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At least 19 recordsLinked to original sources
PubMed1997

Regulation of astrocyte nitric oxide synthase type II expression by ATP and glutamate involves loss of transcription factor binding to DNA.

Pretreatment of astrocytes with either glutamate or ATP, acting via specific receptors, suppresses subsequent cytokine-induced expression of type II nitric oxide synthase (NOS). This effect is downstream from the activation and translocation of nuclear factor (NF)-kappaB, as western blotting revealed no difference in accumulation of the p50 subunit in nuclear extracts from agonist-treated vs. nontreated cells. However, evidence from gel-shift assay suggests that the binding of nuclear protein from agonist-treated cells to NF-kappaB and activator protein-1 consensus oligonucleotides is markedly reduced. Selective inhibitors of protein kinases A and C could not restore either binding of transcription factors to DNA or type II NOS mRNA expression in agonist-pretreated cells. The modulation of proinflammatory cytokine-evoked type II NOS expression by ATP and glutamate may play an important role in CNS pathologies associated with stroke and trauma.

Adenosine Triphosphate↗
PubMed1997

Macrophage triggering with cecropin A and melittin-derived peptides induces type II nitric oxide synthase expression.

Triggering of RAW 264.7 cells with a cecropin A-melittin hybrid peptide (CA(1-8)M(1-18)) promoted a rapid rise in the intracellular calcium concentration that was followed, after a lag period of 6 h, by nitric oxide synthesis through the expression of the cytokine-inducible form of nitric oxide synthase (type II NOS or iNOS). The maximal effect was obtained at peptide concentrations in the 2 to 5-microM range. Simultaneous incubation with the peptide and LPS abrogated the nitric oxide synthesis elicited after LPS treatment of the cells. CA(1-8)M(1-18) induced a rapid activation of nuclear factor kappaB as evidenced by the presence of p50/p65 heterodimers of the nuclear factor kappaB/c-Rel family in the nuclei of activated cells. This peptide also activated the reporter activity of cells transfected with a plasmid harboring a 1-kb fragment corresponding to the 5'-flanking region of the murine iNOS gene. CA(1-8)M(1-18) promoted apoptotic cell death at concentrations below 1 to 2 microM, whereas higher concentrations altered the plasma membrane integrity. These results suggest the involvement of multiple intracellular signaling pathways in the mechanism by which this peptide elicits macrophage triggering.

Animals↗
PubMed2000

Correlation between type II nitric oxide synthase and p53 expression in oral squamous cell carcinoma.

Prolonged nitric oxide (NO) production by the enzyme type II nitric oxide synthase (NOS2) has been implicated in angiogenesis and metastasis of human cancers. In animal models, wild-type p53 (but not mutant) protein results in down-regulation of NOS2 expression, which reduces both tumour growth and dissemination. In the current study, we aimed to find out whether a correlation was present in oral squamous cell carcinoma. Fifty-six cases of squamous cell carcinoma were assessed immunohistochemically using antibodies to NOS2 and p53 (clone DO-7). We also confirmed NOS2 protein expression in selected cases using immunoblotting. The results were correlated with clinicopathological findings. Statistical analysis showed a significant relationship between p53 and NOS2 expression (P= 0.001). No relationship was found between size of tumour or histological degree of differentiation, and NOS2 expression in the primary tumour.

Adult↗
PubMed1997

Neuronal (type I) nitric oxide synthase regulates nuclear factor kappaB activity and immunologic (type II) nitric oxide synthase expression.

Nitric oxide subserves diverse physiologic roles in the nervous system. NO is produced from at least three different NO synthase (NOS) isoforms: neuronal NOS (nNOS), endothelial NOS, and immunologic NOS (iNOS). We show that nNOS is the predominant isoform constitutively expressed in glia. NO derived from nNOS in glia inhibits the transcription factor nuclear factor kappaB (NF kappaB) as NOS inhibitors enhance basal NF kappaB activation. Pyrrolidine dithiocarbamate (PDTC) is an inhibitor of NF kappaB in most cells; however, we show that PDTC is also a potent scavenger of NO through formation of mononitrosyl iron complexes with PDTC. In Jurkat cells, a human T-cell lymphoma cell line, tumor necrosis factor-alpha (TNF-alpha) induces NF kappaB activation that is inhibited by PDTC. Contrary to the results in Jurkat cells, PDTC did not inhibit tumor necrosis factor-alpha-induced NF kappaB activation in astrocytes; instead PDTC itself induces NF kappaB activation in astrocytes, and this may be related to scavenging of endogenously produced NO by the PDTC iron complex. In astrocytes PDTC also dramatically induces the NF kappaB-dependent enzyme, iNOS, supporting the physiologic relevance of endogenous NO regulation of NF kappaB. NF kappaB activation in glia from mice lacking nNOS responds more rapidly to PDTC compared with astrocytes from wild-type mice. Our data suggest that nNOS in astrocytes regulates NF kappaB activity and iNOS expression, and indicate a novel regulatory role for nNOS in tonically suppressing central nervous system, NF kappaB-regulated genes.

Animals↗
PubMed2004

Increased hepatic expression of nitric oxide synthase type II in cirrhotic rats.

AIM: To determine the role and effect of nitric oxide synthase type II (NOSII) in cirrhotic rats. METHODS: Expression of NOSII mRNA was detected by real time RT-PCR. The activity of nitric oxide synthase and serum levels of NO, systemic and portal hemodynamics and degrees of cirrhosis were measured with high sensitive methods. Chinese traditional medicine tetrandrine was used to treat cirrhotic rats and to evaluate the function of NO. Double-blind method was applied during the experiment. RESULTS: The concentration of NO and the activity of NOS were increased markedly at all stages of cirrhosis, and iNOSmRNA was greatly expressed. Meanwhile the portal-venous-pressure (PVP), and portal-venous-flow (PVF) were significantly increased. NO, NOS and iNOSmRNA were positively correlated to the quantity of hepatic fibrosis. Tetrandrine significantly inhibited NO production and the expression of iNOSmRNA. CONCLUSION: Increased hepatic expression of NOSII is one of the important causes of hepatic cirrhosis and portal hypertension.

Alkaloids↗
PubMed2001

Type II nitric oxide synthase (NOS2) expression correlates with lymph node status in oral squamous cell carcinoma.

In tumour biology, nitric oxide (NO) has a complex array of concentration-dependent actions, including both inhibitory and promoting effects. It is thought that the levels of NO found in many human cancers lead to enhanced angiogenesis and tumour dissemination. In the current study, we assessed the immunohistochemical expression of the enzyme type II nitric oxide synthase (NOS2) in 41 cases of oral squamous cell carcinoma and correlated the findings with lymph node status. A significant relationship was found between NOS2 expression and lymph node metastasis (P<0.0002). Furthermore, lymph node metastasis correlated with the degree and intensity of staining seen (P<0.001). No correlation was found between the size of the primary tumour, degree of tumour differentiation or smoking status and NOS2 staining. Western blotting confirmed NOS2 protein expression in select cases. As with many other human tumours, NOS2 is not a ubiquitous finding in oral cancer. Its expression may be of value in assessing lymph node status prior to surgery, and it represents a target for possible therapeutic manipulation.

Adult↗
PubMed1996

Nitric oxide synthase type II mRNA stability is translation- and transcription-dependent.

Nitric oxide synthase (NOS) type II is induced in many cell types in response to cytokines or endotoxin. The duration of type II NOS mRNA expression in astroglial cells and macrophages in vitro is brief, even in the continuous presence of inducers, and their removal dramatically accelerates mRNA decay. Addition of cycloheximide, in the presence or absence of actinomycin D, protected the mRNA from degradation. Whereas type II NOS mRNA was partially stabilized by actinomycin D, manganese superoxide dismutase mRNA was almost completely stabilized. This suggests that type II NOS mRNA stability is regulated via transcription- as well as translation-dependent processes and that the effect of actinomycin D is mRNA specific.

Animals↗
PubMed2007

Effects of a nitric oxide synthase Type II inhibitor on compound action potential thresholds in experimental endolymphatic hydrops.

HYPOTHESIS: Nitric oxide (NO) is likely to be synthesized by nitric oxide synthase Type II (NOS II) action and may partake in the origin of changes of compound action potential (CAP) threshold observed in guinea pigs with induced endolymphatic hydrops. This study aimed to assess the action of a NOS II inhibitor on CAP thresholds in these experimental samples. BACKGROUND: In guinea pigs with experimental endolymphatic hydrops, there are lesions on the cochlea and progressive increase of CAP threshold. NOS II was found in the cochlea of this animal model, and it was inferred that NO can contribute by such alterations. METHODS: The animals were divided into two groups, in which eight received an intake of a NOS II inhibitor, aminoguanidine, and another eight served as a control group. During 16 weeks, CAP thresholds at 1,000, 2,000, 4,000 and 6,000 on electrocochleography were compared between the groups. RESULTS: The group that had an intake of aminoguanidine showed a lower increase on CAP thresholds at 2,000 (p < 0.05) and 6,000 Hz (p < 0.05) at the 12th postoperative week, and at 1,000 (p < 0.05), 2,000 (p < 0.001), 4,000 (p < 0.001), > and 6,000 Hz (p < 0.001) at the 16th week. CONCLUSION: We conclude that NOS II inhibitor reduced the elevation of CAP thresholds in experimentally induced endolymphatic hydrops.

Action Potentials↗
PubMed1999

Localized increase in nitric oxide production and the expression of nitric oxide synthase isoforms in rat uterus with experimental intrauterine infection.

OBJECTIVE: We recently reported that nitric oxide was associated with increased mortality among pregnant rats with intrauterine infection. In our current study we investigated the expression of different isoforms of nitric oxide synthases and nitric oxide in the nonpregnant rat uterus with experimental intrauterine infection. STUDY DESIGN: Pathogenic Escherichia coli was inoculated into the uterine lumen of ovariectomized rats. Animals were killed after inoculation, and uterine horns were collected for assessing nitric oxide production with high-performance liquid chromatography and nitric oxide synthase (type II and type III) protein expression with Western immunoblotting and immunofluorescence methods. RESULTS: (1) Nitric oxide production increased in the infected uterine horn in a time-dependent manner after intrauterine infection but did not increase in the uninfected horn. (2) Nitric oxide synthase type III protein contents did not show a difference between infected and uninfected horns, and type III nitric oxide synthase was expressed by the epithelial cells and smooth muscle cells. (3) Type II nitric oxide synthase was abundantly expressed in infected horns but was not expressed in uninfected horns. Immunofluorescence data indicated that macrophages and natural killer cells, located in the endometrial layer clustering around epithelial cells, expressed type II protein. CONCLUSION: We suggest that localized increase in type II nitric oxide synthase expression and nitric oxide production occurs in response to intrauterine infection and that the nitric oxide system may play a role in host response to restrict the infection.

Animals↗
PubMed2000

A reverse nuclear factor-kappaB element in the rat type II nitric oxide synthase promoter mediates the induction by interleukin-1beta and interferon-gamma in rat aortic smooth muscle cells.

The rat type II nitric oxide synthase (iNOS) promoter contains two nuclear factor-kappaB (NF-kappaB)-binding sites, one upstream (-965 to -956 bp) and one downstream (-107 to -98 bp), which are important for iNOS induction. We have identified a third NF-kappaB site located at -901 to -892 bp whose sequence is identical to that of the upstream site but with the opposite orientation ("the reverse NF-kappaB site"). We hypothesized that the reverse NF-kappaB site, like the other two sites, is important for iNOS induction. With the use of a rat iNOS promoter fragment of -906 to -887 bp as probe, electrophoretic mobility shift assays were performed on nuclear proteins extracted from rat aortic smooth muscle cells (RASMCs) treated with interleukin-1beta (IL-1beta, 100 U/ml) +/- interferon-gamma (IFN-gamma, 250 U/ml) for 30 min. IL-1beta, but not IFN-gamma, induced a DNA-protein complex that was supershifted by either anti-NF-kappaB p50 or anti-NF-kappaB p65 antibody. The functionality of the reverse NF-kappaB site was evaluated by mutation experiments and transfection assays. The wild-type and mutated -1.4 kb rat iNOS promoter-luciferase constructs were transfected into RASMCs. Compared with the wild type, reverse-NF-kappaB site (-901 to -892 bp) deletion, substitution of T for C at -894 bp, and substitution TTT for CCC at -896 to -894 bp decreased the IL-1beta-induced promoter activity by 67% (p < 0.001), 45% (p < 0.001), and 56% (p < 0.001), respectively. These deletion/substitutions also decreased the IL-1beta- and IFN-gamma-induced promoter activity by 74% (p < 0.001), 53%(p < 0. 001), and 63% (p < 0.001), respectively. In conclusion, a p50 and p65 NF-kappaB heterodimer binds to a reverse-NF-kappaB site on the rat iNOS promoter and contributes to iNOS induction by IL-1beta and IFN-gamma in RASMCs.

Animals↗
PubMed1995

Cytokine-induced expression of type II nitric oxide synthase in astrocytes is downregulated by ATP and glutamate.

Combinations of cytokines and/or phorbol ester induce expression of Type II nitric oxide synthase (NOS) mRNA in astrocyte cultures via protein kinase mediated pathways (Simmons and Murphy: GLIA 11:227, 1994; Fernstein et al.: J Neurochem 62:811, 1994). Agonists that activate receptors linked to protein kinases did not reproduce this effect of cytokines in astrocytes. On the contrary, ATP and glutamate treatment of astrocytes prior to a combination of interleukin-1 beta and interferon-gamma markedly reduced (30-50%) subsequent NOS mRNA expression. The effect was not seen if treatment coincided with or followed cytokine activation, suggesting that ATP and glutamate were not destabilizing NOS mRNA. The effects of ATP and glutamate were additive and could be mimicked by selective receptor agonists, but were insensitive to a specific inhibitor of protein kinase C. The inhibition of cytokine-induced NOS mRNA expression caused by these agents was not the result of interference with the activation/translocation of nuclear factor-Kappa Beta by interleukin-1 beta. These results suggest that exposure of astrocytes to ATP and glutamate, both of which increase markedly in a variety of neuropathologies, could modulate the subsequent responsiveness of these cells to NOS-inducing stimuli. As such, this may be an important regulatory mechanism in the expression of Type II NOS in vivo.

Adenosine Triphosphate↗
PubMedDate not supplied

Experimental endolymphatic hydrops under action of a type II nitric oxide synthase inhibitor: otoacoustic emissions evaluation and electrocochleography.

UNLABELLED: In experimental endolymphatic hydrops distortion-products otoacoustic emission (dpoae) amplitudes decrease and there is elevation on electrocochleographic thresholds. Some authors found type ii nitric oxide synthase (nos ii) expression in hydropic cochleas and they suggest nitric oxide (no) may be involved in endolymphatic hydrops pathogenesis. The aim of this study was to evaluate the action of a nos ii inhibitor on dpoae and electrocochleography in experimental endolymphatic hydrops. MATERIAL AND METHODS: endolymphatic hydrops was induced in 16 guinea pigs by obliterating the endolymphatic duct and sac in the right ear. They were divided in two groups: eigth guinea pigs under the action of aminoguanidine, a nos ii inhibitor and eigth control guinea pigs. We compared dpoae amplitudes at geometric means of frequencies 1062, 2187, 4375 and 7000 hz, compound action potential threshold at 1000, 2000, 4000 and 6000 hz and summating potential to action potential (sp/ap) ratio between the groups during the postoperative observation period of 16 weeks. RESULTS: there were no significant changes in the dpoae amplitudes and in the sp/ap ratio. The group that received aminoguanidine had a lower degree of threshold increase at 2000 (p<0.05) And 6000 hz (p<0.05) In 12th postoperative week and at 1000 (p<0.05), 2000 (P<0.001), 4000 (P<0.001) And 6000 hz (p<0.001) At 16th postoperative week. CONCLUSIONS: nos ii inhibitor decreased the electrocochleography threshold elevation on experimental endolymphatic hydrops.

Animals↗
PubMed1996

Expression of nitric oxide synthase type II in the spinal cord under conditions producing thermal hyperalgesia.

There is evidence supporting spinal cord nitric oxide (NO) production in the mechanisms underlying hyperalgesia, presumed to arise from the activity of neuronal nitric oxide synthase type I (NOS I). Intrathecal administration of interleukin-1 beta and interferon-gamma to rats results in a thermal hyperalgesia which peaks at 2 h post-injection but which is undetectable 8 h post-injection. Expression of mRNA for nitric oxide synthase type II (NOS II) was detected by reverse transcription-polymerase chain reaction followed by Southern hybridization utilizing specific oligonucleotides in spinal cord tissue from animals 4 h and 8 h after cytokine injection, but not at longer time points. NOS II protein was detected in soluble fractions of spinal cords from animals 4 h and 8 h after cytokine injection. In situ hybridization for NOS II mRNA revealed positive cells bilaterally in the spinal cord 4 h after cytokine injection in a perivascular distribution and scattered throughout the gray and white matter. Immunohistochemistry for NOS II showed a similar distribution which could only be partially accounted for by macrophages/microglia. These results provide evidence for induction of NOS II expression under conditions producing thermal hyperalgesia and suggest a possible role in this behavior for the production of NO by a variety of cell types in the CNS.

Animals↗
PubMed2002

Antipsychotic, antidepressant, anxiolytic, and anticonvulsant drugs induce type II nitric oxide synthase mRNA in rat brain.

Nitric oxide synthase inhibitors have been regarded as potentially beneficial for psychiatric disorders such as depression and schizophrenia, though little is known about how nitric oxide synthases are affected by psychotropic drugs in the brain. Using reverse transcription-polymerase chain reaction analysis, we investigated the effects of short- and long-term oral treatments with several psychotropics on type II nitric oxide synthase gene expression in the rat brain. With maprotiline and fluvoxamine, enzyme mRNA levels were higher after a 28 day treatment than after 1 and 4 day treatments. Zonisamide, carbamazepine and diazepam also increased mRNA, though differences in levels between 1, 4 and 28 day treatments were not significant. These results suggest that psychotropics modulate the gene expression of type-II nitric oxide synthase in the brain.

Animals↗
PubMed1995

In vivo pharmacological evaluation of two novel type II (inducible) nitric oxide synthase inhibitors.

Selective type II (inducible) nitric oxide synthase (NOS) inhibitors have several potential therapeutic applications, including treatment of sepsis, diabetes, and autoimmune diseases. The ability of two novel, selective inhibitors of type II NOS, S-ethylisothiourea (EIT) and 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT), to inhibit type II NOS function in vivo was studied in lipopolysaccharide (LPS) treated rats. Type II NOS activity was assessed by measuring changes in plasma nitrite and nitrate concentrations ([NOx]). Both EIT and AMT elicited a dose-dependent and > 95% inhibition of the LPS-induced increase in plasma [NOx]. The ED50 values for EIT and AMT were 0.4 and 0.2 mg/kg, respectively. In addition, the administration of LPS and either NOS inhibitor resulted in a dose-dependent increase in animal mortality; neither compound was lethal when administered alone. Pretreatment with L-arginine (but not D-arginine) prevented the mortality, while not affecting the type II NOS-dependent NO production, suggesting the toxicity may be due to inhibition of one of the other NOS isoforms (endothelial or neuronal). Thus, although EIT and AMT are potent inhibitors of type II NOS function in vivo, type II NOS inhibitors of even greater selectivity may need to be developed for therapeutic applications.

Animals↗
PubMed1996

Expression of type II nitric oxide synthase in primary human astrocytes and microglia: role of IL-1beta and IL-1 receptor antagonist.

In this work, we studied the expression of type II nitric oxide synthase (NOS) in primary cultures of human astrocytes and microglia. Cytokine-activated human fetal astrocytes expressed a 4.5-kb type II NOS mRNA that was first evident at 8 h, steadily increased through 48 h, and persisted through 72 h. The inducing signals for astrocyte NOS II mRNA expression were in the order IL-1beta + IFN-gamma > IL-1beta + TNF-alpha > IL-1beta. SDS-PAGE analysis of cytokine-stimulated astrocyte cultures revealed an approximately 130-kDa single NOS II band that was expressed strongly at 48 and 72 h (72 h > 48 h). Specific NOS II immunoreactivity was detected in cytokine-treated astrocytes, both in the cytosol and in a discrete paranuclear region, which corresponded to Golgi-like membranes on immunoelectron microscopy. In human microglia, cytokines and LPS failed to induce NOS II expression, while the same stimuli readily induced TNF-alpha expression. In cytokine-treated human astrocytes, neither NOS II mRNA/protein expression nor nitrite production was inhibited by TGF-beta, IL-4, or IL-10. In contrast, IL-1 receptor antagonist exerted near complete inhibition of NOS II mRNA and nitrite induction. Monocyte chemoattractant peptide-1 mRNA was induced in TGF-beta-treated astrocytes, demonstrating the presence of receptors for TGF-beta in astrocytes. These results confirm that in humans, cytokines stimulate astrocytes, but not microglia, to express NOS II belonging to the high output nitric oxide system similar to that found in rodent macrophages. They also show that the regulation of type II NOS expression in human glia differs significantly from that in rodent glia. A crucial role for the IL-1 pathway in the regulation of human astrocyte NOS II is shown, suggesting a potential role for IL-1 as a regulator of astrocyte activation in vivo.

Astrocytes↗
PubMed2005

Signalling pathway involved in nitric oxide synthase type II activation in chondrocytes: synergistic effect of leptin with interleukin-1.

The objective of the present study was to investigate the effect of leptin, alone or in combination with IL-1, on nitric oxide synthase (NOS) type II activity in vitro in human primary chondrocytes, in the mouse chondrogenic ATDC5 cell line, and in mature and hypertrophic ATDC5 differentiated chondrocytes. For completeness, we also investigated the signalling pathway of the putative synergism between leptin and IL-1. For this purpose, nitric oxide production was evaluated using the Griess colorimetric reaction in culture medium of cells stimulated over 48 hours with leptin (800 nmol/l) and IL-1 (0.025 ng/ml), alone or combined. Specific pharmacological inhibitors of NOS type II (aminoguanidine [1 mmol/l]), janus kinase (JAK)2 (tyrphostin AG490 and Tkip), phosphatidylinositol 3-kinase (PI3K; wortmannin [1, 2.5, 5 and 10 micromol/l] and LY294002 [1, 2.5, 5 and 10 micromol/l]), mitogen-activated protein kinase kinase (MEK)1 (PD098059 [1, 5, 10, 20 and 30 micromol/l]) and p38 kinase (SB203580 [1, 5, 10, 20 and 30 micromol/l]) were added 1 hour before stimulation. Nitric oxide synthase type II mRNA expression in ATDC5 chondrocytes was investigated by real-time PCR and NOS II protein expression was analyzed by western blot. Our results indicate that stimulation of chondrocytes with IL-1 results in dose-dependent nitric oxide production. In contrast, leptin alone was unable to induce nitric oxide production or expression of NOS type II mRNA or its protein. However, co-stimulation with leptin and IL-1 resulted in a net increase in nitric oxide concentration over IL-1 challenge that was eliminated by pretreatment with the NOS II specific inhibitor aminoguanidine. Pretreatment with tyrphostin AG490 and Tkip (a SOCS-1 mimetic peptide that inhibits JAK2) blocked nitric oxide production induced by leptin/IL-1. Finally, wortmannin, LY294002, PD098059 and SB203580 significantly decreased nitric oxide production. These findings were confirmed in mature and hypertrophic ATDC5 chondrocytes, and in human primary chondrocytes. This study indicates that leptin plays a proinflammatory role, in synergy with IL-1, by inducing NOS type II through a signalling pathway that involves JAK2, PI3K, MEK-1 and p38 kinase.

Animals↗