Prevention of cycloheximide-induced apoptosis in hepatocytes by adenosine and by caspase inhibitors

The mechanism by which cycloheximide induces apoptosis in isolated rat hepa tocytes was studied. Cycloheximide (1-300 mu M) induced apoptosis within 3- 4 hr in the hepatocytes. Specific apoptotic characteristics such as blebbin g, phosphatidyl serine (PS) exposure, chromatin condensation, and nuclear f ragmentation were induced. Cycloheximide (CHX) dose dependently activated t he caspase-3-like proteases, but not the caspase-1-like proteases. Pretreat ment of the hepatocytes with 100 mu M of the caspase inhibitors z-Val-Ala-D L-Asp-fluoromethylketone or Ac-Asp-Glu-Val-Asp-aldehyde completely abrogate d the caspase activation and the apoptosis. Addition of adenosine (100 mu M ) reduced phosphatidyl serine exposure and other morphological characterist ics of apoptosis by 50%; however, it did not prevent the activation of the caspases, suggesting that adenosine inhibited downstream of caspase activat ion. The adenosine receptor antagonist 8-[4-[[[[(2-aminoethyl)amino]-carbon yl]methyl]oxy]phenyl]-1,3-dipropylxanthine abolished the capacity of adenos ine to prevent apoptosis, indicating that prevention was receptor-mediated. During apoptosis, the mitochondrial membrane potential in apoptotic cells (cells with PS exposition) was decreased to 50-60% of the control value; in the population viable cells, however, the mitochondrial membrane potential remained stable. Prevention of apoptosis by the caspase inhibitor z-Val-Al a-DL-Asp-fluoromethylketone or adenosine prevented the decrease in mitochon drial membrane potential. In conclusion, CHX rapidly induces apoptosis in i solated rat hepatocytes, which is inhibited by adenosine at a relatively la te step. (C) 1999 Elsevier Science Inc.