2-phenyl-4-4-5-5-tetramethylimidazoline-1-oxyl-3-oxide and 6-anilino-5-8-quinolinedione

Nitrate assimilation in plants and related organisms is a highly regulated and conserved pathway in which the enzyme nitrate reductase (NR) occupies a central position. Although some progress has been made in understanding the regulation of the protein, transcriptional regulation of the NR gene (NIA1) is poorly understood. This work describes a mechanism for the ammonium-mediated repression of NIA1. We report the characterization of a mutant defective in the repression of NIA1 and NR in response to ammonium and show that a gene (CYG56) coding for a nitric oxide (NO)-dependent guanylate cyclase (GC) was interrupted in this mutant. NO donors, cGMP analogs, a phosphodiesterase inhibitor isobutylmethylxanthine (IBMX), and a calcium ionophore (A23187) repress the expression of NIA1 in Chlamydomonas reinhardtii wild-type cells and also repress the expression of other ammonium-sensitive genes. In addition, the GC inhibitors LY83,583 (6-anilino-5,8-quinolinedione) and ODQ (1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one) release cells from ammonium repression. Intracellular NO and cGMP levels were increased in the presence of ammonium in wild-type cells. In the cyg56 mutant, NIA1 transcription was less sensitive to NO donors and A23187, but responded like the wild type to IBMX. Results presented here suggest that CYG56 participates in ammonium-mediated NIA1 repression through a pathway that involves NO, cGMP, and calcium and that similar mechanisms might be occurring in plants.

Topics: Aminoquinolines; Arabidopsis; Arabidopsis Proteins; Calcium; Chlamydomonas reinhardtii; Cyclic GMP; Cyclic N-Oxides; Gene Expression Regulation, Enzymologic; Guanylate Cyclase; Imidazoles; Models, Biological; Molecular Sequence Data; Mutation; NG-Nitroarginine Methyl Ester; Nitrate Reductase; Nitric Oxide; Nitrogen; Oxadiazoles; Quaternary Ammonium Compounds; Quinoxalines; Receptors, Cytoplasmic and Nuclear; Signal Transduction; Soluble Guanylyl Cyclase; Transcription, Genetic

Microglia migrate rapidly to lesions in the central nervous system (CNS), presumably in response to chemoattractants including ATP released directly or indirectly by the injury. Previous work on the leech has shown that nitric oxide (NO), generated at the lesion, is both a stop signal for microglia at the lesion and crucial for their directed migration from hundreds of micrometers away within the nerve cord, perhaps mediated by a soluble guanylate cyclase (sGC). In this study, application of 100 microM ATP caused maximal movement of microglia in leech nerve cords. The nucleotides ADP, UTP, and the nonhydrolyzable ATP analog AMP-PNP (adenyl-5'-yl imidodiphosphate) also caused movement, whereas AMP, cAMP, and adenosine were without effect. Both movement in ATP and migration after injury were slowed by 50 microM reactive blue 2 (RB2), an antagonist of purinergic receptors, without influencing the direction of movement. This contrasted with the effect of the NO scavenger cPTIO (2-(4-carboxyphenyl)-4,4,5,5-teramethylimidazoline-oxyl-3-oxide), which misdirected movement when applied at 1 mM. The cPTIO reduced cGMP immunoreactivity without changing the immunoreactivity of eNOS (endothelial nitric oxide synthase), which accompanies increased NOS activity after nerve cord injury, consistent with involvement of sGC. Moreover, the sGC-specific inhibitor LY83583 applied at 50 microM had a similar effect, in agreement with previous results with methylene blue. Taken together, the experiments support the hypothesis that ATP released directly or indirectly by injury activates microglia to move, whereas NO that activates sGC directs migration of microglia to CNS lesions.

Topics: Adenosine Triphosphate; Aminoquinolines; Analysis of Variance; Animals; Cell Movement; Cyclic GMP; Cyclic N-Oxides; Dose-Response Relationship, Drug; Enzyme Inhibitors; Free Radical Scavengers; Imidazoles; In Vitro Techniques; Leeches; Microglia; Nitric Oxide; Nucleotides; Trauma, Nervous System; Triazines

Nitric oxide (NO) is an important signaling molecule in the CNS, regulating neuronal survival, proliferation and differentiation. Here, we explored the mechanism by which NO, produced from the NO donor S-nitroso-acetyl-d-l-penicillamine (SNAP), exerts its neuroprotective effect in purified cultures of chick retinal neurons. Cultures prepared from 8-day-old chick embryo retinas and incubated for 24 h (1 day in culture, C1) were treated or not with SNAP, incubated for a further 72 h (up to 4 days in culture, C4), fixed, and the number of cells estimated, or processed for cell death estimation, by measuring the reduction of the metabolic dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Experimental cultures were run in parallel but were re-fed with fresh medium in the absence or presence of SNAP at culture day 3 (C3), incubated for a further 24 h up to C4, then fixed or processed for the MTT assay. Previous studies showed that the re-feeding procedure promotes extensive cell death. SNAP prevented this death in a concentration- and time-dependent manner through the activation of soluble guanylate cyclase; this protection was significantly reversed by the enzyme inhibitors 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) or LY83583, and mimicked by 8-bromo cyclic guanosine 5'-phosphate (8Br-cGMP) (GMP) or 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1), guanylate cyclase activators. The effect was blocked by the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO). The effect of NO was also suppressed by LY294002, Wortmannin, PD98059, KN93 or H89, indicating the involvement, respectively, of phosphatidylinositol-3 kinase, extracellular-regulated kinases, calmodulin-dependent kinases and protein kinase A signaling pathways. NO also induced a significant increase of neurite outgrowth, indicative of neuronal differentiation, and blocked cell death induced by hydrogen peroxide. Cyclosporin A, an inhibitor of the mitochondrial permeability transition pore considered an important mediator of apoptosis and necrosis, as well as boc-aspartyl (OMe) fluoromethylketone (BAF), a caspase inhibitor, also blocked cell death induced by re-feeding the cultures. These findings demonstrate that NO inhibits apoptosis of retinal neurons in a cGMP/protein kinase G (PKG)-dependent way, and strengthens the notion that NO plays an important role during CNS development.

Topics: Adenosine; Aminoquinolines; Analysis of Variance; Animals; Cell Survival; Cells, Cultured; Chick Embryo; Cyclic GMP; Cyclic N-Oxides; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Inhibitors; Free Radical Scavengers; Imidazoles; Neurons; Nitrates; Nitric Oxide; Nitric Oxide Donors; Nitrites; Penicillamine; Retina; Signal Transduction; Tetrazolium Salts; Thiazoles; Tritium

In this study, we highlight a role for the nitric oxide-cGMP-dependent protein kinase (NO-G-kinase) signaling pathway in glial intercellular Ca(2+) wave initiation and propagation. Addition of the NO donor molsidomine (100-500 microM) or puffing aqueous NO onto primary glial cell cultures evoked an increase in [Ca(2+)](i) in individual cells and also local intercellular Ca(2+) waves, which persisted after removal of extracellular Ca(2+). High concentrations of ryanodine (100-200 microM) and antagonists of the NO-G-kinase signaling pathway essentially abrogated the NO-induced increase in [Ca(2+)](i), indicating that NO mobilizes Ca(2+) from a ryanodine receptor-linked store, via the NO-G-kinase signaling pathway. Addition of 10 microM nicardipine to cells resulted in a slowing of the molsidomine-induced rise in [Ca(2+)](i), and inhibition of Mn(2+) quench of cytosolic fura-2 fluorescence mediated by a bolus application of 2 microM aqueous NO to cells, indicating that NO also induces Ca(2+) influx in glia. Mechanical stress of individual glial cells resulted in an increase in intracellular NO in target and neighboring cells and intercellular Ca(2+) waves, which were NO, cGMP, and G-kinase dependent, because incubating cells with nitric oxide synthase, guanylate cyclase, and G-kinase inhibitors, or NO scavengers, reduced Delta[Ca(2+)](i) and the rate of Ca(2+) wave propagation in these cultures. Results from this study suggest that NO-G-kinase signaling is coupled to Ca(2+) mobilization and influx in glial cells and that this pathway plays a fundamental role in the generation and propagation of intercellular Ca(2+) waves in glia.

Topics: Aminoquinolines; Animals; Antineoplastic Agents; Apyrase; Astrocytes; Caenorhabditis elegans Proteins; Calcium; Calcium Channel Blockers; Cells, Cultured; Chelating Agents; Cyclic GMP; Cyclic N-Oxides; Egtazic Acid; Enzyme Inhibitors; Estrenes; Free Radical Scavengers; GTP-Binding Proteins; Imidazoles; Ionomycin; Ionophores; Neurons; Nicardipine; Nitric Oxide; Nitric Oxide Synthase; omega-N-Methylarginine; Phosphodiesterase Inhibitors; Potassium Chloride; Prosencephalon; Pyrrolidinones; Rats; Receptor, Insulin; Ryanodine; Ryanodine Receptor Calcium Release Channel; Signal Transduction; Suramin; Thionucleotides; Type C Phospholipases