noc-18 and 1-3-dihydroxy-4-4-5-5-tetramethyl-2-(4-carboxyphenyl)tetrahydroimidazole

Ulcerative colitis is an inflammatory bowel disease characterized by acute inflammation, ulceration, and bleeding of the colonic mucosa. Its cause remains unknown. Increases in adhesion molecules in vascular endothelium, and activated neutrophils releasing injurious molecules such as reactive oxygen species, are reportedly associated with the pathogenesis of dextran sodium sulfate (DSS)-induced colitis. Nitric oxide (NO) production derived from inducible NO synthase (iNOS) via activation of nuclear factor κB (NF-κB) has been reported. It is also reported that stimulation of Toll-like receptor 4 (TLR4) by lipopolysaccharide can activate NF-κB. In this study, we investigated the involvement of NO production in activation of the TLR4/NF-κB signaling pathway in mice with DSS-induced colitis. The addition of 5% DSS to the drinking water of male ICR mice resulted in increases in TLR4 protein in colon tissue and NF-κB p65 subunit in the nuclear fraction on day 3, increases in colonic tumor necrosis factor-α on day 4, and increases in P-selectin, intercellular adhesion molecule-1, NO2(-)/NO3(-), and nitrotyrosine in colonic mucosa on day 5. These activated inflammatory mediators and pathology of colitis were completely suppressed by treatment with a NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, as well as an iNOS inhibitor, aminoguanidine. Conversely, a NO-releasing compound, NOC-18, increased TLR4 levels and nuclear translocation of NF-κB p65 and exacerbated mucosal damage induced by DSS challenge. These data suggest that increases in TLR4 expression induced by drinking DSS-treated water might be directly or indirectly associated with NO overproduction.

Topics: Animals; Benzoates; Cells, Cultured; Colitis, Ulcerative; Colon; Dextran Sulfate; Disease Models, Animal; Female; Free Radical Scavengers; Guanidines; Humans; Imidazoles; Intercellular Adhesion Molecule-1; Intestinal Mucosa; Mice; Mice, Inbred ICR; NF-kappa B; Nitric Oxide; Nitric Oxide Synthase Type II; Nitroso Compounds; P-Selectin; Signal Transduction; Toll-Like Receptor 4; Tumor Necrosis Factor-alpha

Nitric oxide has been investigated widely both during neurodevelopment and in neurological diseases. However, whilst it has been established that nitric oxide-producing enzymes of nitric oxide synthase family are expressed in cerebellar Purkinje neurons, the effects of nitric oxide on the viability and morphology of these neurons remain unknown. Here, we have demonstrated that the activity of neuronal nitric oxide synthase, but not the inducible or endothelial forms of this enzyme, is required to support the survival of a proportion of cerebellar Purkinje neurons in vitro. We discovered that donation of high concentrations of exogenous nitric oxide reduces Purkinje neuron survival in culture and that peroxynitrite is also toxic to these cells. Finally, we demonstrated that exogenous nitric oxide and peroxynitrite reduce both the magnitude and the complexity of the neurite arbour extended by cerebellar Purkinje neurons. Taken together, these findings reveal that whilst a low level of endogenous nitric oxide, released by the activity of neuronal nitric oxide synthase, is beneficial to cerebellar Purkinje neurons in vitro, high levels of exogenous nitric oxide and peroxynitrite are detrimental to both the survival of these neurons and to their ability to extend processes and form functional neural networks.

Topics: Animals; Benzoates; Cell Count; Cells, Cultured; Cerebellar Cortex; Citrulline; Female; Imidazoles; Isothiuronium; Mice; Mice, Inbred C57BL; Nerve Degeneration; Nerve Tissue Proteins; Neurites; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitroso Compounds; Peroxynitrous Acid; Pregnancy; Purkinje Cells; Reactive Nitrogen Species; Thiourea

Recently, it has been reported that inflammatory processes are associated with the pathophysiology of Alzheimer's disease and that treatment of non-steroidal anti-inflammatory drugs reduce the risk for Alzheimer's disease. In the present study, we examined nitric oxide radical quenching activity of non-steroidal anti-inflammatory drugs and steroidal drugs using our established direct in vitro nitric oxide radical detecting system by electron spin resonance spectrometry. The non-steroidal anti-inflammatory drugs, aspirin, mefenamic acid, indomethacin and ketoprofen directly and dose-dependently scavenged generated nitric oxide radicals. In experiments of nitric oxide radical donor, NOC18-induced neuronal damage, these four non-steroidal drugs significantly prevented the NOC18-induced reduction of cell viability and apoptotic nuclear changes in neuronal cells without affecting the induction of inducible nitric oxide synthase-like immunoreactivity. However, ibuprofen, naproxen or steroidal drugs, which had less or no scavenging effects in vitro, showed almost no protective effects against NOC18-induced cell toxicity. These results suggest that the protective effects of the former four non-steroidal anti-inflammatory drugs against apoptosis might be mainly due to their direct nitric oxide radical scavenging activities in neuronal cells. These direct NO. quenching activities represent novel effects of non-steroidal anti-inflammatory drugs. Our findings identified novel pharmacological mechanisms of these drugs to exert not only their anti-inflammatory, analgesic, antipyretic activities but also neuroprotective activities against neurodegeneration.

Topics: Alzheimer Disease; Animals; Anti-Inflammatory Agents, Non-Steroidal; Aspirin; Benzoates; Cell Death; Cell Line; Cell Nucleus; Cell Survival; Dexamethasone; Electron Spin Resonance Spectroscopy; Free Radical Scavengers; Free Radicals; Humans; Hydrocortisone; Imidazoles; Indomethacin; Inflammation; Mefenamic Acid; Naproxen; Neuroblastoma; Neurons; Neuroprotective Agents; Nitric Oxide; Nitric Oxide Donors; Nitroso Compounds; Rats; Tumor Cells, Cultured

The present study was performed to clarify the involvement of nitric oxide (NO) in the expression of LH receptor in porcine granulosa cells. The granulosa cells, prepared from porcine ovarian follicles, were developed in the presence of FSH. LH receptor mRNA was induced to reach a maximal level after a 24-h culture with FSH, as determined by semi-quantitative reverse transcriptase-PCR (RT-PCR). In our previous report (Nishida et al., 2000), we found that NO was released from granulosa cells after a 40-48 h culture with FSH. When 200 microM NO scavenger was added to cultures before the NO release (30 h), the content of LH receptor on the cells decreased to 28% that of the control. However, the receptor content was not influenced by addition of NO scavenger at 46 h, or by 50 microM NO donor at 30 or 46 h. During transformation of mature granulosa cells to luteal cells, LH receptor mRNA was induced after a 24-h culture with LH, which induction was inhibited by removal of endogenous NO. However, the expression was not influenced by addition of either NO scavenger at 46 h or by NO donor. During the period of these treatments, cellular DNA contents were constant. Consequently, the transient generation of NO may play a critical role in expression of the LH receptor at transcription and post-transcription levels.

Topics: Animals; Benzoates; Cyclic AMP; Female; Follicle Stimulating Hormone; Free Radical Scavengers; Granulosa Cells; Imidazoles; Nitric Oxide; Nitric Oxide Donors; Nitroso Compounds; Ovary; Progesterone; Protein Processing, Post-Translational; Receptors, LH; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Swine; Transcription, Genetic

Atherosclerosis is a major cause of morbidity and mortality in chronic renal failure and is associated with the proliferation of macrophages within atherosclerotic lesions.. Because the progression of atherosclerosis as a consequence of decreased nitric oxide synthesis has been described, we investigated the correlation between the inhibition of inducible nitric oxide synthase (iNOS) by urea, macrophage proliferation as assayed by cell counting, tritiated thymidine incorporation and measurement of cell protein, and macrophage apoptosis.. Urea induces a dose-dependent inhibition of inducible nitric oxide synthesis in lipopolysaccharide-stimulated mouse macrophages (RAW 264.7) with concomitant macrophage proliferation. Macrophage proliferation, as determined by cell counting, became statistically significant at 60 mM urea, corresponding to a blood urea nitrogen level of 180 mg/100 ml, concentrations seen in uremic patients. iNOS protein expression showed a dose-dependent reduction, as revealed by immunoblotting when cells were incubated with increasing amounts of urea. The decrease of cytosolic DNA fragments in stimulated macrophages incubated with urea shows that the proliferative actions of urea are associated with a decrease of NO-induced apoptosis.. Our data demonstrate that the inhibition of iNOS-dependent NO production caused by urea enhances macrophage proliferation as a consequence of diminished NO-mediated apoptosis.

Topics: Animals; Apoptosis; Arteriosclerosis; Benzoates; Cell Division; Cell Line; Diuretics, Osmotic; DNA Fragmentation; Enzyme Inhibitors; Hemoglobins; Imidazoles; Lipopolysaccharides; Macrophages; Mice; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitroso Compounds; Urea

We have determined the role of nitric oxide (NO) in the nucleus of the solitary tract (NTS) of normotensive Wistar rats. The unilateral microinjection of Nomega-nitro-l-arginine methyl ester (10 nmol) to block the synthesis of NO into the NTS significantly decreased the arterial pressure, heart rate (HR) and renal sympathetic nerve activity (RSNA) (-19+/-2 mmHg, -23+/-5 beats/min, -30+/-2%, respectively). The microinjection of carboxy-2-phenyl-4,4,5, 5-tetramethylimidazoline-1-oxyl 3-oxide (Carboxy PTIO) (trapper of NO; 0.1 nmol) into the NTS also decreased arterial pressure and RSNA. Conversely, the microinjection of Et2N[N(O)NO]Na (NOC 18) (NO donor; 10 nmol) caused increases in arterial pressure, HR and RSNA (+14+/-2 mmHg, +11+/-2 beats/min, +38+/-7%, respectively), which was inhibited by the pre-microinjection of Carboxy PTIO (0.1 nmol). On the other hand, not only l-arginine (10 nmol) but also d-arginine (10 nmol), which is inactive to produce NO, significantly decreased the arterial pressure and RSNA. These results suggest that (1) NO acts at the NTS to increase the arterial pressure and RSNA, and (2) the microinjection of l-arginine as well as d-arginine led to decreases in arterial pressure and RSNA that were not mediated by the formation of NO in the NTS.

Topics: Animals; Arginine; Benzoates; Blood Pressure; Enzyme Inhibitors; Heart Rate; Imidazoles; Kidney; Male; Microinjections; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitroso Compounds; Rats; Rats, Wistar; Solitary Nucleus; Sympathetic Nervous System

Although active oxygen species and related metabolites, such as nitric oxide (NO), have been postulated to play important roles in the apoptosis of various cells, a precise mechanism leading to cell death remains to be elucidated. Recently we found that the lifetime of NO depends greatly on the concentration of environmental oxygen and that NO reversibly inhibits mitochondrial respiration and ATP synthesis; the inhibitory effect is stronger at physiologically low oxygen tension than under atmospheric conditions (Arch. Biochem. Biophys. 323, 27-32, 1995). The present work describes the effects of the NO-generating agent, 1-hydroxy-2-oxo-3,3-bis(2-aminoethyl)-1-triazene (NOC 18) and oxygen tension on the respiration, ATP synthesis and apoptosis of HL-60 cells. When respiration was inhibited by NOC 18, cellular ATP levels decreased significantly and DNA fragmentation was elicited. Both events were enhanced by decreasing oxygen tension and suppressed by adding NO-trapping agents, such as 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO) and oxyhemoglobin. The fragmentation of cellular DNA was inhibited in a dose dependent manner by herbimycin A, a tyrosine kinase inhibitor. Fragmentation of the DNA of HL-60 cells was also induced either by peroxynitrite, superoxide or hydroxyl radical by some mechanism which was diminished by lowering the oxygen tension. These results indicated that the decrease in cellular ATP and activation of tyrosine kinase might play important roles in NO-induced apoptosis particularly under physiologically low oxygen tensions.

Topics: Adenosine Triphosphate; Apoptosis; Benzoates; Benzoquinones; DNA Fragmentation; Enzyme Inhibitors; Free Radicals; HL-60 Cells; Humans; Imidazoles; Lactams, Macrocyclic; Nitrates; Nitric Oxide; Nitrites; Nitroso Compounds; Oxygen; Quinones; Reactive Oxygen Species; Rifabutin

To investigate the cardiovascular role of nitric oxide (NO) in the rostral ventrolateral medulla (RVLM), NOC 18, an NO donor, was microinjected into the RVLM of rats. NOC 18 significantly decreased mean arterial pressure (MAP). Pre-treatment with an NO trapper, carboxy-PTIO, abolished the NOC 18-induced decrease in MAP. Microinjection of L-NAME, an NO synthase inhibitor, increased MAP. L-Arginine reduced MAP and inhibited the pressor response induced by L-NAME. Results suggest that NO acts on the RVLM neurons and plays an important role in cardiovascular regulation.

Topics: Animals; Benzoates; Blood Pressure; Heart Rate; Imidazoles; Male; Medulla Oblongata; Microinjections; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitroso Compounds; Rats; Rats, Wistar

It is becoming apparent that nitrogen monoxide (NO) such as nitric oxide has regulatory roles for neuronal cell functions. We examined the role of NO using NO donors on [3H]noradrenaline (NA) release from prelabeled rat hippocampal slices. Sodium nitroprusside (SNP), which had no effect by itself, stimulated [3H]NA release in a dose-dependent manner (ED50 = 0.5 mM) in the presence of dithiothreitol (DTT). The stimulatory effect of SNP with DTT, but not high K+, was observed in an extracellular Ca(2+)-free buffer. The maximal effect of SNP was obtained after incubation for 1-2 h with DTT in buffer at physiological pH (7.4). The simultaneous addition of SNP and DTT to the slices induced a small effect, and the effect of SNP declined after 3.5 h. SNP stimulated cyclic GMP accumulation in the slices without DTT. NaNO2 and 1-hydroxy-2-oxo-3,3-bis(2-aminoethyl)-1-triazene (a generator of nitric oxide), which stimulated cyclic GMP accumulation by themselves, did not stimulate [3H]NA release in the presence and absence of DTT. 3-Morpholinosydnonimine HC1 (a generator of peroxynitrite) had no effect on the release. The stimulatory effect of SNP and DTT on NA release was inhibited 40% by nitric oxide scavengers such as oxyhemoglobin and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxy-3-oxide, although cyclic GMP accumulation induced by NO donors was completely inhibited. These findings suggest that SNP reacts with DTT to produce unknown active species, and that cyclic GMP is not a mediator for SNP-stimulated NA release.

Topics: Animals; Benzoates; Calcium; Cyclic GMP; Dithiothreitol; Drug Interactions; Hippocampus; Hydrogen-Ion Concentration; Imidazoles; In Vitro Techniques; Kinetics; Male; Nitric Oxide; Nitroprusside; Nitroso Compounds; Norepinephrine; Oxyhemoglobins; Potassium Chloride; Rats; Rats, Wistar; Sodium Nitrite

The addition of nitric oxide (NO)-releasing agents, S-nitroso-N-acetyl-D,L-penicillamine (SNAP), 1-hydroxy-2-oxo-3,3-bis(2-aminoethyl)-1-triazene (NOC18), or 3-[(+/-)-(E)-ethyl-2'-[(E)-hydroxyimino]-5-nitro-3-hexenecarbam oyl]-pyridine (NOR 4), significantly inhibited natural killer (NK) cell activity against cytomegalovirus (CMV)-infected cells. Inhibition of NK cell activity was due to NO released in the culture medium because the concentration of nitrite in the culture medium correlated with the inhibition of NK cell activity and the addition of an antagonist of NO, [2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide] (carboxy-PTIO), to NK assay restored NK cell activity. The mechanism of inhibition of NK cell activity against CMV-infected cells by NO-releasing agents includes (1) inhibition of the production of interferon (IFN)-alpha by CD16 (Leu11b)-depleted cells cultured with CMV-infected cells and (2) inhibition of the activation process of NK cell by IFN-alpha. It is suggested that the production of NO by an inflammatory process may lead to the inhibition of NK cell-mediated cytotoxicity against CMV-infected cells.

Topics: Benzoates; CD18 Antigens; Cells, Cultured; Cytomegalovirus; Cytotoxicity, Immunologic; Humans; Imidazoles; Interferon-alpha; Killer Cells, Natural; Lymphocyte Function-Associated Antigen-1; Nitric Oxide; Nitroso Compounds; Penicillamine; Pyridines; Receptors, IgG; Receptors, Interleukin-2; S-Nitroso-N-Acetylpenicillamine