Peroxynitrite Anion Stimulates Arginine Release from Cultured Rat Astrocytes

The biosynthesis of the physiological messenger nitric oxide (*NO) in neuronal cells is thought to depend on a glial-derived supply of the *NO synthase substrate arginine. To expand our knowledge of the mechanism responsible for this glial-neuronal interaction, we studied the possible roles of peroxynitrite anion (ONOO-), superoxide anion (O2*-), *NO, and H2O2 in L-[3H]arginine release in cultured rat astrocytes. After 5 min of incubation at 37 degrees C, initial concentrations of 0.05-2 mM ONOO- stimulated the release of arginine from astrocytes in a concentration-dependent way; this effect was maximum from 1 mM ONOO- and proved to be approximately 400% as compared with control cells. ONOO(-)-mediated arginine release was prevented by arginine transport inhibitors, such as L-lysine and N(G)-monomethyl-L-arginine, suggesting an involvement of the arginine transporter in the effect of ONOO-. In situ xanthine/xanthine oxidase-generated O2*- (20 nmol/min) stimulated arginine release to a similar extent to that found with 0.1 mM ONOO-, but this effect was not prevented by arginine transport inhibitors. *NO donors, such as sodium nitroprusside, S-nitroso-N-acetylpenicillamine, or 1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium+ ++-1,2-diolate, and H2O2 did not significantly modify arginine release. As limited arginine availability for neuronal *NO synthase activity may be neurotoxic due to ONOO- formation, our results suggest that ONOO(-)-mediated arginine release from astrocytes may contribute to replenishing neuronal arginine, hence avoiding further generation of ONOO- within these cells.     

Nitric oxide, mitochondria and neurological disease

Damage to the mitochondrial electron transport chain has been suggested to be an important factor in the pathogenesis of a range of neurological disorders, such as Parkinson's disease, Alzheimer's disease, multiple sclerosis, stroke and amyotrophic lateral sclerosis. There is also a growing body of evidence to implicate excessive or inappropriate generation of nitric oxide (NO) in these disorders. It is now well documented that NO and its toxic metabolite, peroxynitrite (ONOO-), can inhibit components of the mitochondrial respiratory chain leading, if damage is severe enough, to a cellular energy deficiency state. Within the brain, the susceptibility of different brain cell types to NO and ONOO- exposure may be dependent on factors such as the intracellular reduced glutathione (GSH) concentration and an ability to increase glycolytic flux in the face of mitochondrial damage. Thus neurones, in contrast to astrocytes, appear particularly vulnerable to the action of these molecules. Following cytokine exposure, astrocytes can increase NO generation, due to de novo synthesis of the inducible form of nitric oxide synthase (NOS). Whilst the NO/ONOO- so formed may not affect astrocyte survival, these molecules may diffuse out to cause mitochondrial damage, and possibly cell death, to other cells, such as neurones, in close proximity. Evidence is now available to support this scenario for neurological disorders, such as multiple sclerosis. In other conditions, such as ischaemia, increased availability of glutamate may lead to an activation of a calcium-dependent nitric oxide synthase associated with neurones. Such increased/inappropriate NO formation may contribute to energy depletion and neuronal cell death. The evidence available for NO/ONOO--mediated mitochondrial damage in various neurological disorders is considered and potential therapeutic strategies are proposed.     

Lipopolysaccharide-induced dopaminergic cell death in rat midbrain slice cultures: role of inducible nitric oxide synthase and protection by indomethacin

Glial cell activation associated with inflammatory reaction may contribute to pathogenic processes of neurodegenerative disorders, through production of several cytotoxic molecules. We investigated the consequences of glial activation by interferon-gamma (IFN-gamma)/lipopolysaccharide (LPS) in rat midbrain slice cultures. Application of IFN-gamma followed by LPS caused dopaminergic cell death and accompanying increases in nitrite production and lactate dehydrogenase release. Aminoguanidine, an inhibitor of inducible nitric oxide synthase (iNOS), or SB203580, an inhibitor of p38 mitogen-activated protein kinase, prevented dopaminergic cell loss as well as nitrite production. SB203580 also suppressed expression of iNOS and cyclooxygenase-2 (COX-2) induced by IFN-gamma/LPS. A COX inhibitor indomethacin protected dopaminergic neurons from IFN-gamma/LPS-induced injury, whereas selective COX-2 inhibitors such as NS-398 and nimesulide did not. Notably, indomethacin was able to attenuate neurotoxicity of a nitric oxide (NO) donor. Neutralizing antibodies against tumour necrosis factor-alpha and interleukin-1beta did not inhibit dopaminergic cell death caused by IFN-gamma/LPS, although combined application of these antibodies blocked lactate dehydrogenase release and decrease in the number of non-dopaminergic neurons. These results indicate that iNOS-derived NO plays a crucial role in IFN-gamma/LPS-induced dopaminergic cell death, and that indomethacin exerts protective effect by mechanisms probably related to NO neurotoxicity rather than through COX inhibition.     

Activation of p38 MAPK induced peroxynitrite generation in LPS plus IFN-gamma-stimulated rat primary astrocytes via activation of iNOS and NADPH oxidase

We have shown that immunostimulated astrocytes produce excess nitric oxide (NO) and eventually peroxynitrite (ONOO(-)) that was closely associated with the glucose deprivation-potentiated death of astrocytes. The present study shows that activated p38 MAPK regulates ONOO(-) generation from lipopolysaccharide (LPS) plus interferon-gamma (IFN-gamma)-stimulated astrocytes. LPS+IFN-gamma-induced p38 MAPK activation and ONOO(-) generation were attenuated by SB203580 or SKF-86002, specific inhibitors of p38 MAPK. ONOO(-) generation was blocked by NADPH oxidase inhibitor, diphenyleneiodonium chloride, and nitric oxide synthase (NOS) inhibitor, N omega-nitro-L-arginine methyl ester, suggesting both enzymes are involved in ONOO(-) generation. Inhibition of p38 MAPK suppressed LPS+IFN-gamma-induced NO production through down-regulating inducible form of NOS expression. It also suppressed LPS+IFN-gamma-induced NADPH oxidase activation and eventually, the inducible form of superoxide production. Transfection with dominant negative vector of p38 alpha reduced LPS+IFN-gamma-induced ONOO(-) generation through blocking both iNOS-derived NO production and NADPH oxidase-derived O2(-) production. Our results suggest that activated p38 MAPK may serve as a potential signaling molecule in ONOO(-) generation through dual regulatory mechanisms, involving iNOS induction and NADPH oxidase activation.     

Tetrandrine suppresses LPS-induced astrocyte activation via modulating IKKs-IκBα-NF-κB signaling pathway

Astrocyte activation has been implicated in the pathogenesis of many neurological diseases. These reactive astrocytes are capable of producing a variety of proinflammatory mediators and potentially neurotoxic compounds, such as nitric oxide (NO), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and interleukin-1beta (IL-1beta). In this study, we examined the suppressive effects of Tetrandrine (TET) on astrocyte activation induced by lipopolysaccharide (LPS) in vitro. We found that TET decreased the release of NO, TNF-alpha, IL-6 and IL-1beta in LPS-activated astrocytes. Also mRNA expression levels of inducible nitric oxide synthase (iNOS), macrophage inflammatory protein-1alpha (MIP-1alpha) and vascular cell adhesion molecule-1 (VCAM-1) were inhibited in TET pretreated astrocytes. Such suppressive effects might be resulted from the inhibition of nuclear factor kappa B (NF-kappaB) activation through downregulating IkappaB kinases (IKKs) phosphoration, which decreased inhibitor of nuclear factor-kappaB-alpha (IkappaBalpha) phosphoration and degradation. Our results suggest that TET acted to regulate astrocyte activation through inhibiting IKKs-IkappaBalpha-NF-kappaB signaling pathway.     

Synergistic depletion of astrocytic glutathione by glucose deprivation and peroxynitrite: correlation with mitochondrial dysfunction and subsequent cell death

Previously we reported that immunostimulated astrocytes were highly vulnerable to glucose deprivation. The augmented death was mimicked by the peroxynitrite (ONOO )-producing reagent 3-morpholinosydnonimine (SIN-1). Here we show that glucose deprivation and ONOO- synergistically deplete intracellular reduced glutathione (GSH) and augment the death of astrocytes via formation of cyclosporin A-sensitive mitochondrial permeability transition (MPT) pore. Astrocytic GSH levels were only slightly decreased by glucose deprivation or SIN-1 (200 microM) alone. In contrast, a rapid and large depletion of GSH was observed in glucose-deprived/ SIN-1-treated astrocytes. The depletion of GSH occurred before a significant release of lactate dehydrogenase (a marker of cell death). Superoxide dismutase and ONOO-scavengers completely blocked the augmented death, indicating that the reaction of nitric oxide with superoxide to form ONOO was implicated. Furthermore, nitrotyrosine immunoreactivity (a marker of ONOO-) was markedly enhanced in glucose-deprived/SIN-1 -treated astrocytes. Mitochondrial transmembrane potential (MTP) was synergistically decreased in glucose-deprived/SIN-1-treated astrocytes. The glutathione synthase inhibitor L-buthionine-(S,R)-sulfoximine markedly decreased the MTP and increased lactate dehydrogenase (LDH) releases in SIN-1-treated astrocytes. Cyclosporin A, an MPT pore blocker, completely prevented the MTP depolarization as well as the enhanced LDH releases in glucose-deprived/SIN-1-treated astrocytes.     

nNOS mediated mitochondrial injury in LPS stimulated oligodendrocytes

Products of inflammation and the activation of nitric oxide synthase have been proposed as a mechanism of oligodendrocyte injury in CNS inflammation. There are currently three well described and known isoforms of NOS. Of these, neuronal NOS (nNOS) was initially discovered in neurons, endothelial NOS (eNOS) in vascular endothelium, while the inducible form of NOS (iNOS) is known to be activated in oligodendrocytes, astrocytes and microglia. We examined the activation of nNOS and the down stream effects of NO production in oligodendrocyte precursor cells (OPC) and MO3.13 cell line following culture with LPS. Our studies show that both MO3.13 cells and OPC are susceptible to the cellular injury resulting from LPS mediated activation and NO production. Activation of the TLR4 receptor with LPS led to decrease in cell viability that was associated with loss of mitochondrial membrane potential and impaired enzymatic activity of complex I and complex IV protein of the respiratory chain. 7-NI, a known inhibitor of nNOS was able to rescue of cells from LPS mediated mitochondrial damage. Loss of mitochondrial function was associated with translocation of cytochrome C and apoptosis inducing factor to the cytosol, setting the stage for apoptosis. Phosphorylation of PI3K and Akt was required for optimal activation of NOS. These studies provide a biochemical basis for nNOS mediated oligodendrocyte injury and suggest similar mechanisms may play a role in diseases characterized by oligodendrocyte loss and demyelination.     

Peroxynitrite and protein tyrosine nitration of prostacyclin synthase

When working on the regulation of prostacyclin synthase (PGIS), we found that PGIS was selectively inhibited by peroxynitrite (ONOO-), a potent oxidant formed by the combination of superoxide anion and nitric oxide (NO) at a rate of diffusion-controlled. None of the cellular antioxidants studied (i.e. GSH, Vitamins C and E, and others) prevented the inhibition of ONOO- on PGIS. This unexpected behavior was explained by a catalytic reaction of the iron-thiolate center of PGIS with ONOO- anion. In contrast, ONOO- activated both thromboxane A2-synthase and cyclooxygenases. In addition, we demonstrated that sub-micromolar levels of ONOO- inhibited PGI2-dependent vasorelaxation and triggered a PGH2-dependent vasospasm, indicating that ONOO- increased PGH2 formation as a consequence of PGIS nitration. We have subsequently demonstrated that endogenous ONOO- caused PGIS nitration and TxA2 activation in several diseased conditions such as atherosclerotic vessels, hypoxia-reperfusion injury, cytokines-treated cells, diabetes, as well as hypertension. Since NO is produced physiologically it seems that excessive formation of superoxide not only eliminates the vasodilatory, growth-inhibiting, anti-thrombotic and anti-adhesive effects of NO and PGI2 but also allows and promotes an action of the potent vasoconstrictor, prothrombotic agent, growth promoter, and leukocyte adherer, PGH2. We conclude that the nitration of PGIS nitration might be a new pathogenic mechanism for superoxide-induced endothelium dysfunction often observed in vascular diseases such as atherosclerosis, hypertension, ischemia, endotoxic shock, and diabetes.     

The contribution of reactive nitrogen and oxygen species to the killing of Francisella tularensis LVS by murine macrophages

Intracellular killing of Francisella tularensis by macrophages depends on interferon-gamma (IFN-gamma)-induced activation of the cells. The importance of inducible nitric oxide synthase (iNOS) or NADPH phagocyte oxidase (phox) for the cidal activity was studied. Murine IFN-gamma-activated peritoneal exudate cells (PEC) produced nitric oxide (NO), measured as nitrite plus nitrate, and superoxide. When PEC were infected with the live vaccine strain, LVS, of F. tularensis, the number of viable bacteria was at least 1000-fold lower in the presence than in the absence of IFN-gamma after 48 h of incubation. PEC from iNOS-gene-deficient (iNOS-/-) mice killed F. tularensis LVS less effectively than did PEC from wild-type mice. PEC from phox gene-deficient (p47phox-/-) mice were capable of killing the bacteria, but killing was less efficient, although still significant, in the presence of NG-monomethyl-L-arginine (NMMLA), an inhibitor of iNOS. A decomposition catalyst of ONOO-, FeTPPS, completely reversed the IFN-gamma-induced killing of F. tularensis LVS. Under host cell-free conditions, F. tularensis LVS was exposed to S-nitroso-acetyl-penicillamine (SNAP), which generates NO, or 3-morpholinosydnonimine hydrochloride (SIN-1), which generates NO and superoxide, leading to formation of ONOO-. During 6 h of incubation, SNAP caused no killing of F. tularensis LVS, whereas effective killing occurred in the presence of equimolar concentrations of SIN-1. The results suggest that mechanisms dependent on iNOS and to a minor degree, phox, contribute to the IFN-gamma-induced macrophage killing of F. tularensis LVS. ONOO- is likely to be a major mediator of the killing.     

Intracerebroventricular injection of neuronal and inducible nitric oxide synthase inhibitors attenuates fever due to LPS in rats.

Nitric oxide (NO) has been shown to be an important mediator of febrile response to lipopolisaccharide (LPS). To clarify the role of different isoforms of NO synthase (NOS) in febrile response to immune challenge, effects of selective iNOS and nNOS inhibitors on fever to LPS were examined in freely moving biotelemetered rats. Vinyl-L-NIO (N(5) - (1-Imino-3-butenyl) - ornithine (vL-NIO), a neuronal nitric oxide synthase (nNOS) inhibitor, and aminoguanidine hydrochloride, an inducible nitric oxide synthase (iNOS) inhibitor, were injected intracerebroventricularly at a dose of 10 microg/rat just before intraperitoneal injection of LPS at a dose of 50 microg/kg. Both inhibitors injected at a selected doses had no effect on normal day-time body temperature (T(b)) and normal night-time T(b). vinyl-L-NIO and aminoguanidine injected intracerebroventricularly at a dose of 10 microg/animal suppressed the LPS-induced fever in rats. The fever index calculated for rats pretreated with v-LNIO or with aminoguanidine and injected with LPS was reduced by 43% and 72%, respectively, compared to that calculated for water-pretreated and LPS-injected rats. Whereas vL-NIO partly attenuated both phases of febrile rise in T(b), administration of aminoguanidine into the brain completely prevented fever induced by LPS. These data indicate that activation of iNOS inside the brain is not only responsible for triggering but also for maintaining of LPS-induced fever in rats. It is, therefore, reasonable to hypothesize that, activation of iNOS inside the brain is more important in fever development than activation of nNOS.     

Dose dependent effects of reactive oxygen and nitrogen species on the function of neuronal nitric oxide synthase

Reactive nitrogen species (RNS) and oxygen species (ROS) have been reported to modulate the function of nitric oxide synthase (NOS); however, the precise dose-dependent effects of specific RNS and ROS on NOS function are unknown. Questions remain unanswered regarding whether pathophysiological levels of RNS and ROS alter NOS function, and if this alteration is reversible. We measured the effects of peroxynitrite (ONOO-), superoxide (O2.-), hydroxyl radical (.OH), and H2O2 on nNOS activity. The results showed that NO production was inhibited in a dose-dependent manner by all four oxidants, but only O2.- and ONOO- were inhibitory at pathophysiological concentrations (50muM). Subsequent addition of tetrahydrobiopterin (BH4) fully restored activity after O2.- exposure, while BH4 partially rescued the activity decrease induced by the other three oxidants. Furthermore, treatment with either ONOO- or O2.- stimulated nNOS uncoupling with decreased NO and enhanced O2.- generation. Thus, nNOS is reversibly uncoupled by O2.- (50muM), but irreversibly uncoupled and inactivated by ONOO-. Additionally, we observed that the mechanism by which oxidative stress alters nNOS activity involves not only BH4 oxidation, but also nNOS monomerization as well as possible degradation of the heme.     

Nitric oxide from neuronal nitric oxide synthase sensitises neurons to hypoxia-induced death via competitive inhibition of cytochrome oxidase

Hypoxia/ischaemia is known to trigger neuronal death, but the role of neuronal nitric oxide synthase (nNOS) in this process is controversial. Nitric oxide (NO) inhibits cytochrome oxidase in competition with oxygen. We tested whether NO derived from nNOS synergises with hypoxia to induce neuronal death by inhibiting mitochondrial cytochrome oxidase. Sixteen hours of hypoxia (2% oxygen) plus deoxyglucose (an inhibitor of glycolysis) caused extensive, excitotoxic death of neurons in rat cerebellar granule cell cultures. Three different nNOS inhibitors (including the selective inhibitor N-4S-4-amino-5-2-aminoethyl-aminopentyl-N'-nitroguanidine) decreased this neuronal death by half, indicating a contribution of nNOS to hypoxic death. The selective nNOS inhibitor did not, however, block neuronal death induced either by added glutamate or by added azide (an uncompetitive inhibitor of cytochrome oxidase), indicating that nNOS does not act downstream of glutamate or cytochrome oxidase. Hypoxia plus deoxyglucose-induced glutamate release and neuronal depolarisation, and the nNOS inhibitor decreased this. Hypoxia inhibited cytochrome oxidase activity in the cultures, but a selective nNOS inhibitor prevented this inhibition, indicating NO from nNOS was inhibiting cytochrome oxidase in competition with oxygen. These data indicate that hypoxia synergises with NO from nNOS to induce neuronal death via cytochrome oxidase inhibition causing neuronal depolarisation. This mechanism might contribute to ischaemia/stroke-induced neuronal death in vivo.     

Nitric oxide mediates anti-inflammatory action of extracorporeal shock waves

Here, we show that extracorporeal shock waves (ESW), at a low energy density value, quickly increase neuronal nitric oxide synthase (nNOS) activity and basal nitric oxide (NO) production in the rat glioma cell line C6. In addition, the treatment of C6 cells with ESW reverts the decrease of nNOS activity and NO production induced by a mixture of lipopolysaccharides (LPS), interferon-gamma (IFN-gamma) plus tumour necrosis factor-alpha (TNF-alpha). Finally, ESW treatment efficiently downregulates NF-kappaB activation and NF-kappaB-dependent gene expression, including inducible NOS and TNF-alpha. The present report suggests a possible molecular mechanism of the anti-inflammatory action of ESW treatment.     

Inducible nitric oxide synthase knockout mouse macrophages disclose prooxidant effect of interferon-gamma on low-density lipoprotein oxidation.

To test our hypothesis that interferon-gamma (IFN-gamma) has a direct prooxidant effect on macrophage-mediated LDL oxidation behind its antioxidant effect via induction of inducible nitric oxide synthase (iNOS), we incubated LDL with wild-type (iNOS(+/+)) or iNOS knockout mouse (iNOS(-/-)) macrophages preincubated with IFN-gamma or IFN-gamma plus lipopolysaccharide (IFN-gamma/LPS) for 24 h. LDL oxidation was measured in terms of formation of thiobarbituric acid reactive substances (TBARS) and electrophoretic mobility. Thiol production, nitrite production, and superoxide production from macrophages were measured by using Ellman's assay, the Griess reagent, and the SOD-inhibitable cytochrome c reduction method, respectively. IFN-gamma alone or combined with LPS induced iNOS expression and increased nitrite production in iNOS(+/+) macrophages, but not in iNOS(-/-) macrophages. TBARS formation from LDL was suppressed in IFN-gamma- and IFN-gamma/LPS-treated iNOS(+/+) macrophages but was increased in IFN-gamma-treated iNOS(-/-) macrophages. In the presence of N(G)-monomethyl-l-arginine (l-NMMA), a NOS inhibitor, the suppressive effect of IFN-gamma and IFN-gamma/LPS was abolished and TBARS formation was even increased to a level above that of untreated iNOS(+/+) macrophage. NOC 18, an NO donor, dose dependently inhibited macrophage-mediated LDL oxidation. IFN-gamma increased superoxide and thiol productions in both types of macrophages. We conclude that IFN-gamma promotes macrophage-mediated LDL oxidation by stimulating superoxide and thiol production under conditions where iNOS-catalyzed NO release is restricted.     

Nitrosylation precedes caspase-3 activation and translocation of apoptosis-inducing factor in neonatal rat cerebral hypoxia-ischaemia

Excessive nitric oxide (NO) production after cerebral hypoxia-ischaemia (HI) may induce cellular injury in various ways, including reaction with superoxide to form the highly reactive peroxynitrite. We characterized the spatial and temporal formation of peroxynitrite through immunohistochemical detection of nitrosylated proteins. Nitrotyrosine immunoreactivity peaked around 3 h post-HI and was detected in areas of injury, as judged by the loss of microtubule-associated protein-2 (MAP-2) staining, in neurones, glia and endothelial cells. Nitrotyrosine staining co-localized with three other cellular markers of injury, active caspase-3, nuclear translocation of apoptosis-inducing factor (AIF) and an oligonucleotide hairpin probe detecting specific DNA strand breaks. The number of nitrotyrosine-positive cells at early time points outnumbered the cells positive for the other three markers of injury, indicating that nitrosylation preceded caspase-3 activation. Pharmacological inhibition of neuronal and inducible nitric oxide synthase (nNOS and iNOS) using 2-iminobiotin, which has been demonstrated earlier to be neuroprotective, significantly reduced nitrotyrosine formation and caspase-3 activation, but not nuclear translocation of AIF, in cortex and striatum of the ipsilatral hemisphere. In summary, nitrotyrosine is an early marker of cellular injury and inhibition of nNOS and iNOS is a promising strategy for neuroprotection after perinatal HI.     

Requirement for Nitric Oxide in Retinal Neuronal Cell Death Induced by Activated Müller Glial Cells

Retinal Müller glial cells express the inducible isoform (-2) of nitric oxide (NO) synthase (NOS) in vitro after stimulation by lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) or in vivo in some retinal pathologies. Because NO may have beneficial or detrimental effects in the retina, we have used cocultures of retinal neurons with retinal Müller glial (RMG) cells from mice disrupted for the gene of NOS-2 [NOS-2 (-/-)] to clarify the role of NO in retinal neurotoxicity. We first demonstrated that NO produced by activated RMG cells was not toxic for RMG cells themselves. Second, the NO released from LPS/IFN-gamma-stimulated RMG cells induced neuronal cell death, because no neuronal cell death has been observed in cocultures with RMG cells from NOS-2 (-/-) mice and because inhibition of NOS-2 induction by transforming growth factor-beta or blockade of NO release by different NOS inhibitors prevented neuronal cell death. Addition of urate, a peroxynitrite scavenger, or superoxide dismutase partially prevented neuronal cell death induced by NO, whereas the presence of a poly(ADP-ribose) synthetase inhibitor, caspase inhibitors, or a guanylate cyclase inhibitor had no significant effect on cell death. These results demonstrated that a large release of NO from RMG cells is responsible for retinal neuronal cell death in vitro, suggesting a neurotoxic role for NO and peroxynitrite during retinal inflammatory or degenerative diseases, where RMG cells were activated.     

Induction of nitric oxide synthase by saponins of heat-processed ginseng

Total saponin of heat-processed ginseng (TSHG) stimulated the production of nitric oxide (NO) in interferon-gamma (IFN-gamma)-primed macrophages through the increased expression of inducible nitric oxide synthase (iNOS). However, TSHG by itself had a very weak effect on the NO synthesis without IFN-gamma priming. The saponins of white ginseng inhibited the NO production in lipopolysaccharide (LPS)/IFN-gamma activated macrophages rather than the stimulation of NO production found in IFN-gamma primed macrophages. The NO production by TSHG-stimulated macrophages was inhibited by the NOS inhibitor (N(G)-monomethyl-L-arginine (L-NMMA)) and nuclear factor-kappaB inhibitor (pyrrolidine dithiocarbamate (PDTC)). TSHG showed different serum-dependence from LPS on the activation of IFN-gamma primed macrophages. This property of TSHG may explain the intensified anti-tumor properties of heat-processed ginseng through its immunostimulating activity.