protein expression in lung homogenates was attenuated by 48±5%. Treatment of conscious mice with a high dose of endotoxin (60 mg/kg, i.p.) resulted in a survival rate of only 10% at 36 h ( n=20). However, therapeutic application of pentoxifylline (3 mg/kg, i.p. at 0, 6, 15 and 24 h after lipopolysaccharide) increased the 36-h survival to 35% ( n=20). Thus, pentoxifylline protects against circulatory failure and improves survival in rodents with severe endotoxaemia. These effects may be due to inhibition of the release of TNF-α and of the induction of inducible NO synthase.

Vasoactive Intestinal Peptide and Pituitary Adenylate Cyclase-Activating Polypeptide Prevent Inducible Nitric Oxide Synthase Transcription in Macrophages by Inhibiting NF-κB and IFN Regulatory Factor 1 Activation

AbstractHigh-output nitric oxide (NO) production from activated macrophages, resulting from the induction of inducible NO synthase (iNOS) expression, represents a major mechanism for macrophage cytotoxicity against pathogens. However, despite its beneficial role in host defense, sustained high-output NO production was also implicated in a variety of acute inflammatory diseases and autoimmune diseases. Therefore, the down-regulation of iNOS expression during an inflammatory process plays a significant physiological role. This study examines the role of two immunomodulatory neuropeptides, the vasoactive intestinal peptide (VIP) and the pituitary adenylate cyclase-activating polypeptide (PACAP), on NO production by LPS-, IFN-γ-, and LPS/IFN-γ-stimulated peritoneal macrophages and the Raw 264.7 cell line. Both VIP and PACAP inhibit NO production in a dose- and time-dependent manner by reducing iNOS expression at protein and mRNA level. VPAC1, the type 1 VIP receptor, which is constitutively expressed in macrophages, and to a lesser degree VPAC2, the type 2 VIP receptor, which is induced upon macrophage activation, mediate the effect of VIP/PACAP. VIP/PACAP inhibit iNOS expression and activity both in vivo and in vitro. Two transduction pathways appear to be involved, a cAMP-dependent pathway that preferentially inhibits IFN regulatory factor-1 transactivation and a cAMP-independent pathway that blocks NF-κB binding to the iNOS promoter. The down-regulation of iNOS expression, together with previously reported inhibitory effects on the production of the proinflammatory cytokines IL-6, TNF-α, and IL-12, and the stimulation of the anti-inflammatory IL-10, define VIP and PACAP as “macrophage deactivating factors” with significant physiological relevance.

Mechanism of adipose tissue iNOS induction in endotoxemia.

The aim of the present study was to investigate the mechanism of adipose tissue inducible nitric oxide synthase (iNOS) induction in endotoxemia. Systemic administration of the bacterial endotoxin lipopolysaccharide (LPS) to rats for </=8 h markedly increased iNOS mRNA and protein levels in white and brown adipose tissues. This effect was comparable to or greater than the induction of iNOS in liver, kidney, or skeletal muscle. iNOS activity was also found to be greatly enhanced in both white and brown adipose tissues of LPS-treated rats (an approximately 12- to 20-fold increase). Treatment of cultured 3T3-L1 adipocytes with LPS, tumor necrosis factor-alpha (TNF-alpha), or interferon-gamma (IFN-gamma) alone failed to induce iNOS activity. However, when used in combination, TNF-alpha, IFN-gamma, and LPS markedly and synergistically increased iNOS activity in these cells. In conclusion, these results suggest that adipose tissue is a major site of iNOS expression in endotoxemia. Our data further indicate that iNOS induction can be reproduced in vitro in cultured adipocytes and that a concerted action of cytokines and endotoxin is needed for maximal activation of the enzyme.

Expression of Cytokines and Activation of Transcription Factors in Lipopolysaccharide-Administered Rats and Their Inhibition by PhenylN-tert-Butylnitrone (PBN)

The spin-trapping compound phenylN-tert-butylnitrone (PBN) affords protection from the lethality of septic shock in rodents. Previous studies have shown that PBN elicits its protection by inhibiting inducible nitric oxide synthase (iNOS) induction. In the present study, using the lipopolysaccharide (LPS) rat septic shock model, we determined the expression of various cytokine genes (tumor necrosis factor (TNF)-α, TNF-β, interferon (IFN)-γ, interleukin (IL)-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, and IL-10) and the activation of transcription factors nuclear factor κB (NF-κB) and activator protein-1 (AP-1) in the liver tissue, 30 min and 3 h after LPS administration. The effects of PBN preadministration on the production levels were also investigated. The results show that LPS (4 mg/kg, ip) induced the production of the cytokine genes and increased the nuclear protein level of NF-κB within 30 min after LPS administration. Preadministration of PBN (150 mg/kg, ip) significantly down-regulated the production of cytokine genes (TNF-α by 94%, IL-1 by 63%, and IL-1 by 70%) and reduced the nuclear protein level of NF-κB by 75% and AP-1 by 72% at 3 h after LPS injection. These results demonstrate that PBN, in addition to its iNOS induction inhibition, also has multiple anti-inflammatory effects in septic shock, via modulation of the production of the key inflammatory mediators.

Theaflavin-3,3′-digallate from black tea blocks the nitric oxide synthase by down-regulating the activation of NF-κB in macrophages

Nitric oxide (NO) plays an important role in inflammation and also in multiple stages of carcinogenesis. We investigated the effects of various tea polyphenols, including theaflavin, a mixture of theaflavin-3-gallate and theaflavin-3′-gallate, theaflavin-3,3′-digallate, thearubigin, and (−)-epigallocatechin-3-gallate on the induction of NO synthase in lipopolysaccharide-activated murine macrophages, RAW 264.7 cells. Theaflavin-3,3′-digallate was found to be stronger than (−)-epigallocatechin-3-gallate in inhibiting NO generation and inducible NO synthase protein in activated macrophages, while theaflavin, a mixture of theaflavin-3-gallate and theaflavin-3′-gallate and thearubigin were less effective. Inhibition of NO production was observed when cells were cotreated with theaflavin-3,3′-digallate and lipopolysaccharide. Western blot and reverse transcriptase-polymerase chain reaction (RT-PCR) analyses demonstrated that significantly reduced 130-kDa protein and mRNA levels of inducible NO synthase were expressed in lipopolysacchride-activated macrophages with theaflavin-3,3′-digallate, compared to those without theaflavin-3,3′-digallate. Electrophoretic mobility shift assay (EMSA) indicated that theaflavin-3,3′-digallate blocked the activation of nuclear factor κB (NF-κB), a transcription factor necessary for inducible NO synthase induction. Theaflavin-3,3′-digallate also blocked phosphorylation of IκB from cytosolic fraction and reduced lipopolysacchride-induced nuclear accumulation of transcription factor NF-κB p65 and p50 subunits. These results suggest that theaflavin-3,3′-digallate decreases the protein levels of inducible NO synthase by reducing the expression of inducible NO synthase mRNA, and the reduction could be via preventing the activation of NF-κB, thereby inhibiting the induction of inducible NO synthase transcription. It was also demonstrated that the gallic acid moiety of theaflavin-3,3′-digallate is essential for their potent anti-inflammation activity.

Pharmacological evidence that inducible nitric oxide synthase is a mediator of delayed preconditioning.

Brief periods of myocardial ischaemia preceding a subsequent more prolonged ischaemic period 24-72 h later confer protection against myocardial infarction ('delayed preconditioning' or the 'second window' of preconditioning). In the present study, we examined the effects of pharmacological modifiers of inducible nitric oxide synthase (iNOS) induction and activity on delayed protection conferred by ischaemic preconditioning 48 h later in an anaesthetized rabbit model of myocardial infarction. Rabbits underwent a myocardial preconditioning protocol (four 5 min coronary artery occlusions) or were sham-operated. Forty-eight hours later they were subjected to a sustained 30 min coronary occlusion and 120 min reperfusion. Infarct size was determined with triphenyltetrazolium staining. In rabbits receiving no pharmacological intervention, the percentage of myocardium infarcted within the risk zone was 43.9+5.0% in sham-operated animals and this was significantly reduced 48 h after ischaemic preconditioning with four 5 min coronary occlusions to 18.5+5.6% (P<0.01). Administration of the iNOS expression inhibitor dexamethasone (4 mg kg(-1) i.v) 60 min before ischaemic preconditioning completely blocked the infarct-limiting effect of ischaemic preconditioning (infarct size 48.6+/-6.1%). Furthermore, administration of aminoguanidine (300 mg kg(-1), s.c.), a relatively selective inhibitor of iNOS activity, 60 min before sustained ischaemia also abolished the delayed protection afforded by ischaemic preconditioning (infarct size 40.0+/-6.0%). Neither aminoguanidine nor dexamethasone per se had significant effect on myocardial infarct size. Myocardial risk zone volume during coronary ligation, a primary determinant of infarct size in this non-collateralized species, was not significantly different between intervention groups. There were no differences in systolic blood pressure, heart rate, arterial blood pH or rectal temperature between groups throughout the experimental period. These data provide pharmacological evidence that the induction of iNOS, following brief periods of coronary occlusion, is associated with increased myocardial tolerance to infarction 48 h later.

Inducible Nitric Oxide Synthase Induction in Thy 1 Glomerulonephritis Is Complement and Reactive Oxygen Species Dependent

Thy 1 glomerulonephritis (GN) is a rat model of complement-dependent immune mesangial injury with induced glomerular nitric oxide (NO) synthesis. To examine mechanisms of inducible nitric oxide synthase (iNOS) induction, we studied the effects of treatment with the antioxidant N-acetyl-cysteine (NAC) and soluble complement receptor 1 (sCR1). Thy 1 GN was induced by intravenous anti-Thy 1 antibody. Glomeruli were isolated and kidney tissue taken from 30 min to 24 h after induction. Nitrite (NO^–₂) synthesis, luminol chemiluminescence for reactive oxygen species (ROS), and iNOS and cytokine mRNA were assayed in isolated glomeruli. Mesangial injury (mesangiolysis) and leucocyte infiltration were quantitated on tissue sections. NAC (i.p. 1,000 mg/kg, 1 h prior to anti-Thy 1) reduced glomerular NO^–₂ synthesis (3.5 ± 0.66 vs. untreated 8.2 ± 1.1, p = 0.02), and iNOS mRNA expression, and abolished enhanced chemiluminescence. In vitro incubation of nephritic glomeruli with 20 mM NAC also suppressed nitrite production (4.7 ± 0.8 vs. untreated 12.2 ± 0.7 nmol NO^–₂/2,000 glomeruli/48 h, p = 0.003), and chemiluminescence. In NAC-treated animals, neutrophil infiltration (0.5 ± 0 vs. untreated 9.6 ± 1.6 glomerulus, p = 0.0005), and macrophage infiltration (1.7 ± 0.4 vs. untreated 12.0 ± 0.1, p = 0.006) were abolished, and mesangiolysis was significantly reduced (45.9 ± 1.3 vs. untreated 34.4 ± 2.1 cells/glomerulus, p = 0.009). NAC did not inhibit anti-Thy 1 antibody deposition. C1q was unaffected, but C3 was reduced. sCR1 treatment prevented iNOS mRNA induction, the enhanced chemiluminescence, and the neutrophil infiltration at 1 h. IL-1β and TNFα mRNAs were not affected by either NAC or sCR1. These results show that NAC inhibits iNOS induction and NO synthesis in this model, and suppresses ROS synthesis and injury. They suggest that complement-dependent ROS generation is the critical initiating event that follows fixation of anti-Thy 1 antibody.

Role of tumour necrosis factor-α and inducible nitric oxide synthase in the prevention of nitro-flurbiprofen small intestine toxicity

The present study compares the intestinal toxicity of nitro-flurbiprofen and flurbiprofen in order to determine their differential properties on tumour necrosis factor-α production and inducible nitric oxide synthase induction. Rats received one s.c. injection of flurbiprofen, nitro-flurbiprofen at equimolar dose of solvent. Twenty-four hours later, the rats were sacrificed and small intestine tissue was taken up for macroscopical quantification of ulceration, ex vivo production of tumour necrosis factor-α and nitrites, and determination of tissue inducible nitric oxide synthase and myeloperoxidase activities. Anti-inflammatory activity was examined in the carrageenan-induced paw edema model. We demonstrated that flurbiprofen induced dose-dependently small intestine production of tumour necrosis factor-α, nitrites, myeloperoxidase and inducible nitric oxide synthase activities. On the other hand, nitro-flurbiprofen did neither induce tumour necrosis factor-α nor nitrite production. Concurrently, no small intestine ulceration was observed with nitro-flurbiprofen whereas flurbiprofen induced dose-dependent ulceration. Nitro-flurbiprofen is devoid of intestinal toxicity despite inhibiting cyclooxygenase activity. This is associated with the absence of tumour necrosis factor-α and inducible nitric oxide synthase induction in normal rats. Nitro-flurbiprofen is an anti-inflammatory drug with a much more favorable gastro-intestinal toxicity profile than flurbiprofen.

Induction of inducible nitric oxide synthase and heme oxygenase-1 in rat glial cells

Recent observations suggest a possible interaction between the nitric oxide (NO)/NO synthases and carbon monoxide (CO)/heme oxygenases systems. We examined the effects of lipopolysaccharide (LPS), interferon-γ (IFN-γ), and NO donor such as S-nitroso-N-acetylpenicillamine (SNAP) on induction of inducible NO synthase (iNOS) and heme oxygenase-1 (HO-1) in mixed glial cells and in rat hippocampus. In in vitro glial cells, treatment with LPS induced the expression of 130-kDa iNOS after 6 h, and NO 2 accumulation and enhancement of the protein level of 33-kDa HO-1 after 12h. In addition, treatment with SNAP induced HO-1 expression after 6 h. Although a NOS inhibitor, such as N G-nitro-L-arginine (NNA), did not change LPS-induced iNOS expression, the inhibitor suppressed both NO 2 accumulation and the enhancement of HO-1. Immunocytochemistry showed that LPS-treatment induced iNOS-immunoreactivity predominantly in microglia, while this treatment induced HO-1-immunoreactivity in both microglia and astrocytes. These results suggest that endogenous NO production by iNOS in microglia causes autocrine- and paracrine-induction of HO-1 protein in microglia and astrocytes in rat brain.

Diethylcarbamazine (DEC) Does Not Induce Nitric Oxide (NO) Synthesis

Rajan, T. V., Shultz, L. D., Babu, S., Doukas, J., Greiner, D., and Porte, P. 1998. Diethylcarbamazine (DEC) does not induce nitric oxide (NO) synthesis.Experimental Parasitology88, 217–222. Diethylcarbomazine (DEC) was discovered in 1947 as a potent therapeutic agent in lymphatic filariasis and has been a mainstay of antifilarial therapy over the past five decades (R. I. Hewitt,et al., 1947,Journal of Laboratory and Clinical Medicine32, 1304–1313). Several hundred million doses of this drug have been administered to people. Despite its widespread and successful use over this prolonged time scale, its mechanism of action remains obscure (R. M. Maizels and D. A. Denham, 1992,Parasitology105Suppl. 549–560). Numerous studies suggest that DEC has no direct effect on the parasite (F. Hawking and W. Laurie, 1949,Lancet2, 146–147) and that it exerts its action by stimulating host immune defense mechanisms (F. Hawkinget al., 1948,Lancet2, 730–731), or by activating host platelets to become microfilaricidal (J. Y. Cesbronet al., 1987,Nature325(6104) 533–536). Recent data from two different laboratories suggest that NO may be involved in host defense against filarial parasites (T. V. Rajanet al., 1996,Infection and Immunity64(8), 3351–3353; M. J. Tayloret al., 1996,Parasitology112, 315–322). We investigated whether DEC stimulates the production of NO from murine macrophages or rat endothelial cells. DEC did not stimulate the synthesis or secretion of NO from either, nor did it synergize with interferon-gamma or tumor necrosis factor-alpha in the induction of inducible NO synthase (iNOS). In addition, there was no consistent increase in the output of inorganic nitrate, the end product of NO metabolism, in the urines of rats treated with DEC. These data suggest that DEC does not achieve its therapeutic efficacy through the induction of host iNOS.