Mechanisms of Protein Kinase C Activatic by Lipid Peroxidation and its Biological Significance

众所周知，细胞膜脂质在氧和其他各种自由基的攻击下发生脂质过氧化，并引起不同程度的细胞损伤。然而，尚未证明某些非常特定的物质（脂质）的脂质过氧化物适当地抑制或激活非常特定的细胞反应。大多数蛋白激酶C（PKC）被多种脂质激活，尤其是甘油二酯（DAG），其特有的PKC激活作用引起了研究者的广泛关注。由于DAG含有易氧化的多不饱和脂肪酸，我们推测，氢过氧化DAG（DAG-OOH）可以通过膜脂质过氧化产生。在这方面，我们尝试并成功地从大豆磷脂酰胆碱制备DAG-OOH，通过酶促裂解形成1，2-DAG（地亚麻酸）并进行有氧氧化。然后，我们应用这些DAG，DAG-OOH和PMA（佛波酯），这是已知的是一个通用的，最有效的， ...更多信息 有效的PKC激活剂，对从大鼠脑中纯化的PKC的作用。结果表明，DAG-OOH的活性是天然DAG的3倍，与PMA的活性相当，但人工合成的1，3-DAG（天然DAG无一例外地为1，2-DAG）的过氧化氢形式并没有表现出任何特殊的PKC活性。在本实验中使用的DAG及其氧化形式是从大豆获得的二亚油酸甘油酯（LL-DAG）。然而，在动物组织中，棕榈酸酯（P）、亚油酸酯（L）-DAG、硬脂酸酯（S）、L-DAG等必须是共同分布的。我们将这些系列DAG的过氧化氢形式应用于大鼠PKC，发现P，L-和S，L-DAG-OOH与L，L-DAG-OOH类似地有效。PMA激活的PKC过度磷酸化诱导微管解体，最终在人或动物培养细胞中引起"神经元缠结"样损伤。我们应用了DAG，DAG-OOH（1，2-和1，3-形式）和PMA对培养的神经元细胞的作用（从大鼠胎脑和NGF刺激的PC 12细胞培养），以及最明显的结构变化（链珠样病变，很可能是由于微管异常）由1，2-DAG-OOH引起，但不是由1，3-DAG-OOH是一种生物可利用的物质，不同于人工合成的PMA，这一发现可能为阐明阿尔茨海默病等神经退行性疾病的发病机制提供了重要线索。少

It is well known fact that cell membrane lipids undergo lipid peroxidation by the attacks of oxygen and other various free radicals, and cell injuries of varying degree pursue. It has, however, not yet proved that lipid peroxides of certain very specific substances (lipids) prohibit or activate very specific cell reactions properly. Most of protein kinase C (PKC) are known to be activated by various lipids, especially diacylglycerols (DAG) whose characteristic PKC activation has been attracting many investigators' attentions. Since DAG contains oxidatively vulnerable polyunsaturated fatty acids, we presumed that hydroperoxidized DAG (DAG-OOH) could be produced through membrane lipid peroxidation. In this regards, we tried and succeeded to prepare the DAG-OOH from soybean phosphatidylcholine through the enzymatic cleavage to form 1,2-DAG (dilinorein) and pursuing aerobic oxidation. And then, we applied these DAG,DAG-OOH and PMA (phorbol ester), which is known to be a general and most ef … More fective PKC activator, on PKC purified from rat brains. As a result, DAG-OOH exhibited 3 fold stronger activation than that of the natural DAG,which was almost comparable to that of PMA.Interestingly, hydroperoxidized form of artificially prepared 1,3-DAG (natural one is unexceptionally 1,2-DAG) did not show any particular PKC activation. DAG and its oxidized forms used in this experiment were dilinolein (LL-DAG) obtained from soybeans. In animal tissues, however, palmitate (P), linoleate (L) -DAG,stearate (S), L-DAG and so on must be commonly distributed. We applied hydroperoxidized forms of those series of DAGs on the rat PKC and P,L- and S,L-DAG-OOHs were found to be similarly effective as L,L-DAG-OOH.Reportedly, microtubule associated proteins (tau, map 2 etc.) phosphorylated excessively by PMA activated PKC induce microtubule disassembly which gives finally rise to "neurofibrillary tangles" like lesions in human or animal cultures cells. We applied DAG,DAG-OOH (both 1,2- and 1,3- forms) and PMA on cultured neuronal cells (cultured from rat fetal brain and NGF stimulated PC 12 cells), and the most conspicuous structural changes (chained beads like lesion which may well be due to microtubule abnormality) were provoked by 1,2-DAG-OOH but not by 1,3-DAG-OOH.This evidence may well e an important clue to elucidate the pathogenesis of neurodegenerative diseases such as Alzheimer's disease, for DAG-OOH,differing from an artificial product PMA,is biologically accessible substance. Less