Binding and metabolism of platelet-activating factor by human neutrophils.

Human polymorphonuclear neutrophils rapidly incorporated radiolabeled platelet-activating factor, 1-O-[hexadecyl-9, 10-3H2]-2-acetyl-sn-glycero-3-phosphocholine ([3H]PAF), and then metabolized it into its sn-2-fatty acyl derivative. Fractionation of radiolabel-pretreated cells over Percoll gradients revealed that virtually all of the intact [3H]PAF was located in nongranule membranes that were enriched with alkaline phosphatase and cell surface glycoproteins. While still membrane associated, the ligand was rapidly converted to its acyl derivative and then more slowly transferred to specific granules and, to a lesser extent, azurophilic granules. In contrast, neutrophils did not metabolize [3H]PAF at 4 degrees C but rather gradually accumulated it in their alkaline phosphatase-enriched membrane subfractions. These same subfractions contained receptors for the ligand, as determined by their capacity to bind [3H]PAF specifically. Binding was readily saturated, partially reversible, and fit a two receptor model; dissociation constant (Kd) values for high and low affinity sites were 0.2 and 500 nM, respectively. Receptors with similar affinities were detected in whole cells. Furthermore, the potencies of several structural analogues in inhibiting binding of [3H]PAF to membranes correlated closely with their respective potencies in stimulating degranulation responses. Finally, quantitative studies suggested all or most of the cell's receptors were membrane associated. We conclude that PAF rapidly enters cellular membranes to bind with specific receptors that trigger function. The intramembranous ligand is also deacetylated, acylated, and then transferred to granules. This metabolism may be sufficiently rapid to limit ligand-receptor binding and distort quantitative analyses of receptors.

Correlation of ether lipid content of human leukemia cell lines and their susceptibility to 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine.

A number of synthetic ether-linked phospholipids are selectively cytotoxic to neoplastic cells. However, the mechanisms underlying this selective cytotoxicity are not known. We have investigated the ether-lipid content of HL-60 and K562 human leukemia cells in relation to their sensitivity to 1-O-alkyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3). HL-60 cells are much more sensitive than K562 cells to the cytotoxic effects of ET-18-OCH3 and, at the same time, they contain nearly twice as much ether lipid as the more resistant K562 cells. These observations suggested a relation between the cellular ether-lipid content and sensitivity to ET-18-OCH3. Further evidence linking these properties was obtained when the ether-lipid content of K562 cells was increased by incubating them in medium containing 1-O-hexadecyl-sn-glycerol. This supplementation not only increased the ether-lipid content of the cells but also increased their sensitivity to ET-18-OCH3. The 50% inhibitory concentration for ET-18-OCH3 decreased from 18.4 microM in the control cells to 9.83 microM in the supplemented cells.

Ether-linked phosphoglyceride content of human leukemia cells.

The glycerolipids of most cells are characterized by a specific proportion of ether linkages at the sn-1 position of the glycerol backbone. A number of tumors are known to have altered concentrations of ether-linked lipids compared to normal tissues. However, no through examination of the ether-lipid content of human leukemia cells has been reported despite the importance of these lipids in normal leukocyte function. In the present study samples were obtained from adults with acute myelogenous leukemia (AML), chronic granulocytic leukemia in blast crisis, and acute lymphocytic leukemia and from healthy human donors. The cellular lipids were extracted, the individual phospholipid classes were isolated, lipid phosphorus content was determined, and the lipids were converted to diglyceride benzoate derivatives for separation and quantitation of the subclasses by high performance liquid chromatography. The data indicate that all the leukemic cells analyzed have an altered phospholipid composition compared to their respective normal leukocytes. Furthermore, among the AML patients both the percentage of the choline-containing phosphoglyceride fraction (PC) which is alkyl linked and the nmoles alkyl-PC/10(6) cells differ significantly by FAB subtype. A positive correlation between the levels of alkyl-PC and the degree of cellular differentiation is observed. Although no differences are observed between chronic granulocytic leukemia in blast crisis and AML lipids, the leukemic cells contain dramatically lower levels of alkyl-linked PC than do normal polymorphonuclear leukocytes. In contrast, no differences are observed between the alkyl-PC content of normal and leukemic lymphocytes. In light of the relations among ether-lipids, protein kinase C, and cell differentiation, these data suggest the ether-linked lipids are important in myeloid cell function and differentiation.

Synthesis of platelet activating factor and metabolism of related lipids in embryonic cells.

Primary cultures of mouse embryo palate mesenchyme (MEPM) cells incubated with 1-O-[3H]alkyl-2-lyso-sn-glycero-3-phosphocholine ([3H])lyso-PAF) incorporated radiolabel into 1-radyl-2-acyl-sn-glycero-3-phosphocholine (PC) and -phosphoethanolamine (PE). The radiolabeled PC was insensitive to hydrolysis with HCl fumes, whereas at least 82% of the 3H found in the PE was hydrolyzed to 3H-aldehydes by such treatment. Treatment of the PC with Vitride produced [3H]alkylglycerol; similar treatment of the PE produced [3H]alk-1-enylglycerol. None of the radiolabeled products yielded fatty alcohol upon reduction with Vitride. These findings indicate the radiolabeled PC was 1-O-alkyl-linked whereas the PE contained predominantly 1-O-alk-1'-enyl species with smaller amounts of 1-O-alkyl species. Homogenates of MEPM cells which had been prelabeled with [3H]lyso-PAF and [14C]arachidonic acid produced 14C-fatty acid, [3H]lyso-PC, and [3H]alkylglycerol when incubated at selected values of pH and concentrations of calcium. There was no accumulation of [3H]lyso-PE in the various incubation mixtures. Stimulation of MEPM cells with the ionophore A23187 in the presence of calcium and [3H]acetate resulted in the production of 3H-platelet-activating factor (PAF), identified by its migration with authentic PAF and its conversion to 1-O-[3H]alkyl-2,3-diacetylglycerol upon treatment with phospholipase C and acetic anhydride. These studies demonstrate that: (i) MEPM cells are able to incorporate [3H]lyso-PAF into 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine, the storage form of PAF, and into 1-O-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine (PE plasmalogen); (ii) endogenous 1-O-[3H]alkyl-2-acyl-sn-glycero-3-phosphocholine can serve as a substrate for phospholipase A2 in homogenates; and (iii) MEPM cells have the ability to synthesize PAF, thus raising the possibility that this compound may play a role in modulating the physiology of these embryonic cells.

The biosynthesis of plasmalogens by rat brain: involvement of the microsomal electron transport system.

The biosynthesis of 1-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine (ethanolamine plasmalogens) was studied using 1-[1-14C]hexadecyl-sn-glycero-3-phosphoethanolamine as the substrate and EDTA-washed microsomes from brains of 14-day-old rats. It was found that the 1-E11-14C]hexadecyl-sn-glycero-3-phosphoethanolamine was first acylated to form 1-[1-14C]hexadecyl-2-acyl-sn-glycero-3-phosphoethanolamine, then was desaturated to form 1-[1-14C]hexadec-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine. The desaturation required O2 and NADH or NADPH and was inhibited by KCN but not by CO. The data indicated that the desaturation is carried out by a mixed-function oxidase system similar to that involved in the desaturation of fatty acids and that the pathway for the biosynthesis of plasmalogens in brain is similar to that previously found in other tissues. The desaturase was not stimulated by ATP and Mg2plus nor inhibited by EDTA. The specific activity of microsomes from brains of rats of different ages was determined; the activity decreased with age until in adults the activity was only 15% that of the 12--14-day-old rats.

Specificity of lysophospholipase D.

The specificity of lysophospholipase D (1-alkyl-sn-glycero-3-phosphoethanolamine ethanolaminehydrolase, EC 3.1.4.39; also works on choline analogs) for 1-alkyl- and 1-acyl-linked substrates was examined using rat liver microsomes. The microsomes were treated with diisopropylphosphorofluoridate to inhibit the hydrolysis of acyl chains from the acyl-linked compounds (1-palmitoyl-sn-glycero-3-phosphocholine and 1-palmitoyl-sn-glycero-3-phosphoethanolamine) and were treated with p-bromophenacyl bromide to block acylation of the compounds tested. In the presence of the inhibitors, 1-alkyl-sn-glycero-3-phosphocholine and 1-alkyl-sn-glycero-3-phosphoethanolamine were hydrolyzed extensively by lysophospholipase D but the corresponding 1-acyl-linked analogs were only negligibly hydrolyzed. Lysophospholipase D therefore appears to be specific for the ether-linked compounds. 1-Alk-1-'-enyl-sn-glycero-3-phosphoethanolamine (lyso plasmalogen) was also tested as a substrate, but a plasmalogenase in the rat liver microsomes rapidly hydrolyzed the compound and we were unable to determine whether it is a substrate for lysophospholipase D. Alkyl-linked substrates containing long-chain acyl groups at the 2-position are not hydrolyzed by the enzymes. We tested 1-alkyl-2-acetoyl-sn-glycero-3-phosphocholine and 1-alkyl-2-acetoyl-sn-glycero-3-phosphoethanolamine to determine if the less bulky, more hydrophilic acetate group would permit hydrolysis by lysophospholipase D; the derivatives did not appear to be attacked, except after hydrolysis of the acetate group. However, in the absence of inhibitors, the acetate groups were rapidly hydrolyzed by microsomal preparations.

Reacylation of platelet activating factor with eicosapentaenoic acid in fish-oil-enriched monkey neutrophils.

Platelet activating factor (PAF) is rapidly metabolized via a deacetylation: reacylation pathway which shows striking specificity for arachidonate at the sn-2 position of the 1-O-alkyl-2-acyl-GPC thus formed. We have now examined the effects of a diet enriched in fish oils on the metabolism of PAF and specificity for arachidonate in the reacylation reaction. [3H]PAF was incubated for various lengths of time with neutrophils from monkeys fed a control diet or one enriched in fish oils. The [3H]PAF added to the cell suspension was rapidly converted to 1-O-alkyl-2-acyl-GPC. Reverse-phase HPLC analysis of the acyl chains added at the sn-2 position revealed that arachidonate was the major fatty acid incorporated into the 1-O-alkyl-2-acyl-GPC formed by neutrophils from monkeys on the control diet. In contrast, both 1-O-alkyl-2-arachidonoyl-GPC and 1-O-alkyl-2-eicosapentaenoyl-GPC were formed by the fish-oil-enriched neutrophils. We also report on the fatty acid composition of neutrophil phospholipids during such a diet.

Comparison of acceptor and donor substrates in the CoA-independent transacylase reaction in human neutrophils.

In human neutrophils (PMN) the ethanolamine-containing phosphoglyceride fraction (PE), principally plasmalogen-linked PE (1-O-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine), is the major store of arachidonic acid (AA). Exogenous AA is initially incorporated into 1-acyl-linked phosphoglycerides and is believed to be transferred into the 1-ether-linked phosphoglycerides via the action of a CoA-independent transacylase (CoA-IT). We have investigated the selectivity for both the "acceptor' lysophospholipids and "donor' AA-containing phospholipid substrates in the CoA-IT reaction. Evidence suggests CoA-IT may also participate in the synthesis of platelet activating factor. The transfer of [3H]AA from endogenously labeled choline-containing phosphoglycerides (PC) to exogenously added alkenyl-lyso-PE (0-50 microM) was examined in saponin-permeabilized PMN. In these "donor' studies, we observed that [3H]AA was transferred from both alkyl- and diacyl-linked PC in a proportional manner. More detailed molecular species analysis showed that [3H]AA was deacylated from all the major AA-containing molecular species in both the alkyl and diacyl subclasses with no selectivity for either subclass. To investigate the "acceptor' selectivity, membrane fractions prelabeled with either [3H]alkyl-arachidonoyl-PE or -PC were utilized as donor substrates. Various unlabeled lysophospholipids (10 microM) were added and the generation of [3H]lyso-PE or -PC was monitored as a measure of CoA-IT activity. Significant subclass preference was observed upon addition of lyso-PE species (1-alkenyl > 1-alkyl > 1-acyl) however, little selectivity was seen with the corresponding lyso-PC species. On the other hand, lysophosphatidylserine, lysophosphatidylinositol, and lysophosphatidic acid all served as poor acceptor substrates in the reaction. These data from PMN are consistent with other evidence that the CoA-IT plays a pivotal role in the enrichment of AA into plasmalogen-linked PE.

Differential actions of diacyl- and alkylacylglycerols in priming phospholipase A2, 5-lipoxygenase and acetyltransferase activation in human neutrophils.

One aspect of human neutrophil (PMN) function during inflammation is formation of platelet-activating factor (PAF), leukotriene B4 (LTB4), and 5-hydroxyeicosatetraenoic acid (5-HETE), but production of these lipid mediators is limited if PMN are directly stimulated with soluble, physiologic agonists. In vitro, PMN activities can be enhanced by the process of primed-stimulation where cells are sequentially treated with non-stimulatory concentrations of different agonists. Many agents that prime PMN also induce production of 1,2-diacyl- and 1-O-alkyl-2-acylglycerols. Therefore, we investigated whether diglycerides were involved in priming PMN for production of lipid mediators. We previously described the ability of the diacylglycerol, 1-oleoyl-2-acetylglycerol (OAG), and its alkylacylglycerol analog, 1-O-octadecenyl-2-acetylglycerol (EAG), to prime phospholipase A2 (PLA2) for subsequent activation by a second stimulus. However, while OAG also primed 5-lipoxygenase activity (LTB4 and 5-HETE production), EAG priming inhibited LTB4 and 5-HETE formation. We now report the effects of diglyceride priming on acetyltransferase activation (PAF formation). PMN, prelabeled with 1-O-[9',10'-3H]hexadecyl-2-lyso-sn-glycero-3-phosphocholine, were primed with OAG or EAG before stimulation. Neither OAG nor EAG induced formation of labeled PAF. Treatment of PMN with the chemotactic peptide, N-formyl-met-leu-phe (FMLP), induced low but significant production of PAF; PAF formation doubled in PMN primed with 20 microM OAG before FMLP stimulation while priming with 20 microM EAG more than tripled the level of PAF. Calcium ionophore strongly induced PAF formation; OAG priming before ionophore challenge had no effect but EAG priming further enhanced PAF formation. These results suggests a role for alkylacylglycerols in modulating the production of lipid mediators of inflammation.

Differential activation of human neutrophil cytosolic phospholipase A2 and secretory phospholipase A2 during priming by 1,2-diacyl- and 1-O-alkyl-2-acylglycerols.

We have shown previously that both 1,2-diacylglycerol (AAG) and 1-O-alkyl-2-acylglycerol (EAG) prime neutrophil release of arachidonic acid via uncharacterized phospholipases A2. Therefore, we investigated the actions of EAG and AAG specifically on neutrophil cytosolic (cPLA2) and secretory (sPLA2) phospholipase A2s. We hypothesized that AAG as a protein kinase activator would activate cPLA2 via phosphorylation events. EAG is antagonistic to the AAG activation of PKC, thus it was not expected to act via phosphorylation of cPLA2. Neutrophils were primed with either AAG or EAG and then stimulated with fMLP. When neutrophils were primed with 5-20 microM 1,2-diacylglycerol, a shift was observed in cPLA2 migration on SDS-PAGE gels, consistent with phosphorylation of the protein. This gel shift was not seen after exposure to EAG. AAG also caused a parallel increase in enzymatic activity of cPLA2 that was not seen with EAG. We also investigated whether either diglyceride would cause similar priming or direct secretion of sPLA2. Both AAG and EAG directly caused significant secretion of neutrophil sPLA2. EAG also increased the release of sPLA2 in cells subsequently stimulated with fMLP. Thus, AAG activated cPLA2 and stimulated secretion of sPLA2. In contrast, EAG did not activate cPLA2, but directly activated secretion of sPLA2. We also demonstrated that human synovial fluid sPLA2 increased AA release from resting and fMLP-stimulated neutrophils. Given that diglycerides prime for release of AA, PAF, and LTB4, these current data support the hypothesis that such priming may be mediated by phosphorylation dependent (cPLA2) or phosphorylation independent (e.g. secretion of sPLA2) events.

Evaluation of phospholipase C and D activity in stimulated human neutrophils using a phosphono analog of choline phosphoglyceride.

A phosphono analog of choline phosphoglyceride was used to examine the relative contributions of phospholipase C and D in the generation of diglycerides in fMLP- and A23187-stimulated human neutrophils. The phosphono analog, 1-O-[3H]alkyl-2-lyso-sn-glycero-3-phosphonocholine, contains a carbon-phosphorus bond adjacent to the base moiety and is resistant to phospholipase D hydrolysis, while remaining susceptible to phospholipase C hydrolysis. fMLP stimulated the production of [3H]phosphatidic acid and subsequently [3H]diglyceride from cells containing 1-O-[3H]alkyl-2-acyl-sn-glycero-3-phosphocholine, but not from cells prelabeled with the phosphono analog. Treatment with A23187 also resulted in the formation of these products from cells containing 1-O-[3H]alkyl-2-acyl-sn-glycero-3-phosphocholine. Additionally, A23187 stimulated the conversion of the phosphono analog to phosphodiester-containing choline phosphoglyceride which then resulted in the generation of [3H]phosphatidic acid and subsequently [3H]diglyceride. This study demonstrates the use of a phosphono analog in assessing phospholipase C and D activity in cells and provides evidence that in fMLP- and A23187-stimulated human neutrophils, diglyceride is generated indirectly from choline phosphoglycerides by the combined activities of phospholipase D and phosphatidate phosphohydrolase.

Evidence for protein-catalyzed transfer of platelet activating factor by macrophage cytosol.

Platelet activating factor (PAF) is a potent, proinflammatory lipid. PAF is synthesized in response to stimuli and is rapidly destroyed by specific acetylhydrolases. In order to express its biological activity, PAF and its metabolites are transported among subcellular membranes by as yet unexplained mechanisms. We report here an assay system using methylcarbamyl-PAF (CPAF, 1-O-hexadecyl-2-O-(N-methylcarbamyl)-sn-glycero-3-phosphocholine) and a vesicle-extrusion technique for examining protein-catalyzed intermembrane transfer of CPAF, and demonstrate the presence of proteins catalyzing the separate transfer of CPAF and diacyl phosphatidylcholine in macrophage cytosol. The CPAF transfer activity is heat- and trypsin-sensitive and elutes from gel-filtration columns well separated from proteins catalyzing the transfer of phosphatidylcholine.

Comparison of 1-O-alkyl-, 1-O-alk-1'-enyl-, and 1-O-acyl-2-acetyl-sn-glycero-3-phosphoethanolamines and -3-phosphocholines as agonists of the platelet-activating factor family.

Four naturally occurring platelet-activating factor (PAF) analogs, 1-alk-1'-enyl-2-acetyl-sn-glycero-3-phosphocholine, 1-hexadecanoyl-2-acetyl-sn-glycero-3-phosphocholine, 1-octadecanoyl-2-acetyl-sn-glycero-3-phosphocholine, and 1-alkyl-2-acetyl-sn-glycero-3-phosphoethanolamine, stimulated human neutrophils (PMN) to mobilize Ca2+, degranulate, and produce superoxide anion. They were, respectively, 5-, 300-, 500-, and 4000-fold weaker than PAF in each assay; inhibited PMN-binding of [3H]PAF at concentrations paralleling their biological potencies; and showed sensitivity to the inhibitory effects of PAF antagonists. PAF and the analogs, moreover, desensitized PMN responses to each other but not to leukotriene B4 and actually increased (or primed) PMN responses to N-formyl-MET-LEU-PHE. Finally, 5-hydroxyeicosatetraenoate-enhanced PMN responses to PAF and the analogs without enhancing the actions of other stimuli. It stereospecifically raised each analog's potency by as much as 100-fold and converted a fifth natural analog, 1-alk-1'-enyl-2-acetyl-sn-glycero-3-phosphoethanolamine from inactive to a weak stimulator of PMN. PAF and its analogs thus represent a structurally diverse family of cell-derived phospholipids which can activate, prime, and desensitize neutrophils by using a common, apparently PAF receptor-dependent mechanism.

Mechanism of arachidonic acid release in human polymorphonuclear leukocytes.

Previous studies have demonstrated that [3H]arachidonic acid is released from prelabeled human neutrophil phospholipids when the cells are stimulated by calcium ionophore A23187 or by opsonized zymosan. Neither lysophospholipid generated by phospholipase A2 activity, diacylglycerol nor monoacylglycerol produced via phospholipase C/diacylglycerol lipase action have been identified following neutrophil challenge. The inability to detect any intermediates during the release of arachidonate is due to either rapid reacylation of lysophospholipid or conversion of diacylglycerol (monoacylglycerol) to cellular acylglycerols. The addition of exogenous [14C]fatty acid at the time of challenge was employed to determine the involvement of either phospholipase A2 or phospholipase C activities. Neutrophil stimulation with calcium ionophore A23187 resulted in an incorporation of exogenous [14C]arachidonate into phosphatidylinositol and phosphatidylcholine, those phospholipids which specifically release arachidonate. When the saturated fatty acid, [14C]stearate, replaced [14C]arachidonate, very little [14C]fatty acid was incorporated into any of the phospholipid species. Lipid phosphorus measurements revealed no significant mass change in any phospholipid class following ionophore challenge. Production of [14C]phosphatidic acid was not detected, as would be expected if diacylglycerol kinase and de novo phospholipid metabolism were significantly involved.

Human neutrophils incorporate arachidonic acid and saturated fatty acids into separate molecular species of phospholipids.

The incorporation of radiolabeled arachidonic acid and saturated fatty acids into choline-linked phosphoglycerides (PC) of rabbit and human neutrophils was investigated by resolving the individual molecular species by reversed-phase high performance liquid chromatography. PC from neutrophils incubated with a mixture of [3H]arachidonic acid and [14C]stearic or [14C]palmitic acid contains both radiolabels; however, double labeling of individual molecular species is minimal. After labeling for 2 h, the [3H]arachidonate is distributed almost equally between diacyl and 1-O-alkyl-2-acyl species, but it is incorporated into diacyl species containing unlabeled stearate or palmitate at the sn-1 position. In contrast, labeled saturated fatty acids are incorporated only into diacyl species and contain predominantly oleate and linoleate at the sn-2 position. Labeled linoleate is not incorporated into ether-linked species, but is found in the same species as labeled stearate. The findings suggest that mechanisms exist in neutrophils for specific shunting of exogenous arachidonic acid into certain phospholipid molecular species and support the concept that the 1-O-alkyl-2-arachidonoyl species may be a functionally segregated pool of arachidonic acid within the PC of neutrophils.

Comparison of alkylacylglycerol vs. diacylglycerol as activators of mitogen-activated protein kinase and cytosolic phospholipase A2 in human neutrophil priming.

In human neutrophils, the choline-containing phosphoglycerides contain almost equal amounts of alkylacyl- and diacyl-linked subclasses. In contrast to phosphatidylinositol hydrolysis which yields diacylglycerol, hydrolysis of choline-containing phosphoglycerides by phospholipase D coupled with phosphohydrolase yields both alkylacyl- and diacylglycerol. While diacylglycerol activates protein kinase C, alkylacylglycerol does not, and its role is unclear. Yet previous studies have shown that exogenous alkylacyl- and diacylglycerols can prime for the release of radiolabeled arachidonic acid (AA) in intact neutrophils stimulated by formyl-methionyl-leucyl-phenylalanine. We have now examined the effects of both diacylglycerol (1-oleoyl-2-acetylglycerol; OAG) and alkylacylglycerol (1-O-hexadecyl-2-acetylglycerol; EAG) on the activation of mitogen-activated protein (MAP) kinase and the 85-kDa cytosolic phospholipase A2 (cPLA2) in human neutrophils. We observed that while OAG could effectively activate p42 and p44 MAP kinases along with cPLA2 in a time- and concentration-dependent manner, EAG could not. A novel p40 MAP kinase isoform is also present and activated in response to OAG treatment; the behavior of this MAP kinase isoform is discussed. The activation of cPLA2 and MAP kinase by 20 microM OAG could be inhibited by pretreatment with 1 microM GF-109203X, a selective inhibitor of protein kinase C. Although only OAG activated cPLA2, both OAG and EAG primed for the release of AA mass as determined by gas chromatography/mass spectrometry. The priming of AA release by OAG may be explained by the phosphorylation of cPLA2 through the activation of protein kinase C linked to MAP kinase. However, priming by EAG appears to involve a separate mechanism that is dependent on a different PLA2. Our results support a role for phospholipase D-derived products modulating the activation of cPLA2, further supporting the idea of cross-talk among various phospholipases.

Comparison of phospholipase D activity in vasopressin- and phorbol ester-stimulated fibroblasts.

Phospholipase D (PLD) activation by vasopressin (VP) was compared to activation by TPA in REF52 cells prelabeled with [3H]glycerol and [14C]myristic acid. Upon VP-treatment, the formation of [3H] and [14C]phosphatidic acid (PA) and phosphatidylethanol (PEt) was accompanied by the loss of radioactivity from PC and PI. However, upon TPA-treatment, radioactivity was lost from PC only. No significant changes of phosphatidylethanolamine and phosphatidylserine were detected in the same samples. The inclusion of 5 microM staurosporine for 10 min diminished the production of [3H]PEt and [14C]PEt by 27% and 53% in VP-treated cells, and by 100% and 75% in TPA-treated cells, respectively. Adding 1 mM EGTA to chelate extracellular Ca2+ inhibited [3H]PEt by approximately 31% and [14C]PEt by 17% after VP-stimulation. In contrast, EGTA had no effect on TPA-stimulation. The data suggest that REF52 cells contain dual PLD activities. The first is stimulated only by VP, requires Ca2+ and hydrolyzes PI. The second is stimulated by both TPA and VP, activated by protein kinase C and hydrolyzes PC.

Receptor-independent metabolism of platelet-activating factor by myelogenous cells.

Human neutrophils incorporate and metabolize platelet-activating factor (PAF). We dissociated these events from PAF binding to its receptors. Cells were pretreated with either pronase, a PAF antagonist (L652731), or excess PAF. This reduced PAF receptor numbers by 70 to almost 100% but had no comparable effect upon the neutrophil's ability to metabolize PAF. Furthermore, HL-60 cells efficiently metabolized, but did not specifically bind, PAF. Thus, PAF receptor availability did not correlate with PAF metabolic capacity and we conclude that myelogenous tissues can process this bioactive ligand by a receptor-independent pathway.

Facile synthesis of platelet-activating factor and racemic analogues containing unsaturation in the sn-1-alkyl chain.

Platelet-activating factor, 1 (PAF, 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine), and octadecyl-PAF were synthesized chemically as the racemates. The sn-1-O-alkyl isomers were isolated after treatment of the racemates with phospholipase A2 and subsequent reacetylation of the 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholines released. Analogues of PAF containing unsaturated alkyl moieties at the sn-1 position (2, 4, 5) were synthesized by utilizing the methoxyethoxymethyl protecting group as a novel method for preparing unsaturated alkyl lipids. This procedure provides a facile means for preparing unsaturated either phospholipids of defined structure that may be tritiated to high radiospecific activity for metabolic studies. Unsaturation in the alkyl chain had minimal effect on the bioactivities examined in this study.

Stereospecific labeling of the glycerol moiety: synthesis of 1,2-dioleoyl-sn-[3-3H]glycero-3-phospho(1-rac-glycerol).

1,2-Dioleoyl-sn-[3-3H]glycero-3-phospho(1-rac-glycerol) was synthesized from 1,2-dioleoyl-sn-glycerol using a new radiosynthetic procedure. 1,2-Dioleoyl-sn-glycerol was oxidized to the corresponding aldehyde using pyridinium dichromate and pyridine. The aldehyde was reduced to the radiolabeled alcohol using tritiated sodium borohydride and crown ether. This material was then converted to the phosphocholine derivative using 2-chloro-2-oxo-1,3,2-dioxaphospholane, followed by displacement with trimethylamine. In the last step, the 1,2-dioleoyl-sn-[3-3H]glycero-3-phosphocholine was converted to 1,2-dioleoyl-sn-[3-3H]glycero-3-phospho-(1-rac-glycerol) via a classic transphosphatidylation reaction using glycerol and cabbage phospholipase D. A theoretical explanation of unusual chemical behavior of the primary alcohol of diglycerides is also given, based on semi-empirical calculations.