Oxidative stress stimulates multiple MAPK signalling pathways and phosphorylation of the small HSP27 in the perfused amphibian heart

We investigated the activation of three subfamilies of MAPKs (ERK, JNKs and p38-MAPK) by oxidative stress in the isolated perfused amphibian heart. Activation of p43-ERK by 100 mumol l(-1) H2O2 was maximally observed within 5 min, remained elevated for 30 min and was comparable with the effect of 1 mumol l(-1) PMA. p43-ERK activation by H2O2 was inhibited by PD98059 but not by SB203580. The p46 and p52 species of JNKs were maximally activated by 2.5- and 2.1-fold, respectively, by 100 mumol l(-1) H2O2 within 2 min. JNK activation was still detectable after 15 min, reaching control values at 30 min of treatment. p38-MAPK was maximally activated by 9.75-fold by 100 mumol l(-1) H2O2 after 2 min and this activation progressively declined thereafter, reaching control values within 45 min of treatment. The observed dose-dependent profile of p38-MAPK activation by H2O2 revealed that 30 mumol l(-1) H2O2 induced maximal phosphorylation, whereas 100-300 mumol l(-1) H2O2 induced considerable activation of the kinase. Our studies also Summary showed that the phosphorylation of MAPKAPK2 by H2O2 followed a parallel time-dependent pattern and that SB203580 abolished this phosphorylation. Furthermore, our experiments clearly showed that 30 mumol l(-1) H2O2 induced a strong, specific phosphorylation of HSP27. Our inummohistochemical studies showed that immune complexes of phosphorylated forms of both p38-MAPK and HSP27 were strongly enhanced by 30 mumol l(-1) H2O2 in the perinuclear region as well as dispersedly in the cytoplasm of ventricular cells and that SB203580 abolished this phosphorylation. These data indicate that oxidative stress is a powerful activator of all three MAPK subfamilies in the amphibian heart. Stimulation of p38-MAPK and the consequent phosphorylation of HSP27 may be important in cardioprotection under such conditions.