s-nitro-n-acetylpenicillamine and thiazolyl-blue

Glutamate signaling in the mature retinal tissue is very important for accurate sensory decoding by retinal neurons and orchestrates the fine-tuned output from the retina to higher-order centers at the cerebral cortex. In this study, we show that glutamate induces a rapid extracellular-regulated kinase and cAMP-responsive element binding protein (CREB) phosphorylation in cultured developing retinal neurons. This process is reliant on alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors and nitric oxide (NO) signaling and independent of NMDA receptors activation, as it is blocked by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate antagonists as well as inhibiting NO synthase with NG-nitro-L-arginine methyl ester but not by the NMDA channel blocker dizocilpine maleate. The effect of NO on extracellular-regulated kinase and CREB is mediated by the classical NO/soluble guanylyl cyclase/protein kinase G pathways as it is inhibited by the soluble guanylyl cyclase blocker 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one and the protein kinase G inhibitor KT5823, respectively. Immunocytochemical data suggest that increased CREB phosphorylation in response to glutamate occurs in glial cell nuclei. We also have supporting evidence suggesting that neuronally produced NO directly reaches the glial cells and stimulates CREB phosphorylation. Hence, the results indicate the importance of neuronal-glial communication and glutamate/NO/CREB linkage during retinal development.

Topics: Animals; Cell Communication; Cell Survival; Cells, Cultured; Chick Embryo; CREB-Binding Protein; Dose-Response Relationship, Drug; Enzyme Inhibitors; Excitatory Amino Acid Agents; Extracellular Signal-Regulated MAP Kinases; Glutamic Acid; In Vitro Techniques; Neuroglia; Neurons; Nitric Oxide; Nitric Oxide Donors; Penicillamine; Phosphorylation; Retina; Signal Transduction; Tetrazolium Salts; Thiazoles; Time Factors

Astrocytes, the most abundant glial cell type in the brain, provide metabolic and trophic support to neurons and modulate synaptic activity. In response to a brain injury, astrocytes proliferate and become hypertrophic with an increased expression of intermediate filament proteins. This process is collectively referred to as reactive astrocytosis. Lipocalin 2 (lcn2) is a member of the lipocalin family that binds to small hydrophobic molecules. We propose that lcn2 is an autocrine mediator of reactive astrocytosis based on the multiple roles of lcn2 in the regulation of cell death, morphology, and migration of astrocytes. lcn2 expression and secretion increased after inflammatory stimulation in cultured astrocytes. Forced expression of lcn2 or treatment with LCN2 protein increased the sensitivity of astrocytes to cytotoxic stimuli. Iron and BIM (Bcl-2-interacting mediator of cell death) proteins were involved in the cytotoxic sensitization process. LCN2 protein induced upregulation of glial fibrillary acidic protein (GFAP), cell migration, and morphological changes similar to characteristic phenotypic changes termed reactive astrocytosis. The lcn2-induced phenotypic changes of astrocytes occurred through a Rho-ROCK (Rho kinase)-GFAP pathway, which was positively regulated by nitric oxide and cGMP. In zebrafishes, forced expression of rat lcn2 gene increased the number and thickness of cellular processes in GFAP-expressing radial glia cells, suggesting that lcn2 expression in glia cells plays an important role in vivo. Our results suggest that lcn2 acts in an autocrine manner to induce cell death sensitization and morphological changes in astrocytes under inflammatory conditions and that these phenotypic changes may be the basis of reactive astrocytosis in vivo.

Topics: Amides; Analysis of Variance; Animals; Animals, Newborn; Annexin A2; Apoptosis; Astrocytes; Autocrine Communication; Brain; Cell Movement; Cells, Cultured; Embryo, Nonmammalian; Flow Cytometry; Green Fluorescent Proteins; Mice; Mice, Inbred ICR; Nitrites; Nitroprusside; Penicillamine; Phagocytes; Propidium; Pyridines; rho GTP-Binding Proteins; Tetradecanoylphorbol Acetate; Tetrazolium Salts; Thiazoles; Transfection; Tumor Necrosis Factor-alpha; Zebrafish

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

1. This study was designed to investigate the effects of the nitric oxide (NO) donors sodium nitroprusside (SNP), 3-morpholinosydnonimine (SIN-1) and S-nitroso-N-acetylpenicillamine (SNAP) on N-formyl-L-methionyl-L-leucyl-phenylalanine (fMLP, 1 x 10(-7) M)-induced human eosinophil chemotaxis, cyclic guanosine-3',5'-monophosphate (cGMP) levels, protein nitration and cytotoxicity. 2. Human eosinophils were exposed to SNP, SIN-1 and SNAP (0.001-1.0 mM) for either short (10 min) or prolonged (90 min) time periods. Exposition of eosinophils with these NO donors significantly inhibited the eosinophil chemotaxis irrespective of whether cells were exposed to these agents for 10 or 90 min. No marked differences were detected among them regarding the profile of chemotaxis inhibition. 3. Exposition of eosinophils to SNP, SIN-1 and SNAP (0.001-1.0 mM) markedly elevated the cGMP levels above basal levels, but the 90-min exposition resulted in significantly higher levels compared with the 10-min protocols (5.3+/-0.6 and 2.6+/-0.2 nM 1.5 x 10(6) cells(-1), respectively). The cGMP levels achieved with SNAP were greater than SNP and SIN-1. 4. The NO donors did not induce cell toxicity in any experimental condition used. Additionally, eosinophils exposed to SNP, SIN-1 and SNAP (1.0 mM each) either for 10 or 90 min did not show any tyrosine nitration in conditions where a strong nitration of bovine serum albumin was observed. 5. Our findings show that inhibitory effects of fMLP-induced human eosinophil chemotaxis by NO donors at short or prolonged exposition time were accompanied by significant elevations of cGMP levels. However, additional elevations of cGMP levels do not change the functional profile (chemotaxis inhibition) of stimulated eosinophils.

Topics: Adolescent; Adult; Blotting, Western; Cell Survival; Chemotaxis, Leukocyte; Cyclic GMP; Eosinophils; Female; Humans; In Vitro Techniques; Male; Middle Aged; Molsidomine; N-Formylmethionine Leucyl-Phenylalanine; Nitric Oxide Donors; Nitroprusside; Penicillamine; Tetrazolium Salts; Thiazoles; Tyrosine

Phospholipase C (PLC)-delta1 protein appears to accumulate aberrantly in Alzheimer's disease brains and its expression is reported to be induced by overstimulation of N-methyl-D-aspartate (NMDA) receptor, but there is little knowledge on its physiological role. To clarify this, we examined the expression profile of PLC-delta1 in primary cultured rat cortical neurons treated with NMDA or peroxynitrite, in comparison with those of PLC-beta1 and -gamma1, the overexpression of both of which protects cells from oxidative stress. Overstimulation of NMDA receptor decreased and increased the expression of PLC-beta1 and -delta1, respectively, but did not affect that of PLC-gamma1, in the neurons. The viability of neurons decreased depending on the period of treatment with S-nitroso-N-acetyl D,L-penicillamine (SNAP), there being a significant decrease on 9 h treatment. On examination of the expression profiles of PLC isozymes after treatment of neurons with SNAP, PLC-beta1 was found to be increased after 1h treatment and decreased after 9 h treatment, while PLC-delta1 was significantly increased, especially after 5 h treatment. Peroxynitrite treatment caused a dose-dependent decrease in the viability of neurons, and expression of PLC-beta1 was increased by a nontoxic level of peroxynitrite and decreased by a toxic level of it, while that of PLC-delta1 was increased by a sublethal level of it. These findings suggested that induction of PLC-beta1 might protect neurons from oxidative stress, but that of PLC-delta1 might have the opposite role, although both isozymes responded to oxidative stress.

Topics: Animals; Blotting, Western; Cell Survival; Cells, Cultured; Cerebral Cortex; Dizocilpine Maleate; Drug Interactions; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Isoenzymes; N-Methylaspartate; Neurons; Penicillamine; Peroxynitrous Acid; Rats; Tetrazolium Salts; Thiazoles; Time Factors; Type C Phospholipases

Airway smooth muscle (ASM) hypertrophy and hyperplasia are important determinants of bronchial responsiveness in asthma, and agents that interfere with these processes may prevent airway remodeling. We tested the hypothesis that activators of soluble and particulate guanylyl cyclases would inhibit human ASM cell (HASMC) proliferation. We report that the nitric oxide (NO) donors S-nitroso-N-acetylpenicillamine (SNAP; 10(-6) to 10(-4) M) and sodium nitroprusside (10(-5) to 10(-3) M) and human atrial natriuretic peptide [ANP-(1-28); 10(-8) to 10(-6) M], which activate soluble and particulate guanylyl cyclases, respectively, inhibited serum- and thrombin-induced proliferation of cultured HASMCs. The antimitogenic effect of SNAP was reversed by hemoglobin (10(-5) M), an NO scavenger, suggesting that NO donation was involved. The antiproliferative effects of SNAP and ANP-(1-28) were potentiated by the cGMP-specific phosphodiesterase zaprinast and mimicked by 8-bromo-cGMP (10(-6) to 10(-3) M), suggesting that cGMP-dependent mechanisms were involved. However, first, ANP-(1-28) produced a smaller antiproliferative effect than SNAP in contrast to their abilities to elevate cGMP, and second, rat ANP-(104-126), which binds selectively to ANP clearance receptors without elevating cGMP, had a small antiproliferative effect, suggesting that cGMP-independent mechanisms were also involved. These results provide evidence for a novel antiproliferative effect of NO and ANP in HASMCs mediated through cGMP-dependent and cGMP-independent mechanisms.

Topics: Asthma; Atrial Natriuretic Factor; Blood Proteins; Cell Division; Cells, Cultured; Coloring Agents; Cyclic GMP; Diuretics; Hemoglobins; Hemostatics; Humans; Hyperplasia; Lung; Mitogens; Muscle, Smooth; Nitric Oxide; Nitric Oxide Donors; Nitroprusside; Penicillamine; Peptide Fragments; Phosphodiesterase Inhibitors; Purinones; Tetrazolium Salts; Thiazoles; Thrombin; Vasodilator Agents