Coinduction of inducible nitric oxide synthase and arginine recycling enzymes in cytokine-stimulated PC12 cells and high output production of nitric oxide

Nitric oxide (NO) is involved in many physiological and pathological processes in the brain. NO is synthesized from arginine by nitric oxide synthase (NOS), and the citrulline generated as a by-product can be recycled to arginine by argininosuccinate synthetase (AS) and argininosuccinate lyase (AL) via the citrulline-NO cycle. When neuronal PC12 cells differentiated with nerve growth factor were treated with interferon-gamma (IFNgamma) and tumor necrosis factor-alpha (TNFalpha), iNOS and AS mRNAs and proteins were markedly induced, with AL mRNA and protein being weakly induced. Cationic amino acid transporter-1 and -2 were not induced. IFNgamma or TNFalpha alone was ineffective. A large amount of NO (190 microM NO(2)(-) plus NO(3)(-) in culture medium in 24 h) was produced from arginine by cytokine-stimulated cells, and arginine could be replaced by citrulline. iNOS induction and NO production were attenuated by dexamethasone and dibutyryl cAMP and even more strongly so when combined. Therefore, a large amount of NO is produced in cytokine-stimulated PC12 cells following to induction of iNOS and citrulline-arginine recycling is important for NO production.     

Co-induction of argininosuccinate synthetase, cationic amino acid transporter-2, and nitric oxide synthase in activated murine microglial cells

Nitric oxide (NO) produced by activated microglia has been implicated in many pathophysiological events in the brain including neurodegenerative diseases. Cellular NO production depends absolutely on the availability of arginine, a substrate of NO synthase (NOS). Murine microglial MG5 cells were treated with bacterial lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma), and expression of inducible NO synthase (iNOS) and arginine-supplying enzymes was investigated by RNA blot analysis. iNOS mRNA was strongly induced after treatment and reached a maximum at 6-12 h. mRNA for argininosuccinate synthetase (AS), a citrulline-arginine recycling enzyme, increased at 6 h and reached a maximum at 12 h. Immunoblot analysis showed that iNOS and AS proteins were also induced. In addition, mRNA encoding the cationic amino acid transporter-2 (CAT-2) was strongly induced shortly after treatment. Induction of mRNAs for iNOS, AS, and CAT-2 by LPS/IFN-gamma was also observed following stimulation of rat primary microglial cells. These results strongly suggest that both arginine transport by CAT-2 and citrulline-arginine recycling are important for high-output production of NO in activated microglial cells.     

Cyclosporin A inhibits activation of inducible nitric oxide synthase in C6 glioma cell line

The effects of immunosuppressant cyclosporin A (CsA) on nitric oxide (NO) production and inducible NO synthase (iNOS) mRNA expression in rat C6 glioma cell line were investigated. CsA applied simultaneously with iNOS activator IFN-γ caused dose-dependent reduction of NO synthesis in confluent C6 cells, as determined by measuring accumulation of nitrite, an indicator of NO production, in 48 h culture supernatants. IFN-γ-induced expression of iNOS, but not interferon regulatory factor-1 (IRF-1) mRNA was reduced in CsA-treated cells. The enzymatic activity of iNOS was not changed by CsA, since it failed to affect NO production in cells in which iNOS had already been induced with IFN-γ and any further induction was blocked by protein synthesis inhibitor cycloheximide (CHX). FK506 was not able to mimic inhibitory effect of CsA on NO production in C6 cells, suggesting calcineurin-independent mechanism of CsA action.     

Agmatine suppresses nitric oxide production in microglia

We investigated the effect of agmatine, an arginine metabolite synthesized in the brain, in cultured microglia obtained from neonatal rat cerebral cortex. Agmatine (1–300 μM) did not affect viability of cultured microglia. Activation of microglia by lipopolysaccharide (LPS, 1 μg/ml) caused the expression of inducible nitric oxide synthase (iNOS) and the production of nitric oxide (NO) assessed as the accumulation of nitrite in the culture supernatants. Agmatine had no effect on the expression of iNOS, but significantly suppressed the LPS-induced NO production in a concentration-dependent manner. Agmatine was also effective in suppressing the production of NO induced by a combination of interferon-γ (500 U/ml) and amyloid β protein (10 μM). In co-cultures of rat cortical neurons and microglia, LPS caused significant loss of neuron viability. The LPS neurotoxicity was not observed in the absence of microglia, and was completely blocked by the NOS inhibitor diphenyleneiodoium chloride. The neuronal death induced by microglia-derived NO was significantly attenuated by the presence of agmatine. These results suggest that agmatine works to protect neurons by inhibiting the production of NO in microglia.     

L-arginine uptake, the citrulline-NO cycle and arginase II in the rat brain: an in situ hybridization study.

Nitric oxide (NO) is synthesized from a unique precursor, arginine, by nitric oxide synthase (NOS). In brain cells, arginine is supplied by protein breakdown or extracted from the blood through cationic amino acid transporters (CATs). Arginine can also be recycled from the citrulline produced by NOS activity, through argininosuccinate synthetase (AS) and argininosuccinate lyase (AL) activities, and metabolized by arginase. NOS, AS and AL constitute the so-called citrulline-NO cycle. In order to better understand arginine transport, recycling and degradation, we studied the regional distribution of cells expressing CAT1, CAT3, AS, AL, neuronal NOS (nNOS) and arginase II (AII) in the adult rat brain by non-radioisotopic in situ hybridization (ISH). CAT1, AL and AII presented an ubiquitous neuronal and glial expression, whereas CAT3 and AS were confined to neurons. nNOS was restricted to scattered neurons and a few brain nuclei and layers. We demonstrate by this study that cells expressing nNOS all appear to express the entire citrulline-NO cycle, whereas numerous cells expressing AL do not express AS. The differential expression of these genes within the same anatomical structure could indicate that intercellular exchanges of citrulline-NO cycle metabolites are relevant. Thus vicinal interactions should be taken into account to study their regulatory mechanisms.     

Time dependency of the action of nitric oxide in lipopolysaccharide-interferon-gamma-induced neuronal cell death in murine primary neuron-glia co-cultures

We investigated the time-dependency of the action of nitric oxide (NO) on glia-mediated neuronal cell death. Cortical neuron–glia co-cultures were treated with lipopolysaccharide and interferon γ (LPS/IFNγ). The production of NO was first detectable 9 h after the exposure to LPS/IFNγ and increased for up to 48 h. A significant neuronal cell death was observed 36–48 h after treatment with LPS/IFNγ. The NO generated at the initial stage of NO synthesis (about 12 h) following exposure to LPS/IFNγ was found to be critical for LPS/IFNγ-induced neurotoxicity. Furthermore, the rate of NO production at the initial stage of NO synthesis was correlated linearly with the extent of neuronal cell death. These findings suggest that the maximal rate of NO synthesis, instead of the accumulated NO₂⁻ level, is a sensitive index for predicting endotoxin-induced cytotoxicity.     

Distinct distribution and time-course changes in neuronal nitric oxide synthase and inducible NOS in the paraventricular nucleus following lipopolysaccharide injection

Nitric oxide (NO) is known to be involved in the modulation of neuroendocrine function. To clarify the role of different isoforms of NO synthase (NOS) in the neuroendocrine response to immune challenge, the expressions of neuronal NOS (nNOS) and inducible NOS (iNOS) genes in the hypothalamus following lipopolysaccharide (LPS) injection were examined using in situ hybridization. NOS activity was also determined by NADPH-diaphorase (NADPH-d) histochemistry. LPS (25 mg/kg) or sterile saline was injected intraperitoneally to male Wistar rats and the rats sacrificed 30 min, or 1, 2, 3, 5, 12 or 24 h after injection. nNOS mRNA expression in the paraventricular nucleus (PVN) was significantly increased 2 h after LPS injection. iNOS mRNA, which was not detected until 2 h after LPS injection, was significantly increased in the PVN 3 h after LPS injection. Both RNA expressions had returned to basal levels by 12 h after LPS injection. The number of NADPH-d positive cells was significantly increased 5 h after LPS injection. iNOS expression was more robust in parvocellular PVN, while nNOS was distributed mainly in the magnocellular PVN. Double in situ hybridization histochemistry revealed that some of the iNOS- (48.4%) or nNOS-positive cells (34. 3%) in the parvocellular PVN expressed CRF mRNA. The results demonstrate that LPS-induced sepsis causes significant increases in nNOS and iNOS gene expression with different time-courses and distributions, and that iNOS mRNA was more frequently co-localized with CRF-producing parvocellular neurons in the PVN. Thus, NO produced by iNOS and nNOS may play an important role in the neuroendocrine response to an immune challenge. Distinct differences in the distribution and time-course changes of iNOS and nNOS suggest different roles for the hypothalamic-pituitary-adrenal axis and/or neurohypophyseal 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.     

Inducible nitric oxide synthase is responsible for nitric oxide release from murine pituicytes

This study investigated whether pituicytes were able to produce and release nitric oxide (NO), and which type of nitric oxide synthase (NOS) would be responsible for this phenomenon. Lipopolysaccharide (LPS) 1 micro g/ml was used as inflammatory mediator. Because pituicytes are known to secrete interleukin (IL)-6 upon stimulation with LPS, this parameter was also investigated. Cultured pituicytes, from 4-week-old male mice, were stimulated with LPS for 6 h or 24 h. At 24 h, there was a significant increase in accumulated nitrite indicating NO formation. In contrast, IL-6 release was already significantly higher 6 h after stimulation and further increased at 24 h. The correlation between accumulated nitrite and secreted IL-6 was 0.84 after 24 h of incubation with LPS. The expression of inducible NOS (iNOS) mRNA in the pituicytes was significantly higher than the control level after 6 h and 24 h of exposure to LPS, with levels at 6 h being significantly higher than those at 24 h. There was no detected expression of endothelial NOS or neuronal NOS mRNA. Cultured pituicytes were also subjected to immunocytochemistry for iNOS protein at 6, 12, and 24 h after stimulation with LPS. Most cells were positive for iNOS, but there were no observable differences with the time points that we used. Collectively, these results show that pituicytes are able to produce NO, and that the inducible form of NOS is responsible for this production. Furthermore, there is a weak correlation between NO and IL-6 released from pituicytes after 24 h of stimulation with LPS.     

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.     

Methylene blue inhibits the increase of inducible nitric oxide synthase activity induced by stress and lipopolysaccharide in the medial basal hypothalamus of rats

In infection bacterial products such as lipopolysaccharides (LPS) induce inducible nitric oxide synthase (iNOS) that produces large quantities of NO toxic to the invading organisms, but also often has toxic effects on host cells. Therefore, inhibition of iNOS activity might be beneficial in combatting these adverse effects. To determine if methylene blue (MB), an oxidizing agent that inactivates iNOS, would reduce the iNOS levels in the medial basal hypothalami (MBH) of conscious male rats, LPS (5 mg/kg) was injected intravenously (i.v.), and after 3 h they were injected i.v. with either MB (3 mg/kg) or saline and the effects on iNOS in the MBH determined. iNOS was measured by conversion of labeled arginine into citrulline by incubating MBH in the absence of calcium (Ca(2+)) since iNOS does not require Ca(2+) for activation. The results indicate that iNOS was induced by the injection of saline, but the induction by LPS was much greater, an increase of 10-fold above that of control sham-operated animals. Both the induction of iNOS from the stress of saline injections and LPS were completely eliminated by MB indicating that MB might be beneficial in preventing injury to brain tissue following LPS injection. There was no effect of either LPS or MB on the Ca(2+)-dependent constitutive NOS activity.     

Nitric oxide and prostaglandin E2 formation parallels blood–brain barrier disruption in an experimental rat model of bacterial meningitis

During meningitis, the host produces a plethora of signaling agents as part of a coordinated defense mechanism against invading pathogens. Nitric oxide (NO) and prostaglandin E₂ (PGE₂) are two such inflammatory mediators produced in response to bacterial endotoxins. Disruption of the blood–brain barrier (BBB) is one of many pathophysiological consequences of meningitis. The present objective was to examine the time-course of NO and PGE₂ production in relationship to BBB permeability alterations during experimentally-induced meningitis. Meningeal inflammation was elicited by intracisternal administration of the bacterial endotoxin, lipopolysaccharides (LPS; 200 μg), and NO, PGE₂, and BBB integrity were monitored over the next 24 h. Meningeal NO production was assessed by headspace chemiluminescence; cerebrospinal fluid PGE₂ was determined by enzyme-linked immunosorbent assay (ELISA) immunoassay; and BBB integrity was determined by the brain accumulation of ¹⁴C-sucrose. Similar time-course profiles for NO and PGE₂ were observed, with a peak effect for both inflammatory mediators observed within 6–8 h after intracisternal LPS dosing. Statistically significant (p < 0.05) disruption of the BBB was observed in various brain regions. Strikingly similar temporal relationships were observed for NO and PGE₂ production and BBB disruption. These results suggest the hypothesis that NO and PGE₂ may act in conjunction to disrupt the BBB during experimental meningitis.     

Acetylcholine receptor-reactive antibody induces nitric oxide production by a rat skeletal muscle cell line: influence of cytokine environment

The monoclonal Lewis rat skeletal muscle cell line, LE1, responded to the acetylcholine receptor (AChR)-reactive antibody mAb35 by up-regulating levels of mRNA for inducible nitric oxide synthase (iNOS/NOS-II), followed by levels of NO. Interferon-gamma (IFN-γ) and interleukin-1 (IL-1) were also each capable of inducing iNOS message, and synergistically with mAb35. Finally, myocyte-derived NO was implicated as a possible source of immunomodulation in experimental autoimmune myasthenia gravis (EAMG), as shown by the ability of the culture fluids from IFN-γ-activated LE1 cells to inhibit the proliferation of AChR-reactive T cells.     

Forskolin inhibits expression of inducible nitric oxide synthase mRNA via inhibiting the mitogen activated protein kinase in C6 cells

This study has demonstrated the mechanism of protein kinase A (PKA)-dependent inhibition of astrocytic nitric oxide production and inducible NO synthase mRNA expression induced by lipopolysaccharide. In C6 glioma cells, the stimulation with lipopolysaccharide (LPS; 1 microg/ml) evoked increases of nitric oxide (NO) production, NO synthase (iNOS) mRNA expression, phosphorylation of p38 mitogen activated protein kinase (p-p38), and the activation of NF kappa B. LPS-induced NO production and iNOS mRNA expression were inhibited by the pretreatment with forskolin (FSK; 5 microM) in a dose-dependent manner, and which were reversed by PKA inhibition by compound H89. Furthermore, LPS-induced increases of p-p38, but not activation of NF kappa B, were also reduced by FSK and H89 reversed the FSK-induced inhibition response. The dose-dependent inhibition of NO production and iNOS mRNA expression by compound SB203580 (p38 inhibitor) suggests the participation of p38 in PKA-dependent inhibition of LPS-induced NO production and iNOS mRNA expression. However, the activation of NF kappa B by LPS also not affected by SB203580. Therefore, our results suggest that, in C6 glioma cells, LPS-induced NO production and iNOS gene expression may be regulated by PKA pathway through the reduction of activity of p38 kinase. This inhibitory role of PKA may not involve the activation of NF kappa B.