Kinetic properties of the purified a, fi, and y subspecies of protein kinase C (PKC) to respond to diacylglycerol, phosphatidylserine (PtdSer), and Ca2l were reinvestigated in the presence of several fatty acids. Although responses of these enzyme subspecies to the lipids slightly differed from one another, the reaction velocity of these subspecies was significantly enhanced by synergistic action of diacylglycerol and a cis-unsaturated fatty acid. Arachidonic, oleic, linoleic, linolenic, and docosahexaenoic acids were active in this role, whereas saturated fatty acids such as palmitic and stearic acids were inactive. Elaidic acid was also inactive. In the presence of both PtdSer and diacylglycerol, the cis-unsaturated fatty acids increased further an apparent affinity of PKC to Ca2' and allowed the enzyme to exhibit almost full activation at nearly basal levels of Ca21 concentration. The concentration of fatty acid giving rise to the maximum activation of enzyme was -20-50 ,uM. The result presented herein implies that the receptor-mediated release of unsaturated fatty acids from phospholipids may take part, in synergy with diacylglycerol, in the activation of PKC even when the Ca2+ concentration is low. A possibility arises, then, that the activation of PKC is an integral part of the signal-induced degradation cascade of various membrane phospholipids, which is initiated by the actions of phospholipase C and phospholipase A2.The hydrolysis of phosphatidylinositol, particularly its 4,5-bisphosphate, catalyzed by phospholipase C is generally accepted to be crucially important to initiate signal transduction for eliciting cellular responses (1-3). Recent studies have suggested that receptor-mediated hydrolysis of phosphatidylcholine may also be involved in transmembrane signaling (for a review, see refs. 4 and 5). In fact, it is becoming clear that both phospholipase A2 (6) and phospholipase D (7-11; see ref. 4 for additional references) are activated in a signaldependent manner.Early reports from this laboratory (12, 13) have described that diacylglycerol produced in membranes activates protein kinase C (PKC) in the presence of Ca2' and phospholipids, especially phosphatidylserine (PtdSer). Kinetic analysis has shown that diacylglycerol increases an apparent affinity of the enzyme for Ca2' and PtdSer and thereby activates PKC in the micromolar range of Ca2+ concentrations (13). Subsequent studies in several laboratories (14-21) have found that, in the absence of PtdSer, unsaturated fatty acids such as arachidonic and oleic acids may activate PKC to various degrees, most efficiently activating the y subspecies, and a potential role ofunsaturated fatty acids as second messengers has been postulated. More recently, synergistic action of fatty acids and diacylglycerol for the activation of PKC has been reported (refs. 22-25; also S. G. Chen and K. Murakami, personal communication). Studies on the interaction of fatty acids with diacylglycerol and Ca2l have revealed, however, conflicting results. In some studie...

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Synaptosomes isolated from the rat hippocampus contain the α‐ and β‐subspecies of protein kinase C (PKC), but not the γ‐subspecies which is abundantly expressed in the pyramidal cells in this brain region. Although the γ‐subspecies is known to respond significantly to free arachidonic acid, it is found that both the α‐ and β‐subspecies are also activated dramatically by arachidonic acid in synergistic action with diacylglycerol. Olcic, linoleic, and linolenic acids are all active. It is possible that unsaturated fatty acids may take part in the activation of α‐ and β‐subspecies of PKC which are present in the presynaptic nerve endings terminating at the hippocampal pyramidal cells.

The expression of protein kinase C (PKC) subspecies in synaptosomes prepared from a number of adult brain regions was compared. Cerebral cortical and thalamic/striatal synaptosomes were found to express three peaks of enzyme activity upon hydroxyapatite chromatography, corresponding to the type I(gamma), type II(beta), and type III(alpha) subspecies. Synaptosomes prepared from either the hippocampus or the cerebellar cortex, however, contained only two major peaks, corresponding to the alpha- and beta-subspecies, with barely detectable levels of the gamma-subspecies, even though these tissue areas were enriched in the latter enzyme. When the ontogenic pattern of hippocampal synaptosomal PKC subspecies was examined, it was found that at postnatal day 7, significant quantities of the gamma-subspecies were present and that this subspecies reached its peak levels at around postnatal day 14, before steadily declining to its adult level. Similar changes were observed also for the gamma-subspecies in cerebellar cortex synaptosomes. The dynamic changes in the synaptosomal PKC subspecies take place at a critical period in the development of the rat brain, concomitant with an active period of synaptogenesis, suggesting that it may play a role in synaptogenesis.