We investigated the effects of different antioxidants such as L-ascorbic acid, catalase, and superoxide dismutase (SOD), on the p38-MAPK activation induced by oxidative stress in the isolated perfused amphibian heart. Oxidative stress was exemplified by perfusing hearts with 30 microM H(2)O(2) for 5 min or with the enzymatic system of xanthine/xanthine oxidase (200 microM/10 mU/ml, respectively) for 10 min. H(2)O(2)-induced activation of p38-MAPK (7.04 +/- 0.20-fold relative to control values) was totally attenuated by L-ascorbic acid (100 microM) or catalase (150 U/ml). These results were confirmed by immunohistochemical studies in which the phosphorylated form of p38-MAPK was localised in the perinuclear region and dispersedly in the cytoplasm of the ventricular cells during H(2)O(2) treatment, a pattern that was abolished by catalase or L-ascorbic acid. p38-MAPK was also activated (2.34 +/- 0.17-fold) by perfusing amphibian hearts with the reactive oxygen species (ROS)-generating system of xanthine/xanthine oxidase and this activation sustained in the presence of 150 U/ml catalase (2.16 +/- 0.26-fold), 50 U/ml SOD (2.02 +/- 0.07) or 100 microM L-ascorbic acid (2.18 +/- 0.10), but was suppressed by the combination of 150 U/ml catalase and 50 U/ml SOD. Finally, our studies showed that xanthine/xanthine oxidase induced the phosphorylation of the potent p38-MAPK substrates MAPKAPK2 (3.14 +/- 0.27-fold) and HSP27 (5.32 +/- 0.83-fold), which are implicated in cell protection, and this activation was reduced by the simultaneous use of catalase and SOD.
Amphibians Animals