ONOO−-induced tubulin oxidation/nitration and its prevention by antioxidants in Caco-2 monolayers
| Treatment | Fraction of Tubulin Nitrated | Fraction of Tubulin Oxidized |
|---|---|---|
| Vehicle | 0 | 0 |
| 0.1 mM ONOO− | 0.73 ± 0.033-a | 0.70 ± 0.043-a |
| 1 mM ONOO− | 0.96 ± 0.053-a | 0.94 ± 0.063-a |
| 3 mM SIN-1 | 0.67 ± 0.013-a | 0.69 ± 0.033-a |
| 5 mM SIN-1 | 0.85 ± 0.093-a | 0.83 ± 0.023-a |
| 3 mM SANP/X/XO | 0.71 ± 0.073-a | 0.67 ± 0.013-a |
| 5 mM SANP/X/XO | 0.82 ± 0.023-a | 0.84 ± 0.033-a |
| Urate + 1 mM ONOO− | 0.06 ± 0.043-b | 0.09 ± 0.013-b |
| Urate + 5 mM SIN-1 | 0.02 ± 0.013-b | 0.07 ± 0.053-b |
| Urate + 5 mM SANP/X/XO | 0.09 ± 0.033-b | 0.11 ± 0.063-b |
| l-Cysteine + 1 mM ONOO− | 0.05 ± 0.043-b | 0.04 ± 0.023-b |
| l-Cysteine + 5 mM SIN-1 | 0.03 ± 0.013-b | 0.02 ± 0.013-b |
| l-Cysteine + 5 mM SANP/X/XO | 0.11 ± 0.073-b | 0.13 ± 0.083-b |
| d-Cysteine alone | 0 | 0 |
| d-Cysteine + 1 mM ONOO− | 0.94 ± 0.033-a | 0.97 ± 0.023-a |
| d-Cysteine + 5 mM SIN-1 | 0.88 ± 0.083-a | 0.84 ± 0.123-a |
| d-Cysteine + 5 mM SANP/X/XO | 0.84 ± 0.103-a | 0.83 ± 0.073-a |
| SOD + 5 mM SIN-1 | 0.07 ± 0.023-b | 0.10 ± 0.043-b |
| SOD + 5 mM SANP/X/XO | 0.09 ± 0.053-b | 0.12 ± 0.063-b |
| iSOD alone | 0 | 0 |
| iSOD + 5 mM SIN-1 | 0.83 ± 0.093-a | 0.86 ± 0.033-a |
| iSOD + 5 mM SANP/X/XO | 0.79 ± 0.023-a | 0.82 ± 0.083-a |
Values are mean ± S.E. Monolayers were exposed to ONOO−or donors or were preincubated (30 min) with ONOO− scavengers 1) l-cysteine (3 mM) and as control d-cysteine, 2) urate (0.5 mM), or 3) SOD (300 U/ml) and as control the heat-inactivated iSOD. Controls were incubated in isotonic saline/vehicle. Cells were then processed for immunoblots to assess tubulin oxidation (carbonylation) and nitration (nitrotyrosination). Oxidation or nitration was expressed as the fraction (ratio) of carbonyl or nitrotyrosine immunoreactivity (absorbance) in the treatment group divided by oxidized or nitrated tubulin standards.