nitroarginine and 5-5-dimethyl-1-pyrroline-1-oxide

The effect of recombinant gp120 HIV envelope glycoprotein on the generation of free radicals by monocyte-derived macrophages (MDM) was measured by EPR spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). After 1 day in culture, MDM produced a spin trap adduct of DMPO with hyperfine splitting constants superimposable on those of DMPO-OH. The addition of gp120 to MDM increased the production of DMPO-OH and after 1 h, the amount of DMPO-OH produced by 40 micrograms/ml gp120 was about 300% that of untreated MDM. The use of selective inhibitors suggested the participation of the nitric oxide/L-arginine oxidative pathway, but did not provide evidence for trapping of hydroxyl radical or other oxygen free radicals. The specificity of gp120 was proven by two different anti-gp120 antibodies that either inhibited (polyclonal) or increased (monoclonal) the production of free radicals. Dexamethasone inhibited the effect of gp120, suggesting the possible involvement of an inducible nitric oxide (NO) synthase. Moreover, treatment of MDM with gp120 for 15 h increased in a dose-dependent manner the production of NO2-, a stable end product of NO. Soluble CD4 did not modify the intensity of the DMPO-OH adduct, whereas yeast mannan and Ca(2+)-chelators abolished the increase in the DMPO-OH signal induced by gp120. These data suggest the possible involvement of mannose-specific endocytotic lectin of MDM. The reaction of DMPO with sodium nitroprusside, an organic nitrate that releases NO, also produced DMPO-OH. Our findings indicate that gp120 increases free radical production from MDM as detected by spin-trapping methods, and that the spin trap adduct results from a reaction involving NO or closely related oxidized derivatives.

Topics: Adult; Arginine; Catalase; Cells, Cultured; Cyclic N-Oxides; Dexamethasone; Dose-Response Relationship, Drug; Electron Spin Resonance Spectroscopy; Female; Free Radical Scavengers; HIV Envelope Protein gp120; Humans; Kinetics; Macrophages; Male; Mannans; Monocytes; Nitric Oxide; Nitroarginine; Recombinant Proteins; Superoxide Dismutase; Time Factors

In this study, we analysed the implication of superoxide (O2-.) and nitric oxide (NO.) free radicals and their resulting product peroxynitrite (ONOO-) in the neuronal death induced by the activation of the glutamatergic receptor of the N-methyl-D-aspartate (NMDA) subtype using cultured cerebellar granule cells. The NOl donor SIN-1 (3-morpholinosydnonimine N-ethylcarbamide), at concentrations which produced a much higher guanylate cyclase activation (i.e. NO. concentration) than NMDA, was not neurotoxic and did not increase the NMDA-induced neuronal death. The absence of involvement of NO. in NMDA-induced neuronal death was confirmed by the ineffectiveness of L-NG-nitroarginine (L-Narg) as a neuroprotective compound. Electron paramagnetic resonance (EPR) experiments, using 5,5-dimethyl pyrroline 1-oxide (DMPO) as a spin trap, indicated that NMDA receptor stimulation led to the generation of O2-. from at least 15-30 min. The generation of O2-. by xanthine (XA)-xanthine oxidase (XO) induced a neuronal death similar to that of NMDA. XA-XO-induced neuronal death was suppressed by addition of either superoxide dismutase (SOD) plus catalase (CAT), or DMPO in the incubation medium. In contrast, NMDA-induced neuronal death was widely blocked by DMPO and other spin trap compounds, but not by SOD +/- CAT. XA-XO-induced neuronal death was not potentiated by SIN-1 indicating that ONOO- is not more toxic than O2-. in our neuronal model.

Topics: Amino Acid Oxidoreductases; Animals; Arginine; Catalase; Cell Death; Cells, Cultured; Cerebellum; Cyclic GMP; Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Mice; Molsidomine; N-Methylaspartate; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Receptors, N-Methyl-D-Aspartate; Superoxide Dismutase; Superoxides; Xanthine Oxidase

Neuronal injury resulting from acute brain insults and some neurodegenerative diseases implicates N-methyl-D-aspartate (NMDA) glutamate receptors. The fact that antioxidants reduce some types of brain damage suggests that oxygen radicals may have a role. It has been shown that mutations in Cu/Zn-superoxide dismutase (SOD), an enzyme catalysing superoxide (O2.-) detoxification in the cell, are linked to a familial form of amyotrophic lateral sclerosis (ALS). Here we report that O2.- is produced upon NMDA receptor stimulation in cultured cerebellar granule cells. Electron paramagnetic resonance was used to assess O2.- production that was due in part to the release of arachidonic acid. Activation of kainic acid receptors, or voltage-sensitive Ca2+ channels, did not produce detectable O2.-. We also find that the nitrone DMPO (5,5-dimethyl pyrroline 1-oxide), used as a spin trap, is more efficient than the nitric oxide synthase inhibitor, L-NG-nitro-arginine, in reducing NMDA-induced neuronal death in these cultures.

Topics: Amino Acid Oxidoreductases; Animals; Arachidonic Acid; Arginine; Calcium Channels; Cell Death; Cells, Cultured; Cerebral Cortex; Computer Simulation; Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Mice; Nitric Oxide Synthase; Nitroarginine; Quinacrine; Receptors, Glutamate; Receptors, Kainic Acid; Receptors, N-Methyl-D-Aspartate; Superoxide Dismutase; Superoxides; Xanthine; Xanthine Oxidase; Xanthines