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

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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