Peroxynitrite generated in the rat spinal cord induces oxidation and nitration of proteins: reduction by Mn (III) tetrakis (4-benzoic acid) porphyrin.
To determine whether peroxynitrite at the concentration and duration present after spinal cord injury induces protein oxidation and nitration in vivo, the peroxynitrite donor 3-morpholinosydnonimine (SIN-1) was administered into the gray matter of the rat spinal cord for 5 hr. The cords were removed at 6, 12, 24, and 48 hr after SIN-1 exposure, immunohistochemically stained with antibodies to dinitrophenyl (DNP) and nitrotyrosine (Ntyr), markers of protein oxidation and nitration, respectively, and the immunostained neurons were counted. The percentages of DNP-positive (P = 0.023-0.002) and Ntyr-positive (P < 0.001 for all) neurons were significantly higher in the SIN-1-exposed groups than in the ACSF controls at each time, suggesting that peroxynitrite induced intracellular oxidation and nitration of proteins. The percentages of DNP- and Ntyr-positive neurons were not significantly different over time in either SIN-1- or ACSF-exposed groups (P = 0.20-1.00). The percentage of DNP-positive neurons was 7.6 +/- 3% to 12 +/- 4.2% at 6-24 hr, and it was 14 +/- 2% to 19 +/- 2% at 6-24 hr for Ntyr-positive neurons after SIN-1-exposure, whereas both ranged over 2-3% in ACSF controls. Mn (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP, a broad-spectrum scavenger of reactive species) significantly reduced the percentages of DNP- and Ntyr-positive neurons (P = 0.04 and 0.002, respectively) compared to a SIN-1-exposed, untreated group at 24 hr after SIN-1 exposure. There were no significant differences between MnTBAP-treated and ACSF controls (P = 0.7 for DNP and 0.2 for Ntyr). These results further demonstrate peroxynitrite-induced protein oxidation and nitration and the efficiency of MnTBAP in scavenging peroxynitrite.
Bao
Feng
F
Department of Neurology, University of Texas Medical Branch, Galveston, Texas 77555-0653, USA.
DeWitt
Douglas S
DS
Prough
Donald S
DS
Liu
Danxia
D
D019297:2,4-Dinitrophenol / Q000378:metabolism; D000818:Animals; D004347:Drug Interactions; D004791:Enzyme Inhibitors / Q000633:toxicity; D016166:Free Radical Scavengers / Q000494:pharmacology; D007150:Immunohistochemistry; D008297:Male; D008665:Metalloporphyrins / Q000494:pharmacology*; D017551:Microdialysis / Q000379:methods; D008981:Molsidomine / Q000031:analogs & derivatives* / Q000633:toxicity; D009474:Neurons / Q000187:drug effects / Q000378:metabolism; D010084:Oxidation-Reduction; D030421:Peroxynitrous Acid / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013116:Spinal Cord / Q000166:cytology / Q000378:metabolism / Q000473:pathology; D013119:Spinal Cord Injuries / Q000139:chemically induced / Q000378:metabolism*; D013997:Time Factors; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
D004791:Enzyme Inhibitors; D016166:Free Radical Scavengers; D008665:Metalloporphyrins; C097284:manganese(III)-tetrakis(4-benzoic acid)porphyrin; D030421:Peroxynitrous Acid; C002744:3-nitrotyrosine; D014443:Tyrosine; C002385:linsidomine; D008981:Molsidomine; D019297:2,4-Dinitrophenol