2-2--(hydroxynitrosohydrazono)bis-ethanamine and Nerve-Degeneration

Axonal degeneration can be an important cause of permanent disability in neurological disorders in which inflammation is prominent, including multiple sclerosis and Guillain-Barré syndrome. The mechanisms responsible for the degeneration remain unclear, but it is likely that axons succumb to factors produced at the site of inflammation, such as nitric oxide (NO). We previously have shown that axons exposed to NO in vivo can undergo degeneration, especially if the axons are electrically active during NO exposure. The axons may degenerate because NO can inhibit mitochondrial respiration, leading to intraaxonal accumulation of Na(+) and Ca(2+) ions. Here, we show that axons can be protected from NO-mediated damage using low concentrations of Na(+) channel blockers, or an inhibitor of Na(+)/Ca(2+) exchange. Our findings suggest a new strategy for axonal protection in an inflammatory environment, which may be effective in preventing the accumulation of permanent disability in patients with neuroinflammatory disorders.

Topics: Anesthetics, Local; Animals; Axons; Bepridil; Calcium; Calcium Channel Blockers; Electrophysiology; Flecainide; Lidocaine; Male; Nerve Degeneration; Nitric Oxide; Nitric Oxide Donors; Nitroso Compounds; Rats; Rats, Sprague-Dawley; Sodium; Sodium Channel Blockers; Sodium-Calcium Exchanger; Spinal Nerve Roots

Exposure of SH-SY5Y neuroblastoma or rat cortical neurons to diethylenetriamine-NO (DETA-NO) rapidly depolarized mitochondria. In SH-SY5Y DETA-NO activated caspase 3 and produced cell death. Mitochondrial depolarization in SH-SY5Y was visualized both with JC-1 accumulation and as dequenching of calcein fluorescence in mitochondria initially loaded with calcein-AM and tetramethylrhodamine methyl ester (TMRM). Calcein/TMRM-visualized mitochondrial depolarization was prevented by cyclosporin A (CsA) or approximately two-fold increased levels of BclXL protein. Dynamic imaging of mitochondrial potential (Deltapsi M) with TMRM showed that DETA-NO induced cycles of mitochondrial depolarization/repolarization ("flickering"). Fifteen-30 min of DETA-NO exposure caused high-frequency flickering with small peak size; 2 h of DETA-NO produced large peaks with prolonged depolarization. NO-induced flickering but not that from Bax was blocked by the calcium uniporter antagonist Ru360. Our findings show rapid-onset, dynamic regulation of Deltapsi M by NO, implying that neuroprotective therapies for brain ischemia target cell death processes downstream of effects of NO on mitochondria.

Topics: Animals; bcl-X Protein; Brain Ischemia; Calcium Channels; Calcium-Binding Proteins; Caspase 3; Caspases; Cell Death; Cell Membrane Permeability; Cyclosporine; Dose-Response Relationship, Drug; Fluorescent Dyes; Humans; Intracellular Membranes; Ion Channels; Membrane Potentials; Mitochondria; Nerve Degeneration; Neurons; Nitric Oxide; Proto-Oncogene Proteins c-bcl-2; Rats; Rhodamines; Ruthenium Compounds; Triazenes; Tumor Cells, Cultured

We have studied the modulation of cyclic AMP (cAMP) accumulation by the human immunodeficiency virus type 1 (HIV 1) protein Tat in microglia and astrocyte cultures obtained from neonatal rat brain. Pretreatment of microglia with recombinant Tat resulted in a dose- and time-dependent decrease of cAMP accumulation induced by subsequent exposure to isoproterenol (1 microM). The inhibitory action of 100 ng/mL Tat approached 50% after 4 h of preincubation and reached a maximum of 70% after 24 h. The Tat-induced time- and dose-dependent decrease of cAMP accumulation was observed also when microglial cultures were stimulated with the adenylyl cyclase activator forskolin (100 microM). In both cases, Tat inhibitory action was 70% reverted by a specific monoclonal anti-Tat antibody, but was not prevented either by the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xantine (100 microM) or by a 16-h pretreatment of microglial cultures with the Gi protein inhibitor pertussis toxin (10 ng/mL). All these results suggested that the viral protein acts at a step of the cAMP transduction pathway other than receptors, G proteins and phosphodiesterases. The target of Tat appeared to be adenylyl cyclase, whose activity was markedly reduced (up to 60%) in membranes prepared from Tat-treated microglial cells, both in basal conditions and after stimulation with isoproterenol and forskolin. The inability of the competitive inhibitor of nitric oxide synthase N(G)-monometyl- L-arginine (20 and 200 microM) to revert Tat action on forskolin-induced cAMP accumulation, and of two potent nitric oxide donors, PAPA and DETA (0.1-2 m M), to alter forskolin-induced cAMP accumulation, excluded an involvement of nitric oxide in Tat-induced adenylyl cyclase inhibition. On the contrary, two inhibitors of nuclear factor kappaB activation, N-tosyl-( L)-phenylalanine chloromethyl ketone (10 microM) and SN50 (25 microM), markedly prevented the reduction of forskolin-evoked cAMP accumulation by Tat, suggesting a possible role for this nuclear transcriptional factor in the regulation of adenylyl cyclase by Tat in microglia. This assumption was strengthened by the ability of lipopolysaccharide (100 ng/mL, 4 h) to mimic the inhibitory effect of the viral protein. Conversely, astrocyte cAMP accumulation was unaffected by the viral protein, as tested at various concentrations and time points. Finally, Tat inhibition of microglial adenylyl cyclase was not due to non-specific cytotoxicity. As cAMP has been

Topics: 1-Methyl-3-isobutylxanthine; Adenylate Cyclase Toxin; Adenylyl Cyclases; Animals; Antibodies, Monoclonal; Apoptosis; Arginine; Astrocytes; Cell Membrane; Cells, Cultured; Colforsin; Cyclic AMP; Energy Metabolism; Enzyme Activation; Gene Products, tat; GTP-Binding Protein alpha Subunits, Gi-Go; HIV-1; Hydrazines; Isoproterenol; Lipopolysaccharides; Microglia; Nerve Degeneration; NF-kappa B; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitroso Compounds; Peptides; Pertussis Toxin; Rats; Rats, Wistar; Recombinant Fusion Proteins; Second Messenger Systems; tat Gene Products, Human Immunodeficiency Virus; Tosylphenylalanyl Chloromethyl Ketone; Virulence Factors, Bordetella