Neuronal apoptosis versus necrosis induced by glutamate or free radicals

The type of cell death encountered in neuronal cell cultures exposed to excitatory amino acids — such as glutamate, the major excitatory neurotransmitter in the central nervous system, or free radicals, such as nitric oxide (NO^.) and superoxide anoin (O2^{. –}), which react to form peroxynitrite (ONOO^–) — appears to depend on the intensity of the exposure and may involve two temporarily distinct phases. Following relatively fulminant insults, an initial phase of necrosis — associated with extreme energy depletion — may simply reflect the failure of neurons to carry out the default apoptotic death program used to efficiently dispose of aged or otherwise unwanted cells. Neurons recovering mitochondrial energy potential after an initial fulminant insult or following a more subtle inciting injury may subsequently undergo apoptosis, possibly associated with a factor released from mitochondria that triggers this death program. The maintenance of balanced energy production may be a decisive factor in detemining the degree, type, and progression of neuronal injury caused by excitotoxins and free radicals. Similar events could possibly occur in vivo after ischemia or other insults.     

Nitric oxide does not promote iron release from ferritin

It has been previously reported that iron release from ferritin could be promoted by nitric oxide (NO) generated from sodium nitroprusside. It was thus proposed that some of the toxic effects of NO could be related to its ability to increase intracellular free iron concentrations and generate an oxidative stress. On the contrary, the iron exchange experiments reported here show that NO from S-nitrosothiols is unable to promote iron release from ferritin. The discrepancy may be explained by the disregarded ability of ferrozine, the ferrous trap used in the previous report, to mobilize iron both from ferritin and from sodium nitroprusside spontaneously.     

Intramolecular electron transfer in the oxidation of amines by methylamine oxidase from Arthrobacter P1

 The intramolecular electron-transfer rate constant for the Cu(II)–topa_NH2⇌ Cu(I)–topa_SQ equilibrium in methylamine oxidase has been measured by temperature-jump relaxation techniques. At pH 7.0 the estimated k_obs = 150±30 s^–1 for both methylamine and benzylamine; assuming the equilibrium constant is ≈0.7–1 at pH 7.0 and 296 K, this would correspond to a forward electron-transfer rate constant k_ET≈ 60–75 s^–1. Although substantially slower than the previously determined k_ET≈ 20 000 s^–1 for pea seedling amine oxidase [5] steady-state kinetics measurements established that k_ET > k_cat≈ 4–10 s^–1. Thus the Cu(I)-semiquinone state is a viable intermediate in methylamine oxidase turnover. Received: 16 August 1995 / Accepted: 21 December 1995     

The Superoxide Dismutase-Mimic Copper-Putrescine-Pyridine Suppresses Lipid Peroxidation in CHO Cells. Implications for its Prooxidative and Antioxidative Mechanisms of Action

Copper-Putrescine-Pyridine (Cu-PuPy) is a membrane-permeable complex which efficiently dismutates superoxide. In excess of 0.1 mM it is highly cytotoxic and oxidizes cellular GSH with concomitant production of H₂O₂. Here we show that treatment of CHO cells with 0.2 mM Cu-PuPy (0-200 min) leads to an accumulation of H₂O₂. Organic hydroperoxides which are also formed at low levels in the presence of Cu-PuPy, increase significantly after removal of the copper complex. We conclude that Cu-PuPy acts as an oxidant until cellular GSH is depleted. However, by interfering with radical chain propagation reactions, it suppresses lipid peroxidation and thus substitutes for consumed physiological antioxidants in a later stage of treatment. This consistently explains our previous, seemingly paradox, finding that longer Cu-PuPy treatments may be significantly less toxic than shorter ones.     

Oxygen-Dependent Inhibition of Neutrophil Respiratory Burst by Nitric Oxide

Effect of nitric oxide (NO) on the respiratory burst of neutrophils was examined under different oxygen tensions. Phorbol myristate acetate (PMA) stimulated oxygen consumption and superoxide (O₂^-) generation in neutrophils by a mechanism which was inhibited reversibly by NO. The inhibitory effect of NO increased significantly with a decrease in oxygen tension in the medium. The inhibitory effect of NO was suppressed in medium containing oxyhemoglobin (HbO₂), a NO scavenging agent. In contrast, 3-morpholinosydnonimine (SIN-1), a compound that rapidly generates peroxynitrite (ONOO^-) from the released NO and O₂^-, slightly stimulated the PMA-induced respiratory burst. These results suggested that NO, but not ONOO, might reversibly inhibit superoxide generation by neutrophils especially at physiologically low oxygen tensions thereby decreasing oxygen toxicity particularly in and around hypoxic tissues.     

Iron and copper in plasma membranes of maize (Zea mays L.) roots investigated by proton induced X-ray emission

Summary Plasma membranes of maize (Zea mays L., cv. Sil Anjou 18) roots were isolated by aqueous two-phase partitioning. Multi elemental analysis by proton induced X-ray emission (PIXE) was used for the investigation of elemental composition of plasma membranes. Fe, Cu, and Zn as well as P, S, and Ca were identified. We did not find significant amounts of V, Mn, Se, Mo, or W.Abbreviations EDTA ethylenediaminetetraacetic acid - HCF III hexacyanoferrate III (ferricyanide, K₃[FeCN₆]) - Hepes 2-[4-(2-hydroxyethyl)-1-piperazine]-ethanesulfonic acid - PIXE proton induced X-ray emission (proton microprobe) - STA siliciotungstic acid - Tris tris (hydroxymethyl)aminomethane     

Dexamethasone Up-Regulates a Constitutive Nitric Oxide Synthase in Cerebellar Astrocytes but Not in Granule Cells in Culture

Abstract: Treatment of rat cerebellar astrocyte-enriched primary cultures with dexamethasone enhances the nitric oxide-dependent cyclic GMP formation induced by noradrenaline in a time-(>6 h) and concentration-dependent manner (half-maximal effect at 1 n M ). Stimulation of cyclic GMP formation by the calcium ionophore A23187 is similarly enhanced. In contrast, cyclic GMP accumulation in cells treated with lipopolysaccharide is inhibited by dexamethasone. The potentiating effect of dexamethasone is prevented by the protein synthesis inhibitor cycloheximide and is not due to increased soluble guanylate cyclase activity. Agonist stimulation of [³H]arginine to [³H]citrulline conversion is enhanced by dexamethasone in astrocytes but not in cerebellar granule cells. These results indicate that glucocorticoids may up-regulate astroglial calcium-dependent nitric oxide synthase while preventing expression of inducible nitric oxide synthase and are the first report of a differential long-term regulation of the expression of neuronal and astroglial constitutive nitric oxide synthase activities.     

Nitric Oxide-Induced [3H]GABA Release from Cerebral Cortical Neurons Is Mediated by Peroxynitrite

Abstract: The functional significance of peroxynitrite in the release of [³H]GABA induced by nitric oxide (NO) liberated from NO generators was investigated using cerebral cortical neurons in primary culture. NO generators such as sodium nitroprusside (SNP) and S -nitroso- N -acetylpenicillamine (SNAP) increased [³H]GABA release in a dose-dependent manner. These increases in [³H]GABA release were significantly inhibited by hemoglobin, indicating that those NO generators evoke the release of [³H]GABA by the formation of NO. Two types of superoxide scavengers, Cu²⁺/Zn²⁺ superoxide dismutase and ceruloplasmin, significantly reduced the increase in [³H]GABA release induced by both SNP and SNAP, which assumes that NO requires superoxide to induce [³H]GABA release from the neurons. In addition, synthesized peroxynitrite induced a dose-dependent increase in [³H]GABA release from the neurons. These results indicate that NO-induced [³H]GABA release is mediated by peroxynitrite formed by the reaction of NO with superoxide.     

Nitric oxide is involved in heat-induced HSP70 accumulation

Heat shock potentiated the nitric oxide production (EPR assay) in the liver, kidney, heart, spleen, intestine, and brain. The heat shock-induced sharp transient increase in the rate of nitric oxide production preceded the accumulation of heat shock proteins (HSP70) (Western blot analysis) as measured in the heart and liver. In all organs the nitric oxide formation was completely blocked by the NO-synthase inhibitor (L-NNA). L-NNA also markedly attenuated the heat shock-induced accumulation of HSP70. The results suggests that nitric oxide is involved in the heat shock-induced activation of HSP70 synthesis.     

Carbon and nitrogen cycling in intertidal sediments near Doel,Scheldt Estuary

Carbon and nitrogen cycling in intertidal mud flat sediments in the Scheldt Estuary was studied using measurements of carbon dioxide, methane and nitrous oxide emission rates and pore-water profiles of CO₂, ammonium and nitrate. A comparison between chamber measured carbon dioxide fluxes and those based on CO₂ pore-water gradients using Fick's First law indicates that apparent diffusion coefficients are 2 to 28 times higher than bulk sediment diffusion coefficients based on molecular diffusion. Seasonal changes in gaseous carbon fluxes or CO₂ pore water concentrations cannot be used directly, or in a simple way, to determine seasonal rates of mineralization, because of marked seasonal changes in pore-water storage and exchange parameters.The annual amount of carbon delivered to the sediment is 42 mol m^–2, of which about 42% becomes buried, the remaining being emitted as methane (7%) or carbon dioxide (50%). Each year about 2.6 mol N m^–2 of particulate nitrogen reaches the sediment; 1.1 mol m^–2 is buried and 1.6 mol m^–2 is mineralized to ammonium. Only 0.42 mol m^–2 yr^–1 of the ammonium produced escapes from the sediments, the remaining being first nitrified (1.2 mol m^–2 yr^–1) and then denitrified (1.7 mol m^–2 yr^–1). Simple calculations indicate that intertidal sediments may account for about 14% and 30% of the total estuarine retention of nitrogen and carbon, respectively.