Peroxynitrite affects exocytosis and SNARE complex formation and induces tyrosine nitration of synaptic proteins.
The reactive species peroxynitrite, formed via the near diffusion-limited reaction of nitric oxide and superoxide anion, is a potent oxidant that contributes to tissue damage in neurodegenerative disorders. Peroxynitrite readily nitrates tyrosine residues in proteins, producing a permanent modification that can be immunologically detected. We have previously demonstrated that in the nerve terminal, nitrotyrosine immunoreactivity is primarily associated with synaptophysin. Here we identify two other presynaptic proteins nitrated by peroxynitrite, Munc-18 and SNAP25, both of which are involved in sequential steps leading to vesicle exocytosis. To investigate whether peroxynitrite affects vesicle exocytosis, we used the fluorescent dye FM1-43 to label a recycling population of secretory vesicles within the synaptosomes. Bolus addition of peroxynitrite stimulated exocytosis and glutamate release. Notably, these effects were strongly reduced in the presence of NaHCO(3), indicating that peroxynitrite acts mainly intracellularly. Furthermore, peroxynitrite enhanced the formation of the sodium dodecyl sulfate-resistant SNARE complex in a dose-dependent manner (100-1000 microm) and induced the formation of 3-nitrotyrosine in proteins of SNARE complex. These data suggest that modification(s) of synaptic vesicle proteins induced by peroxynitrite may affect protein-protein interactions in the docking/fusion steps, thus promoting exocytosis, and that, under excessive production of superoxide and nitric oxide, neurons may up-regulate neuronal signaling.
Di Stasi
A M Michela
AM
Laboratorio di Biologia Cellulare, Istituto Superiore di Sanità, Rome, Italy. mdistasi@iss.it
Mallozzi
Cinzia
C
Macchia
Gianfranco
G
Maura
Guido
G
Petrucci
Tamara C
TC
Minetti
Maurizio
M
D000818:Animals; D001923:Brain Chemistry; D004305:Dose-Response Relationship, Drug; D005089:Exocytosis / Q000187:drug effects* / Q000502:physiology; D005456:Fluorescent Dyes; D018698:Glutamic Acid / Q000737:chemistry / Q000378:metabolism; D046911:Macromolecular Substances; D008297:Male; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D030421:Peroxynitrous Acid / Q000494:pharmacology*; D011506:Proteins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D050600:SNARE Proteins; D017693:Sodium Bicarbonate / Q000494:pharmacology; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry / Q000494:pharmacology; D050825:Synaptosomal-Associated Protein 25; D013574:Synaptosomes / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D033921:Vesicular Transport Proteins
D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D008565:Membrane Proteins; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins; D011506:Proteins; D050600:SNARE Proteins; C494210:Snap25 protein, rat; D050825:Synaptosomal-Associated Protein 25; D033921:Vesicular Transport Proteins; D030421:Peroxynitrous Acid; C002744:3-nitrotyrosine; D012967:Sodium Dodecyl Sulfate; D018698:Glutamic Acid; D014443:Tyrosine; D017693:Sodium Bicarbonate