Protein kinase activation by warm and cold hypoxia-reoxygenation in primary-cultured rat hepatocytes-JNK(1)/SAPK(1) involvement in apoptosis

Ischemia-reperfusion procedures induced severe hepatic damages owing to dif ferent processes related to hypoxia and reoxygenation (H/R) phases, includi ng the consecutive oxygen free radical (OFR) release. Stress-activated prot ein kinases (SAPKs) could be activated by extracellular stimuli. The aim of this study was to show whether H/R stress conditions could stimulate these kinases, and especially c-jun-N-terminal kinase UNK1/SAPK(1)), to reveal a potential role of JNK(1)/SAPK(1) in the control of hepatocyte apoptosis, P rimary cultured rat hepatocytes, isolated from other liver cells and blood flow, were subjected to warm and cold hypoxia-reoxygenation phases mimickin g surgical and transplant conditions. The activation status of SAPKs was ev aluated by immunoprecipitation or Western-blotting experiments, whereas apo ptosis was assessed by measuring caspase activation and internucleosomal DN A fragmentation in vitro and by TUNEL reaction, in vivo. Hypoxia, and espec ially hypoxia-reoxygenation, significantly increased JNK(1)/SAPK(1) activat ion in cultured hepatocytes. Either in warm or cold conditions, OFR scaveng ers (N-Acetylcystein, Di-Phenyleneiodonium, Deferoxamine) decreased this st imulation. Warm ischemia-reperfusion also led to JNK activation. Hypoxia an d especially hypoxia-reoxygenation induced programmed cell death in vivo an d in vitro, This last phenomenon was inhibited when hepatocytes were treate d with SE 202190, which was described as a potent inhibitor of p38 and JNK activities. Altogether, these results confirmed that JNK(1)/SAPK(1) was act ivated during the hypoxia-reoxygenation process, and that this activity par ticipated in the onset of the apoptosis program.