Decreased iNOS synthesis mediates dexamethasone-induced protection of neurons from inflammatory injury in vitro

Brain inflammation is accompanied by transection of axons and death of neurons in the acute lesions of multiple sclerosis. We explored mechanisms of inflammatory damage to neurons in vitro using cocultures of rat embryonal cortical neurons with microglia activated by interferon-gamma (IFNgamma) and lipopolysaccharide (LPS). Previously, we have demonstrated that microglia are highly toxic to neurons and that nitric oxide (NO) derived from inducible nitric oxide synthase (iNOS) is necessary and sufficient to mediate this toxicity. Here, we show that addition of dexamethasone (1 micro M) to activated cocultures provides effective neuroprotection. We demonstrate that dexamethasone down-regulates NO production of primary microglia by approximately 50% and reduces steady-state iNOS protein and mRNA expression by approximately 70%. These changes were reversed by the glucocorticoid receptor blocker RU-486. Furthermore, we analysed the stability of iNOS protein and show that whilst inhibitors of the proteasome blocked iNOS degradation they did not reverse the dexamethasone effect. Our results indicate that the main mechanism of corticosteroid activity on iNOS is reduction in protein synthesis, not destabilization as previously suggested.     

Agmatine reduces infarct area in a mouse model of transient focal cerebral ischemia and protects cultured neurons from ischemia-like injury

Agmatine is a primary amine formed by the decarboxylation of L-arginine synthesized in mammalian brain. In this study, we investigated the neuroprotective effect of agmatine on ischemic and ischemia-like insults. Primary cortical neuronal cultures were subjected to oxygen-glucose deprivation (OGD), a model of ischemia-like injury, and treated with agmatine before or at the start of OGD, or upon reperfusion. Neuronal death was reduced when agmatine was present during OGD, and this protection was associated with a reduction of nitric oxide (NO) and neuronal nitric oxide synthase (nNOS), but not inducible NOS (iNOS). Protection by agmatine was also studied at the in vivo level using a model of middle cerebral artery occlusion (MCAO) in mice. Mice were subjected to 2 h MCAO. Agmatine was administered either 30 min before ischemia, at the start of MCAO, at the start of reperfusion, or 2 or 5 h into reperfusion. Agmatine markedly reduced infarct area in all treatment groups except when treatment was delayed 5 h. The number of nNOS immunopositive cells was correlated with neuroprotection. Interestingly, immunoreactivity for iNOS was reduced only when agmatine was administered before and at the onset of MCAO. Our study suggests that agmatine may be a novel therapeutic strategy to reduce cerebral ischemic injury, and may act by inhibiting the detrimental effects of nNOS.     

Agmatine inhibits matrix metalloproteinase-9 via endothelial nitric oxide synthase in cerebral endothelial cells

OBJECTIVES: Matrix metalloproteinases (MMPs) are up-regulated by ischemic injury and degrade the basement membrane of brain vessels to promote cell death and tissue injury. We previously showed that agmatine has a neuroprotective effect on neurons against ischemic injury. In the present study, we investigated the effect of agmatine on the expression of MMPs and nitric oxide (NO) production in cerebral endothelial cells (CECs) after oxygen-glucose deprivation (OGD)-reperfusion injury and its potential association with endothelial nitric oxide synthase (eNOS). METHODS: Primary cultured endothelial cells from murine brain and bEnd.3 cells were subjected to OGD-reperfusion injury. Protein and mRNA levels of both MMP-2 and MMP-9 were determined by immunocytochemical analysis, Western blot and RT-PCR. Protein levels of eNOS were evaluated by Western blot in the CECs. The production of NO was measured using the Griess reagent. RESULTS: Agmatine attenuated the expression of MMP-2 and MMP-9 induced by ischemic injury at the protein and mRNA level, while agmatine increased the expression of eNOS directly. NO production was decreased in CECs after similar insult and was increased by agmatine treatment. In the presence of a nitric oxide synthase (NOS) inhibitor, N(omega)-nitro-L-arginine methyl ester (L-NAME), the expression levels of MMP-2 were decreased, but the expression of MMP-9 was not decreased by agmatine administration. However, NO production was suppressed by a non-specific NOS inhibitor in the agmatine treatment group. CONCLUSION: Our study supports that the down-regulation of MMP-9 by agmatine runs parallel to the up-regulation of eNOS and the maintenance of functional NO release.     

Recombinant arginine deiminase reduces inducible nitric oxide synthase iNOS-mediated neurotoxicity in a coculture of neurons and microglia

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.     

Luteolin reduces primary hippocampal neurons death induced by neuroinflammation

Abstract

Objectives: This study examined whether luteolin may exert an anti-inflammatory effect in microglia and may be neuroprotective by regulating microglia activation.

Methods: We treated BV2 microglia with 1·0 μg/ml lipopolysaccharide (LPS) after incubation with luteolin for 1 hour, the nitric oxide (NO) levels were determined by a Griess reaction, the inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-alpha (TNF-alpha), and interleukin 1beta (IL-1beta) mRNA expression were determined by real-time PCR analysis, the iNOS and COX-2 protein induction were determined by Western blot analysis, and the levels of prostaglandin E₂ (PGE₂), TNF-alpha, and IL-1beta were determined by enzyme-linked immunosorbent assay (ELISA) kits. Rat primary hippocampal neurons were co-cultured with LPS-activated BV2 microglia with 20 μM luteolin for 24 hours, the hippocampal neurons viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and the number of apoptotic hippocampal neurons was determined by immunofluorescence detection.

Results: Luteolin significantly inhibited the expression of iNOS and COX-2 in LPS-induced BV2 microglia. Moreover, the compound down-regulated the proinflammatory cytokines (TNF-alpha and IL-1beta) as well as the production of NO and PGE₂ in these cells. When hippocampal neurons were co-cultured with LPS-stimulated BV2 microglia, the administration of 20 μM luteolin increased the neurons viability and reduced the number of apoptotic neurons.

Conclusion: These data demonstrate that anti-inflammatory activity of luteolin in microglia contributes to its neuroprotective effect and suggest that it may have a potential therapeutic application in the treatment of neurodegenerative diseases.     

Expression of citrulline–nitric oxide cycle in lipopolysaccharide and cytokine-stimulated rat astroglioma C6 cells

Nitric oxide (NO) is involved in many physiological and pathological processes in the brain. NO is synthesized from arginine by nitric oxide synthase (NOS), with citrulline generated as a by-product of the reaction. Thus, citrulline can by recycled to arginine by argininosuccinate synthetase (AS) and argininosuccinate lyase (AL) via the citrulline–NO cycle. Rat astroglioma C6 cells were treated with bacterial lipopolysaccharide (LPS), interferon-γ (IFNγ) and tumor necrosis factor-α, and the expression of the enzymes of the citrulline–NO cycle was investigated by RNA blot and immunoblot analyses. NO production from arginine and citrulline was also assessed. iNOS mRNA and protein were induced 6–12 h after stimulation with LPS and cytokines and decreased at 24 h. AS mRNA increased up to 12 h and decreased at 24 h. AS protein increased gradually up to 48 h. On the other hand, AL mRNA remained unchanged by stimulation. NO production from arginine was enhanced by the treatment with LPS and cytokines. NO production was also observed when arginine was replaced by citrulline. These results indicate that NO production is enhanced in LPS- and cytokine-stimulated C6 cells due to induction of iNOS and that the citrulline–arginine recycling is important for NO production.     

Induction of nitric oxide synthase activity in human astrocytes by interleukin-1β and interferon-γ

Nitric oxide (NO) is a short-lived, diffusible molecule that has a variety of biological activities including vasorelaxation, neurotransmission, and cytotoxicity. In the central nervous system, a constitutive form of nitric oxide synthase (NOS) has been localized in a subset of neurons and in endothelial cells. In addition, both constitutive and LPS-inducible NOS has been demonstrated in rat astrocytes and microglia in vitro. In this report, we present evidence for the production of NO, as measured by the production of nitrite, in highly enriched human fetal astrocyte cultures stimulated with IL-1β. The production of nitrite paralleled the induction of NADPH diaphorase enzyme activity in the perikarya of the majority of stimulated astrocytes. The IL-1β-induced nitrite production by astrocytes was markedly enhanced when cells were co-stimulated with IFN-γ or TNF-α (IFN-γ > TNF-α); LPS had no effect used as a single agent or in combination with other cytokines. N^GMMA and N^G-nitro-arginine, competitive inhibitors of NOS, diminished the accumulation of nitrite, but calmodulin antagonists (trifluoperazine, W-5 and W-7) had little or no inhibitory effect. Human fetal microglia, in contrast to astrocytes, failed to secrete significant amounts of nitrite in response to various stimuli. The results demonstrate the presence of an inducible form of NOS in human fetal astrocytes; human microglia, in turn, may control astrocyte NO production by providing IL-1β as an activating signal.     

Silymarin protects dopaminergic neurons against lipopolysaccharide-induced neurotoxicity by inhibiting microglia activation

An inflammatory response in the central nervous system mediated by activation of microglia is a key event in the early stages of the development of neurodegenerative diseases. Silymarin is a polyphenolic flavanoid derived from milk thistle that has anti-inflammatory, cytoprotective and anticarcinogenic effects. In this study, we first investigated the neuroprotective effect of silymarin against lipopolysaccharide (LPS)-induced neurotoxicity in mesencephalic mixed neuron-glia cultures. The results showed that silymarin significantly inhibited the LPS-induced activation of microglia and the production of inflammatory mediators, such as tumour necrosis factor-alpha and nitric oxide (NO), and reduced the damage to dopaminergic neurons. Therefore, the inhibitory mechanisms of silymarin on microglia activation were studied further. The production of inducible nitric oxide synthase (iNOS) was studied in LPS-stimulated BV-2 cells as a model of microglia activation. Silymarin significantly reduced the LPS-induced nitrite, iNOS mRNA and protein levels in a dose-dependent manner. Moreover, LPS could induce the activation of p38 mitogen-activated protein kinase (MAPK) and c-jun N-terminal kinase but not extracellular signal-regulated kinase. The LPS-induced production of NO was inhibited by the selective p38 MAPK inhibitor SB203580. These results indicated that the p38 MAPK signalling pathway was involved in the LPS-induced NO production. However, the activation of p38 MAPK was not inhibited by silymarin. Nevertheless, silymarin could effectively reduce LPS-induced superoxide generation and nuclear factor kappaB (NF-kappaB) activation. It suggests that the inhibitory effect of silymarin on microglia activation is mediated through the inhibition of NF-kappaB activation.     

Female sex steroids: effects upon microglial cell activation

Multiple sclerosis occurs more commonly in females than males. However, the mechanisms resulting in gender differences in multiple sclerosis are unknown. Activated microglia are believed to contribute to multiple sclerosis pathology, perhaps in part due to production of nitric oxide (NO) and TNF-alpha, molecules which can be toxic to cells including oligodendrocytes. The current study demonstrates that the female sex steroids estriol, beta-estradiol and progesterone inhibit lipopolysaccharide (LPS) induction of nitric oxide (NO) production by primary rat microglia and by the mouse N9 microglial cell line. These hormones act by inhibiting the production of inducible nitric oxide synthase (iNOS) which catalyses the synthesis of NO. Estriol likely inhibits iNOS gene expression since the hormone blocks LPS induction of iNOS RNA levels. The pro-inflammatory cytokines IFN-gamma and TNF-alpha are believed to be important modulators of multiple sclerosis. Here, we demonstrate that estrogens and progesterone also inhibit NO production by microglial cells activated in response to these cytokines. Activated microglia elicit TNF-alpha in addition to NO and we further demonstrate that estrogens and progesterone repress TNF-alpha production by these cells. Finally, estriol and progesterone, at concentrations consistent with late pregnancy, inhibit NO and TNF-alpha production by activated microglia, suggesting that hormone inhibition of microglial cell activation may contribute to the decreased severity of multiple sclerosis symptoms commonly associated with pregnancy.     

Primary murine microglia are resistant to nitric oxide inhibition of indoleamine 2,3-dioxygenase

Indoleamine 2,3-dioxygenase (IDO) is an intracellular heme-containing enzyme that is activated by proinflammatory cytokines, including interferon-γ (IFNγ), and metabolizes tryptophan along the kynurenine pathway. Activation of murine macrophages induces not only IDO but also nitric oxide synthase (iNOS), and the ensuing production of nitric oxide (NO) inhibits IDO. To determine the sensitivity of primary cultures of murine microglia to NO, microglia were stimulated with recombinant murine IFNγ (1 ng/ml) and lipopolysaccharide (LPS) (10 ng/ml). This combination of IFNγ + LPS synergized to produce maximal amounts of nitrite as early as 16 h. Steady-state mRNAs for both iNOS and IDO were significantly increased by IFNγ + LPS at 4 h post-treatment, followed by an increase in IDO enzymatic activity at 24 h. Murine microglia (>95% CD11b⁺) were pretreated with the iNOS inhibitor, L-NIL hydrochloride, at a dose (30 μM) that completely abrogated production of nitrite. L-NIL had no effect on IDO mRNA at 4 h or IDO enzymatic activity at 24 h following stimulation with IFNγ + LPS. These data establish that IDO regulation in murine microglia is not restrained by NO, thereby permitting the accumulation of kynurenine and its downstream metabolites in the central nervous system.     

Increased inducible nitric oxide synthase protein but limited nitric oxide formation occurs in astrocytes of the hph-1 (tetrahydrobiopterin deficient) mouse

It has been suggested that decreased tetrahydrobiopterin (BH₄) availability may be a useful tool for limiting excessive nitric oxide (NO) formation. In order to test this hypothesis we utilised cultured astrocytes derived from the brain of the hph-1 (BH₄ deficient) mouse. In response to treatment with lipopolysaccharide and interferon-γ (LPS/γIFN) levels of BH₄ doubled in both wild type and hph-1 astrocytes. However, levels of BH₄ in hph-1 astrocytes remained only 25% of the wild type astrocytes. Nitric oxide formation, measured with an NO-electrode, was 45% less from LPS/γIFN stimulated hph-1 astrocytes compared with wild type stimulated astrocytes. In contrast, iNOS specific activity and iNOS protein were enhanced in hph-1 stimulated astrocytes by 40 and 60%, respectively when compared with wild type. In conclusion it appears that whilst a decrease in BH₄ may limit NO release per se, the possibility and consequences of long term `over' induction of iNOS protein requires further consideration.     

Characterization of a novel brain-derived microglial cell line isolated from neonatal rat brain.

We observed highly aggressively proliferating immortalized (HAPI) cells growing in cultures that had been enriched for microglia. The cells were initially obtained from mixed glial cultures prepared from 3-day-old rat brains. HAPI cells are typically round with few or no processes when cultured in 10% serum containing medium. As the percentage of serum in the medium is decreased, the HAPI cells have more processes. HAPI cells stain for the isolectin B4, OX-42, and GLUT5, which are markers for microglial cells, but the cells do not immunolabel with A2B5, a marker of cells in the oligodendroglial cell lineage, or with the astrocyte-specific marker, glial fibrillary aciidic protein (GFAP). In addition, HAPI cells are capable of phagocytosis. We conclude that HAPI cells are of microglia/macrophage lineage. Exposing HAPI cells to lipopolysaccharide (LPS) induces the mRNAs for tumor necrosis factor-alpha (TNF-alpha) and inducible nitric oxide synthase (iNOS). LPS exposure also induces secretion of TNF-alpha and production of nitric oxide (NO) in HAPI cells. Because activation of microglia is associated with an increase in iron accumulation and ferritin expression, we tested the hypothesis that iron status affects the production of TNF-alpha and NO. Our studies demonstrate that both iron chelation and iron loading diminished the LPS-induced effect of TNF-alpha and NO. The results of this study indicate that HAPI cells possess the characteristics of microglia/brain macrophages, providing an alternative cell culture model for the study of microglia. In addition, we demonstrate that the activation of microglial cells could be modified by iron.     

Gradual inhibition of inducible nitric oxide synthase but not of interleukin-1β production in rat microglial cells of endotoxin-treated mixed glial cell cultures

In cultures of purified microglial cells and astrocytes from newborn rats, the immunocytochemical localization of interleukin-1β (IL-1β) and inducible nitric oxide synthase (iNOS) using recently developed antibodies, as well as the release of IL-1β and nitric oxide (NO), was studied following exposure of the cells to endotoxin [lipopolysaccharide (LPS)]. In the absence of LPS, IL-1β- and iNOS-immunoreactive microglial cells and IL-1β or NO release were not observed, whereas in the presence of the endotoxin, the production of NO and IL-1β by microglial cells dramatically exceeded their synthesis and release by astrocytes. Interestingly, microglial cells cultured for 4–8 days in the presence of astrocytes appeared to lose their ability to produce iNOS, whereas the release of IL-1β remained unaltered. Moreover, endotoxin-stimulated microglial cells appeared to regain their ability to synthesize iNOS following their separation from astrocytes. These data show that microglia are primarily responsible for NO and IL-1β production in mixed glial cell cultures upon endotoxin stimulation. Moreover, in the presence of astrocytes the induction of iNOS, but not that of IL-1β in microglial cells is gradually inhibited. © 1996 Wiley-Liss, Inc.     

脂多糖对多巴胺能神经元的损毁作用及其机制研究

目的观察脂多糖(1ipopolysaccharide．LPs)对黑质多巴胺(dopamine，DA)能神经元的损毁作用，探讨免疫机制与帕金森病(Parkinsondisease，PD)发病的相关性。方法采用立体定向术将LPS注人大鼠单侧黑质后分别于注射后2、3、4周经腹腔注射阿朴吗啡诱发动物旋转行为；采用高效液相色谱一电化学法(high performanee liquid chromatography，HPLC)测定纹状体和黑质部位DA等单胺类递质含量；采用免疫组化法检测黑质酪氨酸羟化酶(tyrosine hydroxylase，TH)阳性细胞数；采用尼(氏)染色(Nissl)观察小胶质细胞的活化和以双重免疫酶染色法观察小胶质细胞活化和诱导型一氧化氮合酶(inducible nitric oxide，iNOS)合成。结果大鼠单侧黑质注人LPS后2、3、4周，以阿朴吗啡诱发大鼠均出现旋转行为，其损伤侧纹状体和黑质DA及其代谢物含量降低了30%～70%，注射侧的黑质TH阳性细胞数减少，尤以3周和4周为甚．尼(氏)染色和双重免疫酶染色法也分别显示LPS注射侧小胶质细胞活化，同时伴有iNOS合成的增加。结论LPS对DA能神经元具有一定的损毁作用，小胶质细胞的活化及其释放的NO有可能参与该细胞死亡，提示免疫机制与PD的发病可能存在相关性。     

Role of nitric oxide in the brain during lipopolysaccharide-evoked systemic inflammation

Although the inducible isoform of nitric oxide synthase (iNOS) is a well-established source of nitric oxide (NO*) during inflammation of the central nervous system (CNS), little is known about the involvement of constitutive isoforms of NOS (cNOS) in the inflammatory process. The aim of this study was to compare the responses of the expression and activity of iNOS and the two cNOS isoforms, neuronal and endothelial (nNOS and eNOS, respectively), in the brain to systemic inflammation and their roles in the cascade of events leading to degeneration and apoptosis. A systemic inflammatory response in C57BL/6 mice was induced by intraperitoneal injection of lipopolysaccharide [LPS; 1 mg/kg body weight (b.w.)]. The relative roles of the NOS isoforms were evaluated after injection of NG-nitro-L-arginine (NNLA; 30 mg/kg b.w.), which preferentially inhibits cNOS, or 1400W (5 mg/kg b.w.), an inhibitor of iNOS. Biochemical and morphological alterations were analyzed up to 48 hr after administration of LPS. Systemic LPS administration evoked significant ultrastructural alterations in brain capillary vessels, neuropils, and intracellular organelles of neurons, astrocytes, and microglia. Apoptotic/autophagic processes occurred in many neurons of the substantia nigra (SN), which coincided with exclusive enhancement of iNOS expression and activity in this brain region. Moreover, inhibitors of both iNOS and cNOS prevented LPS-evoked release of apoptosis-inducing factor (AIF) from SN mitochondria. Collectively, the results indicate that synthesis of NO* by both the inducible and constitutive NOS isoforms contribute to the activation of apoptotic pathways in the brain during systemic inflammation.     

In vitro and in vivo induction of heme oxygenase-1 in rat glial cells: Possible involvement of nitric oxide production from inducible nitric oxide synthase

To determine whether heme oxygenase-1 (HO-1) protein is induced by endogenous nitric oxide (NO) in rat glial cultures, we examined the effects of lipopolysaccharide (LPS), interferon-γ (IFN-γ), and NO donors such as S-nitroso-N-acetylpenicillamine (SNAP), in mixed glial cells and in vivo rat hippocampus. In cultured glial cells, treatment with LPS induced the expression of 130-kd inducible NO synthase (iNOS) after 6 h, and NO₂⁻accumulation and enhancement of the protein level of 33-kd HO-1 after 12 h. In addition, treatment with SNAP induced HO-1 expression after 6 h. Although NOS inhibitors such as N^G-nitro-L-arginine (NNA) and N^G-methyl-L-arginine did not change LPS-induced iNOS expression, these inhibitors suppressed both NO₂⁻ accumulation and the enhancement of HO-1. Immunocytochemistry showed that treatment with LPS for 24 h induced iNOS immunoreactivity predominantly in ameboid microglia, while this treatment induced HO-1-immunoreactivity in both microglia and astrocytes. In in vivo rat hippocampus, microinjection of LPS plus IFN-γ, or SNAP after 24 h also induced HO-1 immunoreactivity in reactive microglia and astrocytes. In addition, intraperitoneal administration of NNA inhibited HO-1 immunoreactivity induced by the microinjection of LPS plus IFN-γ. 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 vitro and in rat brain. GLIA 22:138–148, 1998.© 1998 Wiley-Liss, Inc.     

Hypothermia suppresses inducible nitric oxide synthase and stimulates cyclooxygenase-2 in lipopolysaccharide stimulated BV-2 cells

Hypothermia is neuroprotective, possibly through suppression of microglial activation. We investigated the effects of hypothermia on lipopolysaccharide (LPS) stimulated BV-2 cells. At 37 degrees C, LPS elicited strong increases in inducible nitric oxide synthase (iNOS), nitric oxide (NO), cyclooxygenase-2 (COX-2), tumour necrosis factor-alpha (TNF-alpha), and interleukin-6 (IL-6), accompanied by translocation of nuclear factor-kappaB (NF-kappaB) to the nucleus. Hypothermia (33 degrees C) caused complete suppression of iNOS and NO, a partial reduction of IL-6 but did not prevent TNF-alpha production or NF-kappaB translocation. In contrast, LPS induced cyclooxygenase-2 (COX-2) to higher levels under hypothermic conditions. These results show that hypothermia selectively suppresses iNOS in microglia.     

Lipopolysaccharide and proinflammatory cytokines require different astrocyte states to induce nitric oxide production

Nitric oxide (NO) production by astrocytes is a significant factor affecting brain physiology and pathology, but the mechanism by which it is regulated is not known. Previous studies using different specimens and stimuli might have described different aspects of a complex system. We investigated the effect of culture and stimulus conditions on NO production by cultured astrocytes and identified two combinations of these allowing NO production. Lipopolysaccharide (LPS)-induced NO production required a high seeding cell density and was independent of the serum concentration, whereas that induced by proinflammatory cytokines required simultaneous treatment with interleukin-1beta, tumor necrosis factor-alpha, and interferon-gamma and low-serum conditions but was less affected by the seeding density. These two pathways showed differential sensitivity to protein kinase inhibitors. Both LPS and cytokines induced expression of inducible nitric oxide synthase (iNOS). Although LPS-induced iNOS expression required a high seeding cell density, cytokine-induced iNOS expression, in contrast to NO production, was not affected by the serum concentration. These results suggest that astrocytes interact with the environment and alter their responsiveness to NO production-inducing stimuli by regulating iNOS expression and activity. This is the first evidence for the selective use of two different regulatory pathways in any cell type.