Peroxynitrite generated in the rat spinal cord induces oxidation and nitration of proteins: Reduction by Mn (III) tetrakis (4‐benzoic acid) porphyrin

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Abstract
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.