Fujita Hirofumi, Ogino Tetsuya, Kobuchi Hirotsugu, Fujiwara Takuzo, Yano Hiromi, Akiyama Jitsuo, Utsumi Kozo, Sasaki Junzo
Institute of Medical Science, Kurashiki Medical Center, Kurashiki 710-8522,
Brain Res. 2006 Oct 3;1113(1):10-23. doi: 10.1016/j.brainres.2006.06.079. Epub 2006 Aug 30.
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.
尽管环磷酸腺苷（cAMP）可保护神经元细胞免受各种凋亡刺激，但其机制尚未完全阐明。我们在此报告6-羟基多巴胺（6-OHDA）诱导嗜铬细胞瘤PC12细胞凋亡的分子机制，以及膜通透性cAMP类似物8-（4-氯苯硫基）腺苷3',5'-环一磷酸（pCPT-cAMP）对其的抑制作用。用6-OHDA处理PC12细胞导致半胱天冬酶激活和凋亡，通过染色质凝聚检测到。6-OHDA还诱导超氧化物生成、Bid裂解和线粒体膜去极化。此外，6-OHDA使有利于细胞存活的Akt磷酸化降低，p38丝裂原活化蛋白激酶（MAPK）磷酸化增加。pCPT-cAMP、Z-VAD-fmk（一种广谱半胱天冬酶抑制剂）和替诺（一种超氧化物清除剂）可抑制PC12细胞凋亡，尽管环孢素A（一种线粒体膜通透性转换抑制剂）不能抑制PC12细胞凋亡。此外，pCPT-cAMP促进Akt磷酸化，但不能阻止超氧化物生成和线粒体膜去极化。相反，Akt上游分子PI3-激酶的抑制剂LY294002增强了6-OHDA诱导的凋亡。这些结果表明，6-OHDA诱导的PC12细胞凋亡是由超氧化物生成引发，随后是半胱天冬酶级联激活，这与Akt磷酸化受抑制和p38磷酸化增加有关。pCPT-cAMP可能通过激活PI3-激酶/Akt途径阻止6-OHDA诱导的凋亡，而对超氧化物生成或线粒体膜去极化没有任何影响。
