Cell-permeable cAMP analog suppresses 6-hydroxydopamine-induced apoptosis in PC12 cells through the activation of the Akt pathway.
Although cAMP protects neuronal cells from various apoptotic stimulations, its mechanism is not fully elucidated. We report here the molecular mechanism of the 6-hydroxydopamine (6-OHDA)-induced apoptosis of pheochromocytoma PC12 cells and its suppression by 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (pCPT-cAMP), which is a membrane permeable cAMP analog. Treatment of PC12 cells with 6-OHDA resulted in the activation of caspases and apoptosis, as detected by chromatin condensation. 6-OHDA also induced superoxide generation, Bid cleavage and mitochondrial membrane depolarization. In addition, Akt phosphorylation that was favorable to cell survival was decreased and p38 MAPK phosphorylation was increased by 6-OHDA. PC12 cell apoptosis was inhibited by pCPT-cAMP, Z-VAD-fmk (a broad-range caspase inhibitor) and tiron (a superoxide scavenger), although PC12 cell apoptosis was not inhibited by cyclosporine A (an inhibitor of mitochondrial membrane permeability transition). Moreover, pCPT-cAMP promoted Akt phosphorylation, but it did not prevent superoxide generation and mitochondrial membrane depolarization. Conversely, LY294002, an inhibitor of Akt upstream molecule PI3-kinase, enhanced 6-OHDA-induced apoptosis. These results indicated that the 6-OHDA-induced apoptosis of PC12 cells was initiated by superoxide generation followed by caspase cascade activation, which was associated with the suppressed Akt phosphorylation and increased p38 phosphorylation. It is likely that pCPT-cAMP prevented the 6-OHDA-induced apoptosis via activation of the PI3-kinase/Akt pathway without any effect on superoxide generation or mitochondrial membrane depolarization.
Fujita
Hirofumi
H
Institute of Medical Science, Kurashiki Medical Center, Kurashiki 710-8522, Japan.fujita@md.okayama-u.ac.jp
Ogino
Tetsuya
T
Kobuchi
Hirotsugu
H
Fujiwara
Takuzo
T
Yano
Hiromi
H
Akiyama
Jitsuo
J
Utsumi
Kozo
K
Sasaki
Junzo
J
D018663:Adrenergic Agents / Q000494:pharmacology*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D015153:Blotting, Western / Q000379:methods; D020169:Caspases / Q000378:metabolism; D000242:Cyclic AMP / Q000031:analogs & derivatives* / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005434:Flow Cytometry / Q000379:methods; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D016627:Oxidopamine / Q000494:pharmacology*; D016716:PC12 Cells; D019869:Phosphatidylinositol 3-Kinases; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D013873:Thionucleotides / Q000494:pharmacology*; D013997:Time Factors
D018663:Adrenergic Agents; D004791:Enzyme Inhibitors; D017382:Reactive Oxygen Species; D013873:Thionucleotides; C015929:8-((4-chlorophenyl)thio)cyclic-3',5'-AMP; D016627:Oxidopamine; D000242:Cyclic AMP; D019869:Phosphatidylinositol 3-Kinases; D020169:Caspases