s-nitro-n-acetylpenicillamine and 3-nitrotyrosine

Age-related changes in the effects of nitric oxide (NO) on neurons of the auditory cortex have not been determined. We therefore evaluated the anatomical changes and neurophysiological characteristics of these neurons in rats as a function of age. The numbers of cresyl violet stained cells, the numbers and areas of NADPH-d-positive neuronal cell bodies, and their optical density, were measured in Sprague-Dawley rats aged 24 months (aged group) and 4 months (control group). The modulatory effects of NO on K(+) currents of acutely isolated rat auditory cortical neurons were also assessed. There were no between-group differences in the distribution patterns of glial cells and neurons, or in the numbers and areas of NADPH-d-positive neuronal cell bodies. However, the optical density of NADPH-d-positive neuronal cell bodies was significantly greater in the aged group than in the control group. In addition, voltage-gated K(+) currents of rat auditory cortical neurons were activated by increased levels of NO. As activation of the K(+) current likely suppresses neuronal excitability, age-associated increases in NO production can hinder the function of the acoustic center by inhibiting neuron excitability.

Topics: Age Factors; Aging; Animals; Auditory Cortex; In Vitro Techniques; Male; Membrane Potentials; NADP; Neurons; Nitric Oxide; Nitric Oxide Donors; Patch-Clamp Techniques; Penicillamine; Rats; Rats, Sprague-Dawley; Tyrosine

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

Protein kinase C (PKC) is an important intracellular signaling molecule whose activity is essential for a number of aspects of neuronal function including synaptic plasticity. We investigated the regulation of PKC activity by reactive nitrogen species in order to examine whether such species regulate PKC in neurons. Neither autonomous nor cofactor-dependent PKC activity was altered when either hippocampal homogenates or rat brain purified PKC were incubated briefly with three different nitric oxide donor compounds. However, brief incubation of either hippocampal homogenates or purified PKC with peroxynitrite (ONOO(-)) inhibited cofactor-dependent PKC activity in a manner that correlated with the nitration of tyrosine residues on PKC, suggesting that this modification was responsible for the inhibition of PKC. Consistent with this idea, reducing agents had no effect on the inhibition of PKC activity caused by ONOO(-). Because there are numerous PKC isoforms that differ in the composition of the regulatory domain, we studied the effect of ONOO(-) on various PKC isoforms. ONOO(-) inhibited the cofactor-dependent activity of the alpha, betaII, epsilon, and zeta isoforms, indicating that inhibition of enzymatic activity by ONOO(-) was not PKC isoform-specific. We also were able to isolate nitrated PKCalpha and PKCbetaII from ONOO(-)-treated hippocampal homogenates via immunoprecipitation. Collectively, our findings support the hypothesis that ONOO(-) inhibits PKC activity via tyrosine nitration in neurons.

Topics: Animals; Brain; Cysteine; Isoenzymes; Male; Neurodegenerative Diseases; Nitrates; Nitric Oxide Donors; Nitroprusside; Nitroso Compounds; Oxidants; Oxidation-Reduction; Penicillamine; Protein Kinase C; Protein Kinase C beta; Protein Kinase C-alpha; Rats; Rats, Sprague-Dawley; S-Nitrosothiols; Tissue Extracts; Tyrosine

We exposed adherent neutrophils to the nitric oxide (NO)-radical donors S-nitroso-N-acetylpenicillamine (SNAP), S-nitrosoglutathione (GSNO), and sodium nitroprusside (SNP) to study the role of NO in morphology and Ca(2+) signaling. Parallel to video imaging of cell morphology and migration in neutrophils, changes in intracellular free Ca(2+) ([Ca(2+)](i)) were assessed by ratio imaging of Fura-2. NO induced a rapid and persistent morphological hyperpolarization followed by migrational arrest that usually lasted throughout the 10-min experiments. Addition of 0.5-800 microM SNAP caused concentration-dependent elevation of [Ca(2+)](i) with an optimal effect at 50 microM. This was probably induced by NO itself, because no change in [Ca(2+)](i) was observed after treatment with NO donor byproducts, i.e. D-penicillamine, glutathione, or potassium cyanide. Increasing doses of SNAP (>/=200 microM) attenuated the Ca(2+) response to the soluble chemotactic stimulus formyl-methionyl-leucyl-phenylalanine (fMLP), and both NO- and fMLP-induced Ca(2+) transients were abolished at 800 microM SNAP or more. In kinetic studies of fluorescently labeled actin cytoskeleton, NO markedly reduced the F-actin content and profoundly increased cell area. Immunoblotting to investigate the formation of nitrotyrosine residues in cells exposed to NO donors did not imply nitrosylation, nor could we mimic the effects of NO with the cell permeant form of cGMP, i.e., 8-Br-cGMP. Hence these processes were probably not the principal NO targets. In summary, NO donors initially increased neutrophil morphological alterations, presumably due to an increase in [Ca(2+)](i), and thereafter inhibited such shape changes. Our observations demonstrate that the effects of NO donors are important for regulation of cellular signaling, i.e., Ca(2+) homeostasis, and also affect cell migration, e.g., through effects on F-actin turnover. Our results are discussed in relation to the complex mechanisms that govern basic cell shape changes, required for migration.

Topics: Actins; Calcium; Calcium Signaling; Cell Adhesion; Cell Movement; Cell Size; Cyclic GMP; Dose-Response Relationship, Drug; Homeostasis; Humans; Indicators and Reagents; Neutrophils; Nitric Oxide; Nitric Oxide Donors; Nitroprusside; Penicillamine; Tyrosine

Overexpression of Cu,Zn superoxide dismutase (SOD1) reduces ischemic injury in some stroke models but exacerbates injury in a neonatal stroke model and in other settings. The current study used a SOD1 transgenic (SOD1-Tg) murine cortical culture system, derived from the same mouse strain previously used for the stroke models, to identify conditions that determine whether SOD1 overexpression in neurons is protective or detrimental. The nitric oxide (NO) donors S-nitroso-N-acetylpenicillamine, spermine-NONOate, and diethylamine-NONOate produced less death in SOD1-Tg neurons than in wild-type neurons (p < 0.01). Also, NO produced markedly less 3-nitrotyosine in SOD1-Tg cells. In contrast, the superoxide generator menadione produced significantly greater death and nearly twice as much 2'7'-dichlorofluorescein fluorescence in SOD1-Tg neurons than in wild-type neurons, suggesting increased peroxide formation in the SOD1-Tg cells. No significant difference was observed in the vulnerability of the two cell types to H2O2, the product of the SOD reaction. Overexpression of SOD1 also had no effect on neuronal vulnerability to glutamate, N-methyl-D-aspartate, or kainate. These observations suggest that SOD1 overexpression can reduce neuronal death under conditions where peroxynitrite formation is a significant factor, but may exacerbate neuronal death under conditions of rapid intracellular superoxide formation or impaired H2O2 disposal.

Topics: Animals; Astrocytes; Cell Death; Cells, Cultured; Cerebral Cortex; Excitatory Amino Acid Agonists; Gene Expression Regulation, Enzymologic; Glutamic Acid; Humans; Hydrazines; Kainic Acid; Mice; Mice, Transgenic; N-Methylaspartate; Neurons; Neurotoxins; Nitric Oxide; Nitric Oxide Donors; Nitrogen Oxides; Penicillamine; Reactive Oxygen Species; Spermine; Superoxide Dismutase; Tyrosine; Vitamin K

Human polymorphonuclear leukocytes (PMN) produce nitric oxide (NO), superoxide (O2.-) and peroxynitrite (ONOO-) upon stimulation. We investigated the role of ONOO- in PMN-induced injury to cultured bovine aortic endothelial cells (BAEC).. BAEC were cocultured with phorbol 12-myristate 13-acetate (PMA)-activated human PMN (effector-to-target ratio, 10:1) and injury to BAEC was evaluated at intervals by 51Cr release assay. The levels of NO, O2.-, ONOO- and nitrotyrosine, a reaction product of ONOO-, were also measured, and the influence of NO synthase inhibitors, O2.- and hydroxyl radical scavengers and other effectors was examined.. In BAEC cocultured with PMA-activated PMN, 51Cr release was significantly increased [14.6 +/- 2.2% at 2 h (p < 0.05) and 42.6 +/- 2.7% at 4 h (p < 0.01); control (nonactivated PMN), < 4%]. Superoxide dismutase (100 U/ml) reduced 51Cr release to 4.6 +/- 2.2% at 2 h (p < 0.05). N-Iminoethyl-L-ornithine (L-NIO, 0.1 mM) potentiated 51Cr release (30.6 +/- 3.8% at 2 h, p < 0.01), and the potentiation was eliminated by anti-CD18 monoclonal antibody. The 51Cr release was completely prevented by dimethyl sulfoxide or by deferoxamine. Treatment of PMN with L-NIO inhibited NO generation and increased O2.- production. The nitrotyrosine level did not increase in BAEC cocultured with PMA-activated PMN.. NO-derived ONOO- is not a major cytotoxic mediator in BAEC injury by activated PMN. NO may have a cytoprotective effect by inhibiting PMN adherence to endothelial cells.

Topics: Animals; Antibodies, Monoclonal; Cattle; CD18 Antigens; Cells, Cultured; Coculture Techniques; Deferoxamine; Dimethyl Sulfoxide; Endothelium, Vascular; Humans; Neutrophil Activation; Neutrophils; Nitrates; Nitric Oxide Synthase; Nitrites; Ornithine; Penicillamine; Superoxide Dismutase; Tetradecanoylphorbol Acetate; Tyrosine