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 different processes related to hypoxia and reoxygenation (H/R) phases, including the consecutive oxygen free radical (OFR) release. Stress-activated protein 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 (JNK(1)/SAPK(1)), to reveal a potential role of JNK(1)/SAPK(1) in the control of hepatocyte apoptosis. Primary cultured rat hepatocytes, isolated from other liver cells and blood flow, were subjected to warm and cold hypoxia-reoxygenation phases mimicking surgical and transplant conditions. The activation status of SAPKs was evaluated by immunoprecipitation or Western-blotting experiments, whereas apoptosis was assessed by measuring caspase activation and internucleosomal DNA fragmentation in vitro and by TUNEL reaction, in vivo. Hypoxia, and especially hypoxia-reoxygenation, significantly increased JNK(1)/SAPK(1) activation in cultured hepatocytes. Either in warm or cold conditions, OFR scavengers (N-Acetylcystein, Di-Phenyleneiodonium, Deferoxamine) decreased this stimulation. Warm ischemia-reperfusion also led to JNK activation. Hypoxia and especially hypoxia-reoxygenation induced programmed cell death in vivo and in vitro. This last phenomenon was inhibited when hepatocytes were treated with SB 202190, which was described as a potent inhibitor of p38 and JNK activities. Altogether, these results confirmed that JNK(1)/SAPK(1) was activated during the hypoxia-reoxygenation process, and that this activity participated in the onset of the apoptosis program.
Crenesse
D
D
Laboratoires de Physiologie, Université de Nice-Sophia Antipolis, 06107 Nice cedex 2, France.
Gugenheim
J
J
Hornoy
J
J
Tornieri
K
K
Laurens
M
M
Cambien
B
B
Lenegrate
G
G
Cursio
R
R
De Souza
G
G
Auberger
P
P
Heurteaux
C
C
Rossi
B
B
Schmid-Alliana
A
A
D000818:Animals; D017209:Apoptosis / Q000502:physiology; D002478:Cells, Cultured; D003080:Cold Temperature; D004789:Enzyme Activation; D016166:Free Radical Scavengers / Q000494:pharmacology; D022781:Hepatocytes / Q000201:enzymology* / Q000502:physiology; D006358:Hot Temperature; D000860:Hypoxia / Q000201:enzymology* / Q000503:physiopathology; D048031:JNK Mitogen-Activated Protein Kinases; D008297:Male; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology; D010100:Oxygen / Q000494:pharmacology*; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism
D016166:Free Radical Scavengers; D017382:Reactive Oxygen Species; D011494:Protein Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D010100:Oxygen