Okadaic acid and anisomycin are protective and stimulate the SAP/JNK pathway

We report that okadaic acid (OA), a known inhibitor of Ser/Thr phosphatases , protects pig myocardium against ischemic injury in an in vivo model and s timulates the activities of stress-activated protein kinases/c-Jun N-termin al kinases (SAPKs/JNKs). When OA was directly infused into the subsequently ischemic myocardium for 60 min before a 60-min period of coronary occlusio n followed by reperfusion, infarct size was reduced from a control value of 83.4 +/- 2.8% of the risk region to 40.7 +/- 9.1%. When OA was infused for 10 min before a 5-min occlusion and during 45 min thereafter, infarct size was reduced to 26.5%. In a separate set of similar experiments, we pretrea ted pig hearts in vivo with the protein-synthesis inhibitor and known activ ator of SAPK/JNK, anisomycin (AN), and found that this compound also signif icantly reduced infarct size from 83.4 +/- 2.8.1% to 48.1 +/- 5.1%. For in vitro assays, OA (600 nM), AN (500 mu M), or solvent (KHB) were locally inf used into the left ventricular myocardium, and biopsies from in situ beatin g hearts were obtained after 10, 30, and 60 min of infusion. The activities of Ser/Thr phosphatases (PPases), especially PP-2A, were significantly dec reased after OA infusion. OA infusion increased the activity tin-gel phosph orylation of N-terminal c-Jun(1-135)) of both 46- and 55-kDa SAPK/JNKs (two fold to threefold, 30 and 60 min of infusion), and this increase correlated well with the observed decrease of PPase activities. Western blot analysis with a phosphospecific SAPK/JNK (Thr 183/Tyr 185) antibody showed an incre ased content of the phosphorylated forms after OA treatment. We observed si gnificant stimulation of SAPK/JNK activity also after AN treatment (threefo ld to fourfold, after 30 min of infusion). In contrast to the SAPK/JNKs, th e infusion of both OA and AN did not significantly change the activities an d phosphorylation of extracellular signal-related kinases (ERKs) and p38-MA PK. The findings that the protective effect of both OA and AN correlates wi th increased activity of SAPK/JNKs suggest the involvement of these enzymes in the mechanism of cardioprotection.