Rapid activation of neutral sphingomyelinase by hypoxia-reoxygenation of cardiac myocytes

Elevated levels of oxygen free radicals have been implicated in the pathway s of reperfusion injury to myocardial tissue. The targets for free radicals may include specific as well as random intracellular components, and part of the cellular response is the induction of extracellularly activated and stress-activated kinases. The intermediate signals that initiate these stre ss responses are not known. Here we show that one of the earliest responses of cardiac myocytes to hypoxia and reoxygenation is the activation of neut ral sphingomyelinase and accumulation of ceramide. Ceramide increased abrup tly after reoxygenation, peaking at 10 minutes with 225+/-40% of the contro l level. Neutral sphingomyelinase activity was induced with similar kinetic s, and both activities remained elevated for several hours. c-Jun N-termina l kinase (JNK) was also activated within the same time frame. Treatment of cardiac myocytes with extracellular ceramides also activated JNK. Pretreati ng cells with antioxidants quenched sphingomyelinase activation, ceramide a ccumulation, and JNK activation. Ceramide did not accumulate in reoxygenate d nonmuscle fibroblasts, and JNK was not activated by reoxygenation in thes e cells. The results identify neutral sphingomyelinase activation as one of the earliest responses of cardiac myocytes to the redox stress imposed by hypoxia-reoxygenation. The results are consistent with a pathway of ceramid e-mediated activation of JNK.