Diacylglycerols release LH: structure-activity relations reveal a role for protein kinase C

A series of diacylglycerols were synthesized with varying lengths and substituents in order to establish the structure-activity relationship between each with activation of protein kinase C and stimulation of a biological response system (pituitary luteinizing hormone release). This approach enables distinction between actions mediated by direct activation of protein kinase C and those due to other, presumably nonspecific, actions. The ability of diacylglycerols to function as regulators of a biological response system (pituitary luteinizing hormone release) and of protein kinase C was investigated with a series of sn-1,2 diacylglycerols containing fatty acids 4-10 carbons in length and with analogs in which the 3' hydroxyl was replaced with a chloro, hydrogen, or sulfhydryl moiety. Several diacylglycerols stimulated LH release in a saturable, time and dose dependent manner that was independent of extra-cellular calcium. Dioctanoylglycerol (diC8) was the most effective of the diacylglycerols tested; 3' analogs lacking the hydroxyl were inactive. The diacylglycerols activated protein kinase C in vitro whereas the 3' analogs did not. These data implicate protein kinase C in the mechanism of LH release, demonstrate that unsaturated fatty acyl moieties within the diacylglycerol are not required for protein kinase C activation, and establish diacylglycerol-protein kinase C structure-function relationships that should prove useful for investigations in other systems.     

The role of calcium, diglyceride ester bindings and a synthetic polypeptide in protein kinase C activation

Protein kinase C, which plays a significant role in the polyphosphoinositide pathway of transmembrane signaling, is activated by a large class of extracellular ligands including neurotransmitters, hormones and growth factors. Diacylglycerols are the intracellular mediators of protein kinase C activation. Tumor promoting phorbol esters mimic the diacylglycerol action in binding to the same site. Active diacylglycerols have the 1.2 sn configuration and saturated short chain or unsaturated long chain fatty acids. Alkyl analogs of diacylglycerols were devoid of activity when an ether bond was present in position 1, whereas activity of the alkyl analog in position 2 was retained. Protein kinase C activation and 3H-TPA binding to the enzyme occurred in the presence of 0.5 mM EGTA. Moreover it has been shown in vivo that full activation of the enzyme was obtained in the intact platelets loaded with an excess of Quin 2, prior to stimulation by phorbol esters. A peptide (residues 499-513) was synthesized which enhanced the affinity of protein kinase C for histone. It is suggested that it may be the receptor site for another peptide of the enzyme (residues 19 to 36) which behaves as a pseudosubstrate.     

Protein kinase C activation by cis-fatty acid in the absence of Ca2+ and phospholipids

Protein kinase C has been shown to be a phospholipid/Ca2+-dependent enzyme activated by diacylglycerol (Nishizuka, Y. (1984) Nature 308, 693-697; Nishizuka, Y. (1984) Science 225, 1365-1370). We have reported that unsaturated fatty acids (oleic acid and arachidonic acid) can activate protein kinase C independently of Ca2+ and phospholipid (Murakami, K., and Routtenberg, A. (1985) FEBS Lett. 192, 189-193). This study shows that other cis-fatty acids such as linoleic acid also fully activate protein kinase C in the same manner. None of the saturated fatty acids (C:4 to C:18) nor the detergents (sodium dodecyl sulfate and Triton X-100) tested here were as effective as oleic acid. Unlike oleic acid, these detergents strongly inhibited protein kinase C activity induced by Ca2+/phosphatidylserine (PS) and diacylglycerol. Lowering the critical micelle concentration of oleic acid by increasing ionic strength also strongly inhibited oleic acid activation of protein kinase C activity. Dioleoylphosphatidylserine activated protein kinase C effectively (Ka = 7.2 microM). On the other hand, dimyristoylphosphatidylserine, which contains saturated fatty acids at both acyl positions, failed to activate protein kinase C even in the presence of Ca2+. These observations suggest that: protein kinase C activation by free fatty acid is specific to the cis-form and is not due to their detergent-like action, cis-fatty acid activation is due to the direct interaction of protein kinase C with the monomeric form of cis-fatty acids and not with the micelles of fatty acids, and cis-fatty acids at acyl positions in PS are also important for Ca2+/PS activation of protein kinase C.     

Hydrolysis of novel diacylglycerol analogs and phorbol diesters by serum lipase

Rat serum, active in the hydrolysis of the tumor-promoting phorbol diester, 12-O-tetradecanoylphorbol-13-acetate (TPA), was examined with regard to lipid interferences of [3H]TPA hydrolysis and enzyme substrate specificity. The enzymatic hydrolysis of TPA could be enhanced 8-fold, over crude serum, by using a lipid-free acetone powder of rat serum. Addition of lipid to the lipid-free acetone powder produced potent inhibition of TPA hydrolysis. The inclusion of multilamallar liposomes resulted in similar inhibition, and isolation of liposomes by high-speed centrifugation showed that 95% of the radiolabeled TPA was associated with the fatty pellet. Substrate specificity studies demonstrated that the serum activity hydrolyzes the long-chain ester of TPA and the long-chain primary acyl group of diacylglycerols. TPA was hydrolyzed at approximately twice the rate of dioleoylglycerol; however, the most reactive substrates were those synthetic analogs of diacylglycerol containing a short-chain ester group at the sn-2 position. Palmitic acid was liberated from [1-14C]palmitoyl-2-acetyl-sn-glycerol and [1-14C]palmitoyl-2-butyryl-sn-glycerol at 120- and 33-times the rate of TPA hydrolysis, respectively. Lipase resistant 1-hexadecyl-2-[3H]acetylglycerol was also used as substrate, but the sn-2 ester moiety showed poor lability. The diacylglycerol analogs are new lipase substrates and, in view of their similarities to the fatty acyl portion of TPA, it is thought that these compounds could serve as protein kinase C activators.     

Further studies on the specificity of diacylglycerol for protein kinase C activation

Specificity of 1,2-diacylglycerol for the activation of protein kinase C was investigated with various synthetic products. 1-Stearoyl-2-arachidonylglycerol, a major species of diacylglycerol derived from the receptor-mediated hydrolysis of inositol phospholipids, was most active, but many other diacylglycerols having naturally occurring fatty acids were almost equally active in this role. Hormone-sensitive lipase could produce potentially active diacylglycerols during lipolysis. The lack of the specificity may be reconciled with the possibility that the stearoyl-arachidonyl species is the diacylglycerol with which protein kinase C indeed comes in contact in the membrane when the receptor is stimulated, and that diacylglycerols from other sources are produced in distinct compartments and are not intercalated into the phospholipid bilayer.     

Incorporation of newly synthesized fatty acids into cytosolic glycerolipids in pea leaves occurs via acyl editing

In expanding pea leaves, over 95% of fatty acids (FA) synthesized in the plastid are exported for assembly of eukaryotic glycerolipids. It is often assumed that the major products of plastid FA synthesis (18:1 and 16:0) are first incorporated into 16:0/18:1 and 18:1/18:1 molecular species of phosphatidic acid (PA), which are then converted to phosphatidylcholine (PC), the major eukaryotic phospholipid and site of acyl desaturation. However, by labeling lipids of pea leaves with [(14)C]acetate, [(14)C]glycerol, and [(14)C]carbon dioxide, we demonstrate that acyl editing is an integral component of eukaryotic glycerolipid synthesis. First, no precursor-product relationship between PA and PC [(14)C]acyl chains was observed at very early time points. Second, analysis of PC molecular species at these early time points showed that >90% of newly synthesized [(14)C]18:1 and [(14)C]16:0 acyl groups were incorporated into PC alongside a previously synthesized unlabeled acyl group (18:2, 18:3, or 16:0). And third, [(14)C]glycerol labeling produced PC molecular species highly enriched with 18:2, 18:3, and 16:0 FA, and not 18:1, the major product of plastid fatty acid synthesis. In conclusion, we propose that most newly synthesized acyl groups are not immediately utilized for PA synthesis, but instead are incorporated directly into PC through an acyl editing mechanism that operates at both sn-1 and sn-2 positions. Additionally, the acyl groups removed by acyl editing are largely used for the net synthesis of PC through glycerol 3-phosphate acylation.     

Cerebral ischemia induced quantitative changes in rat brain membrane lipids involved in phosphoinositide metabolism

The content and acyl group composition of phosphatidylinositol, poly-phosphoinositides, diacylglycerols, phosphatidic acids, and free fatty acids in rat brain homogenates of cerebral cortex and subcellular fractions were examined with respect to a 2 min post-decapitative ischemic treatment. With the exception of free fatty acids, these lipids are involved in the cyclic event associated with the receptor-mediated poly-phosphoinositide turnover. The ischemic treatment elicited a decrease in poly-phosphoinositide level in brain homogenates, synaptosomes, plasma membranes, and microsomes but not in myelin, and an increase in diacylglycerols, which was observed in brain homogenates and synaptosomes but not in other subcellular fractions. On the other hand, the level of phosphatidylinositol was not altered. The acyl groups of phosphoinositides are enriched in stearic and arachidonic acids. The diacylglycerols and free fatty acids that accumulated during the ischemic treatment are also enriched with the same fatty acids. There is a decrease in phosphatidic acid level after the ischemic treatment, but the change was only found in brain homogenates and synaptosomes. Therefore, the diacylglycerols increased during the ischemic treatment may be derived from hydrolysis of poly-phosphoinositides and phosphatidic acids. However, the amount of poly-phosphoinositides degraded is not enough to account for both diacylglycerols and free fatty acid increase.     

Hydrolysis of novel diacylglycerol and phorbol diesters by serum lipase

Rat serum, active in the hydrolysis of the tumor-promoting phorbol diester, 12-O-tetradecanoylphorbol-13-acetate (TPA), was examined with regard to lipid interferences of [³H]TPA hydrolysis and enzyme substrate specificity. The enzymatic hydrolysis of TPA could be enhanced 8-fold, ever crude serum, by using a lipid-free acetone powder of rat serum. Addition of lipid to the lipid-free acetone powder produced potent inhibition of TPA hydrolysis. The inclusion of multilamallar liposomes resulted in similar inhibition, and isolation of liposomes by high-speed centrifugation showed that 95% of the radiolabeled TPA was associated with the fatty pellet. Substrate specificity studies demonstrated that the serum activity hydrolyzes the long-chain ester of TPA and the long-chain primary acyl group of diacylglycerols. TPA was hydrolyzed at approximately twice the rate of dioleoylglycerol; however, the most reactive substrates were those synthetic analogs of diacylglycerol containing a short-chain ester group at the sn-2 position. Palmitic acid was liberated from [1-¹⁴C]palmitoyl-2-acetyl-sn-glycerol and [1-¹⁴C]palmitoyl-2-butyryl-sn-glycerol at 120- and 33-tinies the rate of TPA hydrolysis, respectively. Lipase resistant 1-hexadecyl-2-[³H]acetylglycerol was also used as substrate, but the sn-2 ester moiety showed poor lability. The diacylglycerol analogs are new lipase substrates and, in view of their similarities to the fatty acyl portion of TPA, it is thought that these compounds could serve as protein kinase C activators.     

Structural studies on the diglyceride-mediated activation of protein kinase C

Diglyceride analogs were studied with respect to their abilities to activate protein kinase C (Ca2+- and phospholipid-dependent protein kinase) in the presence of low calcium and phospholipid. Analogs which lacked either a free hydroxyl group at the 3 position or an ester moiety at the 1 position were without activity. It was concluded that the hydrophilic moieties of the active diglycerides are crucial for activity. However, diglyceride analogs containing additional hydrophilic moieties in one of the acyl side chains did not exhibit enhanced activity when compared to diglycerides containing two fatty acyl groups. Diglyceride analogs with a modified glycerol backbone were also studied. Homologous diglycerides with either one or two methylene groups between the 3-methylene group of the diglyceride and the hydroxyl group possessed markedly reduced activities when compared to the appropriate unmodified diglyceride. Isomers of these homologues which contained either a methyl group at the 1 position, or dimethyl groups incorporated at the 1 and 3 positions, were virtually without activity. Where studied, none of the diglyceride analogs prepared possessed antagonist activity. The results of these experiments are discussed with respect to the extreme specificity observed.     

Role of protein kinase C in transmembrane signaling

Many extracellular signals elicit Ca2+ mobilization and diacylglycerol formation in their target cells. Diacylglycerol is derived from the receptor-linked phosphoinositide turnover and serves as a second messenger for the activation of protein kinase C in the presence of Ca2+ and phosphatidylserine. Unique diacylglycerols such as 1-oleoyl-2-acetyl-glycerol, which activate intracellular protein kinase C when added to intact cells, have been synthesized. Tumor-promoting phorbol esters substitute for such diacylglycerols and directly activate protein kinase C in both intact cell and cell-free systems. Under appropriate conditions, the synthetic diacylglycerols and phorbol esters induce protein kinase C activation without Ca2+ mobilization, whereas Ca2+ ionophore A23187 induces Ca2+ mobilization without protein kinase C activation. Using these substances, we have obtained evidence that both protein C and Ca2+ are involved in and play a synergistic role in exocytosis, cell division, and other cellular functions. In this article, the role of protein kinase C in transmembrane signaling is discussed.     

The dissimilar effect of diacylglycerols on Ca(2+)-induced phosphatidylserine vesicle fusion.

We have studied the effect of physiological concentrations of different diacylglycerols on Ca(2+)-induced fusion between phosphatidylserine vesicles. We monitored vesicle fusion as mixing of membrane lipids under conditions where the limiting factor was the aggregation and also in conditions where this aggregation was not the limiting factor. We found that diacylglycerols have a different modulating effect on the Ca(2+)-induced fusion: i) depending on their interfacial conformation, so that 1,2-isomers of diacylglycerols containing unsaturated or short saturated acyl chains stimulated fusion and their 1,3-isomers did not, and ii) depending on their specific type of bilayer interior perturbation, so that diacylglycerols containing unsaturated or short chain saturated acyl chains stimulated fusion but those containing long-chain saturated acyl chains did not. These requirements resembled those required for the diacylglycerol activation of protein kinase C, suggesting that diacylglycerol acts in both the specific activation of this enzyme and the induction of membrane fusion through the same perturbation of lipid structure. We found that polylysine affected the stimulatory role of 1,2-dioleoylglycerol differently, depending on whether aggregation was the limiting factor of fusion. When we studied the effect of very low concentrations of diacylglycerols on the bulk structural properties of phosphatidylserine, we found that they neither significantly perturbed the thermotropic transitions of phosphatidylserine nor affected the interaction of Ca2+ with the phosphate group of phosphatidylserine. The underlying mechanism of fusion between phosphatidylserine vesicles is discussed.     

The nature of protein kinase C activation by physically defined phospholipid vesicles and diacylglycerols

Protein kinase C is activated by a 1,2-sn-diacylglycerol and phospholipid at low calcium concentrations. Of the various phospholipids studied, phosphatidylserine has been shown to be the most effective one and is usually used in assaying the enzyme (Kaibuchi, K., Takai, Y., and Nishizuka, Y. (1981) J. Biol. Chem. 256, 7146-7149). It is shown here that under the conditions of the enzymatic assay, phosphatidylserine does not form typical fluid bilayer structures as seen by electron microscopy and fluorescence polarization. On the other hand, 1:4 phosphatidylserine/phosphatidylcholine bilayer vesicles can be formed which support protein kinase C activation. They have the advantage in that they are characterizable, form physiologically relevant bilayer structures, and are readily and reproducibly formed. In addition, they do not support protein kinase C activation in the absence of added diacylglycerol, a property that makes them invaluable in studying the role of diacylglycerol structure in protein kinase C activation. It is further demonstrated that the rat brain enzyme is activated by 1,2-sn-diolein but not by 2,3-sn-diolein nor 1,3-diolein, demonstrating the high specificity of the kinase toward the glycerol backbone. 1,2-rac-Dielaidin, 1,2-rac-distearin, and 1,2-sn-dipalmitin are all active, which is consistent with the idea that the specificity of protein kinase C is not directed toward the fatty acid side chain of the diacylglycerols.     

Intersubunit transfer of fatty acyl groups during fatty acid reduction

Fatty acid reduction in Photobacterium phosphoreum is catalyzed in a coupled reaction by two enzymes: acyl-protein synthetase, which activates fatty acids (+ATP), and a reductase, which reduces activated fatty acids (+NADPH) to aldehyde. Although the synthetase and reductase can be acylated with fatty acid (+ATP) and acyl-CoA, respectively, evidence for acyl transfer between these proteins has not yet been obtained. Experimental conditions have now been developed to increase significantly (5-30-fold) the level of protein acylation so that 0.4-0.8 mol of fatty acyl groups are incorporated per mole of the synthetase or reductase subunit. The acylated reductase polypeptide migrated faster on sodium dodecyl sulfate-polyacrylamide gel electrophoresis than the unlabeled polypeptide, with a direct 1 to 1 correspondence between the moles of acyl group incorporated and the moles of polypeptide migrating at this new position. The presence of 2-mercaptoethanol or NADPH, but not NADP, substantially decreased labeling of the reductase enzyme, and kinetic studies demonstrated that the rate of covalent incorporation of the acyl group was 3-5 times slower than its subsequent reduction with NADPH to aldehyde. When mixtures of the synthetase and reductase polypeptides were incubated with [3H] tetradecanoic acid (+ATP) or [3H]tetradecanoyl-CoA, both polypeptides were acylated to high levels, with the labeling again being decreased by 2-mercaptoethanol or NADPH. These results have demonstrated that acylation of the reductase represents an intermediate and rate-limiting step in fatty acid reduction. Moreover, the activated acyl groups are transferred in a reversible reaction between the synthetase and reductase proteins in the enzyme mechanism.     

The diacylglycerols dioctanoylglycerol and oleoylacetylglycerol enhance prostaglandin synthesis by inhibition of the lysophosphatide acyltransferase.

Prostanoids are synthesized by resident macrophages upon stimulation with diacylglycerols. Oleoylacetylglycerol and dioctanoylglycerol induced prostaglandin E and thromboxane synthesis in a time- and concentration-dependent manner. Both diacylglycerols inhibited the lysophosphatide acyltransferase, which is the key enzyme in the reacylation of arachidonic acid. By this mechanism the pool of free arachidonic acid available for prostanoid synthesis is increased. Both diacylglycerols were able to inhibit the membrane-bound lysophosphatide acyltransferase by a direct interaction independent of protein kinase C. Thus lysophosphatide acyltransferase could be shown to be a new target of these diacylglycerols, known as activators of protein kinase C.     

Role of sphingomyelin and ceramide in the regulation of the activity and fatty acid specificity of group V secretory phospholipase A2

We previously showed that group V secretory phospholipase A(2) (sPLA(2)V) is inhibited by sphingomyelin (SM), but activated by ceramide. Here, we investigated the effect of sphingolipid structure on the activity and acyl specificity of sPLA(2)V. Degradation of HDL SM to ceramide, but not to ceramide phosphate, stimulated the activity by 6-fold, with the release of all unsaturated fatty acids being affected equally. Ceramide-enrichment of HDL similarly stimulated the release of unsaturated fatty acids. Incorporation of SM into phosphatidylcholine (PC) liposomes preferentially inhibited the hydrolysis of 16:0-20:4 PC. Conversely, SMase C treatment or ceramide incorporation resulted in preferential stimulation of hydrolysis of 16:0-20:4 PC. The presence of a long chain acyl group in ceramide was essential for the activation, and long chain diacylglycerols were also effective. However, ceramide phosphate was inhibitory. These studies show that SM and ceramide in the membranes and lipoproteins not only regulate the activity of phospholipases, but also the release of arachidonate, the precursor of eicosanoids.     

Lipid biosynthesis in developing mustard seed: formation of triacylglycerols from endogenous and exogenous Fatty acids

Cotyledons of developing mustard (Sinapis alba L.) seed have been found to synthesize lipids containing the common plant fatty acids and very long-chain monounsaturated (icosenoic, erucic, and tetracosenic) and saturated (icosanoic, docosanoic, and tetracosanoic) fatty acids from various radioactive precursors. The in vivo pattern of labeling of acyl lipids, either from fatty acids synthesized `endogenously' from radioactive acetate or malonate, or from radioactive fatty acids added `exogenously', indicates the involvement of the following pathways in the biosynthesis of triacylglycerols. Palmitic, stearic, and oleic acid, synthesized in the acyl carrier protein-track, are channeled to the Coenzyme A (CoA)-track and converted to triacylglycerols via the glycerol-3-phosphate pathway. Pools of stearoyl-CoA and oleoyl-CoA are elongated to very long-chain saturated and monounsaturated acyl-CoA, respectively. Most of the very long-chain saturated acyl-CoAs acylate preformed diacylglycerols. Very long-chain monounsaturated acyl-CoAs are converted to triacylglycerols, partly via phosphatidic acids and diacylglycerols, and partly by acylation of preformed diacylglycerols.     

Exogenous sn-1,2-diacylglycerols containing saturated fatty acids function as bioregulators of protein kinase C in human platelets

The ability of exogenous sn-1,2-diacylglycerols and analogs to function as bioregulators of protein kinase C in human platelets was investigated. The activation of protein kinase C in platelets is indicated by specific phosphorylation of a 40,000-dalton protein. Dihexanoylglycerol, dioctanoylglycerol (diC8), didecanoylglycerol, and sn-1-oleoyl-2-acetylglycerol were active in stimulating 40,000-dalton protein phosphorylation. Only a trace of phosphorylation was elicited by dibutyrylglycerol. Phosphorylation was not induced by analogs of diC8 in which an -H, -SH, or -Cl group replaced the free -OH, nor by monoacylglycerols or long chain diacylglycerols. Maximum phosphorylation was induced by dihexanoylglycerol, diC8, and didecanoylglycerol at concentrations from 5 to 20 microM and between 5 and 30 S after exposure of platelets to these diacylglycerols. Under conditions of maximal phosphorylation of the 40,000-dalton protein, these diacylglycerols did not induce phosphatidylinositol turnover, or platelet aggregation, or stimulate release of ATP or serotonin. A small degree of aggregation was evident with platelets isolated in the absence of prostacyclin, and release of serotonin was observed when 1 mM Ca2+ or submaximal concentrations of ionophore A23187 were included. These results are consistent with a model in which platelet activation requires the simultaneous formation of two intracellular signals, diacylglycerols and Ca2+. These diacylglycerols and diacylglycerol analogs provide useful tools to investigate the function of diacylglycerols as bioregulators in intact cells.     

A rapid effect of thyroxine on accumulation of diacylglycerols and on activation of protein kinase C in liver cells

L-Thyroxine rapidly stimulated the accumulation of diacylglycerols in isolated hepatocytes and in liver when lipids were prelabeled with [14C]oleic acid or with [14C]CH3COONa. Perfusion of the liver of hypothyroid animals with L-thyroxine-containing solution or incubation of liver fragments with the hormone increased the content of diacylglycerols in the liver cells. The increase in [14C]diacylglycerol level in the liver cells was accompanied by a decrease in the level of [14C]phosphatidylcholine, whereas contents of other 14C-labeled phospholipids, such as phosphatidylethanolamine, sphingomyelin, lysophosphatidylcholine, phosphatidylinositol (PtdIns), phosphatidylinositol-4-phosphate (PtdIns4P), and phosphatidylinositol-4,5-bis-phosphate (PtdIns(4,5)P2), and of 14C-labeled fatty acids were the same as in the control. The L-thyroxine-induced accumulation of diacylglycerols in hepatocytes was not affected by neomycin but was inhibited by propranolol. Incubation of hepatocytes prelabeled with [14C]oleic acid with L-thyroxine and ethanol (300 mM) was accompanied by generation and accumulation of [14C]phosphatidylethanol that was partially suppressed by 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7). L-Thyroxine was responsible for the translocation of protein kinase C from the cytosol into the membrane fraction and for a many-fold activation of the membrane-bound enzyme. D-Thyroxine failed to affect the generation of diacylglycerols in hepatocytes and the activity of protein kinase C.     

Biosynthesis of triacylglycerols containing very long chain monounsaturated acyl moieties in developing seeds

Particulate (15,000g) fractions from developing seeds of honesty (Lunaria annua L.) and mustard (Sinapis alba L.) synthesize radioactive very long chain monounsaturated fatty acids (gadoleic, erucic, and nervonic) from [1-¹⁴C]oleoyl-CoA and malonyl-CoA or from oleoyl-CoA and [2-¹⁴C]malonyl-CoA. The very long chain monounsaturated fatty acids are rapidly channeled to triacylglycerois and other acyl lipids without intermediate accumulation of their CoA thioesters. When [1-¹⁴C]oleoyl-CoA is used as the radioactive substrate, phosphatidylcholines and other phospholipids are most extensively radiolabeled by oleoyl moieties rather than by very long chain monounsaturated acyl moieties. When [2-¹⁴C]malonyl-CoA is used as the radioactive substrate, no radioactive oleic acid is formed and the newly synthesized very long chain monounsaturated fatty acids are extensively incorporated into phosphatidylcholines and other phospholipids as well as triacylglycerols. The pattern of labeling of the key intermediates of the Kennedy pathway, e.g. lysophosphatidic acids, phosphatidic acids, and diacylglycerols by the newly synthesized very long chain monounsaturated fatty acids is consistent with the operation of this pathway in the biosynthesis of triacylglycerols.     

Tumor necrosis factor stimulates human neutrophils to release leukotriene B4 and platelet-activating factor. Induction of phospholipase A2 and acetyl-CoA:1-alkyl-sn-glycero-3-phosphocholine O2-acetyltransferase activity and inhibition by antiproteinase

Tumor necrosis factor stimulates polymorphonuclearneutrophils to synthesize leukotriene B4 and platelet-activating factor (PAF), but alpha 1-proteinase inhibitor and alpha 1-antichymotrypsin block this response. However, proteinases such as elastase and cathepsin G induce preferentially synthesis of PAF. An acetyltransferase required, together with phospholipase A2, in the remodeling pathway of PAF synthesis is activated in polymorphonuclearneutrophils stimulated by tumor necrosis factor and elastase. In contrast, 1-oleyl-2-acetylglycerol, a protein kinase C activator, promotes PAF formation by the de novo biosynthetic pathway without activating the acetyltransferase. Staurosporine, an inhibitor of protein kinase C, blocks PAF production apparently by inhibiting phospholipase A2. This suggests that diacylglycerols are involved in activating both pathway of PAF synthesis.