Larsen E C, Hatcher J F, Adibhatla R M
Department of Neurological Surgery, H4-330, Clinical Science Center, 600 Highland Avenue, University of Wisconsin-Madison, Madison, WI 53792-3232, USA.
Neuroscience. 2007 May 25;146(3):946-61. doi: 10.1016/j.neuroscience.2007.02.022. Epub 2007 Apr 16.
Alterations in lipid metabolism play an integral role in neuronal death in cerebral ischemia. Here we used an in vitro model, oxygen-glucose deprivation (OGD) of rat pheochromocytoma (PC12) cells, and analyzed changes in phosphatidylcholine (PC) and sphingomyelin (SM) metabolism. OGD (4-8 h) of PC12 cells triggered a dramatic reduction in PC and SM levels, and a significant increase in ceramide. OGD also caused increases in phosphatidylcholine-phospholipase C (PC-PLC) and phospholipase D (PLD) activities and PLD2 protein expression, and reduction in cytidine triphosphate:phosphocholine cytidylyltransferase-alpha (CCTalpha, the rate-limiting enzyme in PC synthesis) protein expression and activity. Phospholipase A2 activity and expression were unaltered during OGD. Increased neutral sphingomyelinase activity during OGD could account for SM loss and increased ceramide. Surprisingly, treatment with PC-PLC inhibitor tricyclodecan-9-yl potassium xanthate (D609) aggravated cell death in PC12 cells during OGD. D609 was cytotoxic only during OGD; cell death could be prevented by inclusion of sera, glucose or oxygen. During OGD, D609 caused further loss of PC and SM, depletion of 1,2-diacylglycerol (DAG), increase in ceramide and free fatty acids (FFA), cytochrome c release from mitochondria, increases in intracellular Ca2+ ([Ca2+]i), poly-ADP ribose polymerase (PARP) cleavage and phosphatidylserine externalization, indicative of apoptotic cell death. Exogenous PC during OGD in PC12 cells with D609 attenuated PC, SM loss, restored DAG, attenuated ceramide levels, decreased cytochrome c release, PARP cleavage, annexin V binding, attenuated the increase in [Ca2+]i, FFA release, and significantly increased cell viability. Exogenous PC may have elicited these effects by restoring membrane PC levels. A tentative scheme depicting the mechanism of action of D609 (inhibiting PC-PLC, SM synthase, PC synthesis at the CDP-choline-1,2-diacylglycerol phosphocholine transferase (CPT) step and causing mitochondrial dysfunction) has been proposed based on our observations and literature.
脂质代谢改变在脑缺血所致神经元死亡中起重要作用。在此，我们使用体外模型，即大鼠嗜铬细胞瘤（PC12）细胞的氧 - 葡萄糖剥夺（OGD），并分析磷脂酰胆碱（PC）和鞘磷脂（SM）代谢的变化。PC12细胞的OGD（4 - 8小时）引发PC和SM水平显著降低，神经酰胺显著增加。OGD还导致磷脂酰胆碱 - 磷脂酶C（PC - PLC）和磷脂酶D（PLD）活性增加以及PLD2蛋白表达增加，同时胞苷三磷酸：磷酸胆碱胞苷转移酶 - α（CCTα，PC合成中的限速酶）蛋白表达和活性降低。OGD期间磷脂酶A2活性和表达未改变。OGD期间中性鞘磷脂酶活性增加可解释SM损失和神经酰胺增加。令人惊讶的是，用PC - PLC抑制剂三环癸烷 - 9 - 基黄原酸钾（D609）处理会加重OGD期间PC12细胞的死亡。D609仅在OGD期间具有细胞毒性；加入血清、葡萄糖或氧气可预防细胞死亡。在OGD期间，D609导致PC和SM进一步损失，1,2 - 二酰基甘油（DAG）耗竭，神经酰胺和游离脂肪酸（FFA）增加，细胞色素c从线粒体释放，细胞内Ca2 +（[Ca2 +]i）增加，聚 - ADP核糖聚合酶（PARP）裂解以及磷脂酰丝氨酸外化，表明细胞凋亡死亡。在含D609的PC12细胞OGD期间加入外源性PC可减轻PC、SM损失，恢复DAG，降低神经酰胺水平，减少细胞色素c释放、PARP裂解、膜联蛋白V结合，减轻[Ca2 +]i增加、FFA释放，并显著提高细胞活力。外源性PC可能通过恢复膜PC水平产生这些作用。基于我们的观察和文献，已提出一个描绘D609作用机制（抑制PC - PLC、SM合酶、在CDP - 胆碱 - 1,2 - 二酰基甘油磷酸胆碱转移酶（CPT）步骤的PC合成并导致线粒体功能障碍）的初步方案。
