Prevention of cycloheximide-induced apoptosis in hepatocytes by adenosine and by caspase inhibitors.
The mechanism by which cycloheximide induces apoptosis in isolated rat hepatocytes was studied. Cycloheximide (1-300 microM) induced apoptosis within 3-4 hr in the hepatocytes. Specific apoptotic characteristics such as blebbing, phosphatidyl serine (PS) exposure, chromatin condensation, and nuclear fragmentation were induced. Cycloheximide (CHX) dose dependently activated the caspase-3-like proteases, but not the caspase-1-like proteases. Pretreatment of the hepatocytes with 100 microM of the caspase inhibitors z-Val-Ala-DL-Asp-fluoromethylketone or Ac-Asp-Glu-Val-Asp-aldehyde completely abrogated the caspase activation and the apoptosis. Addition of adenosine (100 microM) reduced phosphatidyl serine exposure and other morphological characteristics of apoptosis by 50%; however, it did not prevent the activation of the caspases, suggesting that adenosine inhibited downstream of caspase activation. The adenosine receptor antagonist 8-[4-[[[[(2-aminoethyl)amino]-carbonyl]methyl]oxy]phenyl]-1,3-dipropylxa nthine abolished the capacity of adenosine 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-Ala-DL-Asp-fluoromethylketone or adenosine prevented the decrease in mitochondrial membrane potential. In conclusion, CHX rapidly induces apoptosis in isolated rat hepatocytes, which is inhibited by adenosine at a relatively late step.
Blom
W M
WM
Division of Toxicology, Leiden Amsterdam Centre for Drug Research, Leiden University, The Netherlands.
de Bont
H J
HJ
Meijerman
I
I
Mulder
G J
GJ
Nagelkerke
J F
JF
D000241:Adenosine / Q000494:pharmacology*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D061945:Caspase Inhibitors; D003513:Cycloheximide / Q000037:antagonists & inhibitors* / Q000494:pharmacology; D004347:Drug Interactions; D004791:Enzyme Inhibitors / Q000494:pharmacology; D066298:In Vitro Techniques; D008099:Liver / Q000166:cytology / Q000187:drug effects*; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects; D008930:Mitochondria, Liver / Q000187:drug effects / Q000502:physiology; D020011:Protective Agents / Q000494:pharmacology; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar
D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D020011:Protective Agents; D011500:Protein Synthesis Inhibitors; D003513:Cycloheximide; D000241:Adenosine