Evidence that hydrolysis of ethanolamine plasmalogens triggers synthesis of platelet-activating factor via a transacylation reaction

Addition of 1-O-alk-1'-enyl-2-lyso-sn-glycero-3-phosphoethanolamine (alkenyl-lyso-GPE) to human neutrophil membrane preparations containing 1-O-[3H]hexadecyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (1-O-[3H]alkyl-2-arachidonoyl-GPC) resulted in rapid deacylation of the 1-O-[3H]alkyl-2-arachidonoyl-GPC to 1-O-[3H]alkyl-2-lyso-GPC (lyso-platelet-activating factor, lyso-PAF). When acetyl-CoA was included in the incubation mixture, the [3H]lyso-PAF was converted to [3H]PAF. Studies of [3H]arachidonate-labeled neutrophils permeabilized with Staphlococcus aureus alpha-toxin revealed a major shift of labeled [3H]arachidonate from the choline to the ethanolamine-containing phosphoglycerides upon addition of alkenyl-lyso-GPE. The studies indicated that lyso-PAF is formed in the system by the transfer of arachidonate from 1-O-alkyl-2-arachidonoyl-GPC to the alkenyl-lyso-GPE by a CoA-independent transacylase reaction. Mass measurements revealed a rapid loss of arachidonate from 1-radyl-2-acyl-GPE and a concomitant increase in alkenyl-lyso-GPE upon stimulation of the neutrophils by ionophore A23187. Based on these and other findings, a pathway is proposed that may play a significant, if not obligatory, role in the synthesis of PAF in intact stimulated neutrophils. It has been widely accepted that phospholipase A2 acts directly on 1-O-alkyl-2-arachidonoyl-GPC as the first step in the synthesis of PAF via formation of lyso-PAF. In the proposed scheme, phospholipase A2, upon stimulation, acts rapidly on ethanolamine plasmalogen selectively releasing arachidonic acid and generating alkenyl-lyso-GPE. The CoA-independent transacylase then selectively transfers arachidonate from 1-radyl-2-arachidonoyl-GPC to the alkenyl-lyso-GPE generating lyso-PAF, which is then acetylated to form PAF. The interactions outlined can account for the synthesis of 1-acyl-2-acetyl-GPC, 1-O-alk-1'-enyl-2-acetyl-GPE, and eicosanoids, in parallel with PAF.     

Metabolism of platelet-activating factor by rat alveolar macrophages: lyso-PAF as an obligatory intermediate in the formation of alkylarachidonoyl glycerophosphocholine species

1-Alkyl-2-acetyl-sn-glycero-3-phosphocholine (alkylacetyl-GPC; platelet-activating factor; PAF) is actively taken up and metabolized by rat alveolar macrophages maintained in culture. The major metabolic products are lyso-PAF (alkyllyso-GPC) and alkylacyl-GPC. Lyso-PAF accumulates primarily in the media, whereas alkylacyl-GPC is predominantly associated with cellular lipids. The addition of unlabeled lyso-PAF to incubations initiated with [3H]PAF results in an increase in the amount of lyso-[3H]PAF product formed and a decrease in the final product, [3H]alkylacyl-GPC; however, the total amount of [3H]PAF metabolized remains unchanged. Unlabeled lyso-PAF thus enters the metabolic pool of the cell and competes with the deacetylated product of [3H]PAF, i.e., lyso-PAF, for acylation. High-performance liquid chromatography demonstrated that the reacylated product derived from lyso-PAF consisted primarily of the arachidonoyl-containing species that exists as the 16:0-20:4 molecular species. These results document that PAF is inactivated in rat alveolar macrophages via a deacetylation-reacylation reaction with lyso-PAF as an obligatory intermediate. The sequestering of arachidonic acid into the PAF precursor pool and the substantial amount of lyso-PAF secreted by macrophages into the extracellular fluid appear to be significant events in the inactivation process.     

Estimation of platelet-activating factor receptors in the endometrium of the pregnant rabbit: regulation of ligand availability and catabolism by bovine serum albumin.

High affinity receptors have been demonstrated for the potent phospholipid autacoid, platelet-activating factor (PAF C18:0; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine) in a variety of tissues, including the endometrium. Because of the relative instability of PAF and our previous demonstration that lyso-PAF (1-O-alkyl-2-lyso-sn-glycero-3-phosphorylcholine), the major metabolite of PAF, displaced [3H]PAF from endometrial PAF receptor sites, we have examined the ability of bovine serum albumin (BSA) to prevent degradation of PAF and have characterized PAF and lyso-PAF binding sites in purified rabbit endometrial membranes isolated on Day 6 of pregnancy. In buffer containing the phospholipase A2 inhibitors, quinacrine (10 microM) and dibromoacetophenone (2 microM), and 0.25% BSA, 87.4 +/- 3.2% of added [3H]PAF C18:0 remained intact after incubation at 25 degrees C for 150 min. The metabolic products, lyso-PAF and 1-O-alkyl-2-acyl-sn-glycero-3-phosphorylcholine (alkylacyl-GPC), only amounted to 5.2 +/- 3.2 and 3.3 +/- 1.1, respectively. At the same concentration, rabbit serum albumin (RSA) also significantly protected [3H]PAF C18:0 from metabolism, but bovine gamma globulin (BGG) was ineffective. The presence of 0.25% BSA, however, did not protect [3H]lyso-PAF C18:0 from extensive catabolism: the major product formed was [3H]alkylacyl-GPC. Insignificant amounts of [3H]PAF were formed. Under the same conditions (25 degrees C, 150 min) in the presence of 0.25% BSA, saturation analysis revealed the presence of two types of PAF C18:0 receptors in the endometrial membranes. Type 1 sites had a Kd of 0.42 +/- 0.03 nM (mean +/- SD; n = 3) and binding capacity of 0.11 +/- 0.01 pmol/mg protein. Type 2 receptor sites had a Kd of 5.96 +/- 0.35 nM and a binding capacity of 1.59 +/- 0.22 pmol/mg protein. Thus, in the presence of BSA, the binding capacities of the two classes of receptors were markedly reduced compared to values generated previously in its absence. The Kd of the Type 1 sites was not significantly changed by the presence of BSA. A single class of saturable high-affinity binding sites was demonstrable for lyso-PAF C18:0: Kds ranged from 0.76 +/- 0.58 to 11.1 +/- 0.62 nM, depending on which method of analysis was used (Eadie-Hofstee, Scatchard-Rosenthal, or the Lundon nonlinear method). The binding capacities were equally varied, ranging from 0.15 +/- 0.08 to 15.17 +/- 4.95 pmol/mg protein.(ABSTRACT TRUNCATED AT 400 WORDS)     

Human endothelial cells are target for platelet-activating factor. II. Platelet-activating factor induces platelet-activating factor synthesis in human umbilical vein endothelial cells.

Platelet-activating factor (PAF), a phospholipid mediator with broad and potent biologic activities, is synthesized by several inflammatory cells including endothelial cells (EC). PAF is also an effective stimulating agent for EC leading to increased cell permeability and adhesivity. We examined the synthesis of PAF in human umbilical cord vein EC after stimulation of EC with PAF or with its nonmetabolizable analog 1-O-alkyl-2-N-methyl-carbamyl-sn-glycero-3-phosphocholine (C-PAF). PAF (1 to 100 nM) induced a dose- and time-dependent increase of PAF synthesis as detected by [3H]acetate incorporation into PAF fraction. Stimulation of PAF synthesis occurred via activation of the "remodeling pathway" as the 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine (lyso-PAF):acetyl-CoA acetyltransferase was dose-dependently increased after PAF treatment. The de novo pathway of PAF synthesis was not activated under these conditions. C-PAF was able to mimic the effect of authentic PAF on [3H] acetate incorporation. The inactive metabolite lyso-PAF (100 nM) had no influence on PAF synthesis in EC. CV-3988, BN 52021, and WEB 2086, potent and specific antagonists of PAF suppressed PAF effects on the remodeling pathway completely. The PAF- and C-PAF-induced [3H]PAF remained 93% cell-associated and was not degraded up to 10 min after stimulation. Characterization of the [3H]acetate-labeled material co-migrating with authentic PAF revealed that a significant proportion (approximately 57%) was actually 1-acyl-2-acetyl-sn-glycero-3-phosphocholine. PAF-induced PAF synthesis might be an important mechanism for amplifying original PAF signals and potentiating adhesive interactions of circulating cells with the endothelium.     

Stimulation of the de novo pathway for the biosynthesis of platelet-activating factor (PAF) via cytidylyltransferase activation in cells with minimal endogenous PAF production

Treatment of Ehrlich ascites cells with 2 mM oleic acid causes a greater than 10-fold increase in the formation of platelet-activating factor (PAF; 1-[3H]alkyl-2-acetyl-sn-glycero-3-phosphocholine) from the de novo precursor of PAF, 1-[3H]alkyl-2-acetyl-sn-glycerol. Under these conditions, CTP:phosphocholine cytidylyltransferase activity, which is known to catalyze the rate-limiting step in phosphatidylcholine biosynthesis, was stimulated 32% (p less than 0.001) over control cells. Surprisingly, the dithiothreitol-insensitive choline-phosphotransferase activity, which catalyzes the final step in PAF biosynthesis, was reduced approximately 95% in membranes isolated from cells that were pre-treated with 2 mM oleic acid. However, calculations of product formation at this reduced cholinephosphotransferase activity revealed that it was still sufficient to accommodate the increased synthesis of PAF observed in the intact oleic acid-treated cells. Kinetic studies and experiments done with cells treated with phenylmethylsulfonyl fluoride (an acetylhydrolase inhibitor) indicate the various metabolic products formed are derived through the following sequence of reactions: 1-alkyl-2-acetyl-sn-glycerol----1-alkyl-2-acetyl-sn-glycero-3- phosphocholine----1-alkyl-2-lyso-sn-glycero-3-phosphocholine----1-alkyl- 2(long-chain) acyl-sn-glycero-3-phosphocholine. These results indicate PAF is the source of alkylacylglycerophosphocholine through the action of an acetylhydrolase and a transacylase as shown in other cell systems. The relative amounts of PAF, lyso-PAF, and alkylacylglycerophosphocholine produced after treatment of the cells with oleic acid in the absence of the phenylmethylsulfonyl fluoride inhibitor indicate that the acylation rate for lyso-PAF is considerably slower (i.e. rate-limiting) than the deacetylation of PAF by acetylhydrolase. We further conclude that the final step in the de novo pathway for PAF biosynthesis is under the direct control of CTP:phosphocholine cytidylyltransferase, which emphasizes the importance of this regulatory (rate-limiting) step in the biosynthesis of both phosphatidylcholine and PAF.     

A novel CoA-independent transacetylase produces the ethanolamine plasmalogen and acyl analogs of platelet-activating factor (PAF) with PAF as the acetate donor in HL-60 cells.

In this study, we demonstrate the presence of a unique membrane-associated transacetylase that transfers the acetate group from platelet-activating factor (PAF) to lysoplasmalogen (in the presence of EDTA and sodium acetate) with the formation of 1-alk-1-enyl-2-acetyl-sn-glycero-3-phosphoethanolamine (alk-1-enylacetyl-GPE). The identity of alk-1-enylacetyl-GPE was confirmed by acid hydrolysis, phospholipases A2 or C treatment and derivatization by fluorodinitrobenzene. The transacetylase has no requirement for Ca2+, Mg2+, or CoA and a broad pH optimum (7.0-8.0) with Km values of 12.0 microM for PAF and 106.4 microM for lysoplasmalogens. The enzyme activity from the isolated membrane fraction is not changed when whole cells are supplemented with 20:4, induced to differentiate into granulocytes, or treated with ionophore A23187. Radyllyso-sn-glycero-3-phosphocholine (GPC), radyllyso-GPE, acyllyso-sn-glycero-3-phosphoserine (GPS), acyllyso-sn-glycero-3-phosphoinositol (GPI), alkyllyso-sn-glycero-3-phosphate (GP), acyllyso-GP, or cis-9-octadecen-1-ol can also serve as acetate acceptors, whereas alkylglycerol, acylglycerol, or cholesterol are inactive. Differences in substrate acceptor specificity, sensitivity toward phenylmethylsulfonyl fluoride, and response to temperature suggest that the CoA-independent transacetylase and the CoA-independent transacylase that transfers long-chain acyl moieties are two separate enzymes. With intact differentiated HL-60 cells, [3H]acetate from [3H]PAF can be incorporated into alk-1-enylacetyl-GPE in the presence of ionophore A23187, but not in its absence. Moreover, phospholipase A2 inhibitors (p-bromophenacyl bromide and mepacrine) block the transacetylation process in whole cell system. These results indicate the production of alk-1-enyllyso-GPE is a rate-limiting factor for the subsequent transacetylation step during cell activation. We conclude that the transacetylase may participate in the biosynthesis of ethanolamine plasmalogen and acyl analogs of PAF, in vivo, fine-tuning of PAF biological responses, and cross-talk between de novo and remodeling pathways of PAF biosynthesis.     

Biosynthesis and metabolism of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine in rat glomerular mesangial cells

The ability of rat mesangial cells to synthesize 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (1-O-alkyl-2-acetyl-GPC), also known as platelet activating factor (PAF), was studied in mesangial cell cultures originating from isolated rat glomeruli. In response to the phospholipase A2 agonist A23187 mesangial cells synthesized PAF primarily via an acetyltransferase utilizing either [3H]lyso-PAF or [3H]acetate/[3H]acetyl-CoA substrates. The major PAF species synthesized was 1-O-hexadecyl-2-acetyl-GPC. PAF was also synthesized from 1-O-[3H]alkyl-2-acetyl-sn-3-glycerol, indicating the presence of a CDP-cholinephosphotransferase. Mesangial cells incorporated [3H]lyso-PAF to 1-O-[3H]alkyl-2-acyl-GPC. Subsequent stimulation with A23187 (2 microM) resulted in formation and release of [3H]PAF following 3 h, and this was associated with concomitant decrements in intracellular 1-O-[3H]alkyl-2-acyl-GPC and [3H]lyso-PAF levels, indicating a precursor-product relationship among these alkyl ether lipids. Mesangial cells rapidly converted exogenous [3H]PAF to [3H]lyso-PAF and 1-O-[3H]alkyl-2-acyl-GPC, and this process was inhibited by diisopropyl fluorophosphate (10 microM). The demonstration of PAF activation-inactivation pathways in mesangial cells may be of importance in regulating their function and in glomerular injury.     

Subcellular localization of enzyme activities involved in the metabolism of platelet-activating factor in rainbow trout leukocytes

The subcellular distribution of an alkyllyso-GPC: acetyl-CoA acetyltransferase (EC 2.3.1.67) and transacylase, two important enzyme activities involved in the remodeling pathway for the biosynthesis of platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, PAF) have been examined in leukocytes isolated from the pronephros of the rainbow trout, Oncorhynchus mykiss. Contrary to mammalian systems, in which the acetyltransferase is localized to intracellular membranes, the subcellular distribution of an acetyltransferase activity in rainbow trout leukocytes was localized to the plasma membrane. Analysis of the acetyltransferase products by thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC) confirmed synthesis of two subclasses of PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine and 1-acyl-2-acetyl-sn-glycero-3-phosphocholine. The transacylase activity in this study was detected in membrane fractions in two domains of the intermediate density region which also contained the NADH dehydrogenase activity, a marker enzyme for the endoplasmic reticulum. Acylation of lysoPAF (1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine) exhibited approximately 95% specificity for omega-3 fatty acids. Acylation patterns were not significantly different in either domain of the endoplasmic reticulum. A model is proposed herein for the metabolism of PAF in rainbow trout leukocytes.     

Development of a novel scintillation proximity radioimmunoassay for platelet-activating factor measurement: comparison with bioassay and GC/MS techniques

A novel, facile and sensitive scintillation proximity radioimmunoassay (SPRIA) for quantitation of PAF has been developed. No separation of antibody bound [3H]PAF from free [3H]PAF is required as the assay employs protein A - coated fluomicrospheres (beads containing scintillant). The assay system was suitable for the quantitation of 0.03 to 2 pmol of 1-hexadecyl-2-acetyl-sn-glycero-3- phosphocholine. The cross-reactivity was high with 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine but was very low with PAF analogs such as 1-alkyl- and 1-acyl-2-lyso-sn-glycero-3-phosphocholine, 1-acyl-2-acetyl-sn-glycero-3-phosphocholine, and 1-alk-1'-enyl-2-acetyl-sn-glycero-3-phosphocholine. The specificity of SPRIA was higher than that of bioassay (platelet degranulation assay). PAF receptor antagonists (L-652,731, WEB2086, and FR900452) at up to 10 nmol per tube had no affect on the SPRIA. These observations indicate that the specificity of the PAF antibody is quite different from that of the platelet receptor. The values obtained using SPRIA for the measurement of PAF produced in polymorphonuclear leukocytes with stimuli are comparable to those obtained by SIM/GC/MS analysis.     

Metabolism of platelet-activating factor by arachidonic acid-depleted rat polymorphonuclear leukocytes

1-O-[3H]Alkyl-2-acetyl-sn-glycero-3-phosphocholine ([3H]PAF) and 1-O-[3H]alkyl-2-lyso-sn-glycero-3-phosphocholine ([3H]lyso-PAF) when incubated with rat polymorphonuclear leukocytes (PMN) were rapidly metabolized to 1-O-[3H]alkyl-2-acyl-sn-glycero-3-phosphocholine ([3H]alkyl-acyl-GPC) containing long chain acyl groups in the sn-2 position. The specificity and the absolute requirements of arachidonate (20:4) for acylation into PAF and lyso-PAF were investigated by comparing the rate of [3H]PAF and [3H]lyso-PAF metabolism by control rat PMN with that by rat PMN depleted of 20:4. Comparable rates of metabolism of [3H]PAF and [3H]lyso-PAF by both control and 20:4-depleted PMN were observed at all the concentrations of PAF and lyso-PAF studied. The nature of the fatty acyl group incorporated into the sn-2 position of the [3H]alkyl-acyl-GPC formed was analyzed by argentation chromatography. Dienoic fatty acids were the major fatty acid incorporated into the alkyl-acyl-GPC by both control and 20:4-depleted PMN at all the incubation times studied. At 3 min of incubation with [3H]PAF and [3H]lyso-PAF, control PMN had small but significant amounts of [3H]alkyl-acyl-GPC containing tetraenoic fatty acids, the concentration of which gradually increased as the incubation time progressed. On the other hand, under similar conditions, 20:4-depleted PMN had only trace amounts of the [3H]alkyl-acyl-GPC with tetraenoic fatty acid and the concentration of which remained at the low level throughout the incubation time. At 3 min of incubation, the 20:4-depleted PMN had small but significant amounts of [3H]alkyl-acyl-GPC with saturated fatty acids, the amount of which declined by 10 min and remained at that level as the incubation time progressed. While the concentration of [3H]alkyl-acyl-GPC with dienoic fatty acids in the 20:4-depleted cells gradually increased with the progress of incubation time, these molecular species of GPC in the control PMN remained more or less constant. In spite of a very high concentration (equivalent to that of 20:4 in control PMN) of eicosatrienoic acid (20:3 delta 5,8,11) in the 20:4-depleted PMN, no significant amounts of [3H]alkyl-acyl-GPC with trienoic fatty acid were formed by these cells. The rate of metabolism of [3H]PAF and [3H]lyso-PAF by the resident macrophages isolated from control and 20:4-depleted rats was similar.(ABSTRACT TRUNCATED AT 400 WORDS)     

Transacylase-mediated alkylacyl-GPC synthesis and its hydrolysis by phospholipase D occur in separate cell compartments in the human neutrophil

Subcellular localizations of CoA-independent transacylase and phospholipase D enzymes have been investigated in human neutrophils performing a two-step gradient system to separate plasma membranes from internal membranes and from the bulk of granules. The internal membranes were constituted by endoplasmic reticulum and by a subpopulation of specific and tertiary granules. The enzymes activities were assayed in vitro on gradient fractions using exogenous substrates. Following cell prelabelling with [³H]alkyllyso-GPC, we also analyzed the in situ localization of labelled products involving the action of both enzymes. The CoA-independent transacylase activity, together with the CoA-dependent transacylase and acyltransferase activities were only located in the internal membranes. Following 15 min cell labelling, part of the [³H]alkylacyl-GPC was recovered in plasma membranes indicating a rapid redistribution of the acylated compound. Very high contents in arachidonate containing [³H]alkylacyl-GPC were recovered both in plasma membranes and internal membranes. Phospholipase D activity being assayed in the presence of cytosol, GTPγS and gradient fractions, only the plasma membrane fractions from resting or stimulated cells allowed the enzyme to be active. The [³H]alkylacyl-GP and [³H]alkylacyl-GPethanol, phospholipase D breakdown products from [³H]alkylacyl-GPC, obtained after neutrophil prelabelling and activation by phorbol myristate acetate, were exclusively present in the plasma membranes. In contrast, the secondary generated [³H]alkylacylglycerols were equally distributed between plasma and internal membranes. No labelled product was recovered on azurophil granules. These data demonstrate that internal membranes are the site of action of the CoA-independent transacylase and plasma membranes are the site of action of the phospholipase D. This topographical separation between CoA-independent transacylase which generated substrate and phospholipase D which degraded it, suggested that subcellular localisation and traffic of substrates within the cell can be important to regulate the enzymes. © 1996 Wiley-Liss, Inc.     

The role of Ca2+ in regulating the catabolism of PAF-acether (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) in rabbit platelets.

In the present study we have investigated the effect of changes in the concentration of cytosolic free Ca2+ ([Ca2+]i) on the deacetylation-reacylation of PAF-acether (alkylacetylglycerophosphocholine, alkylacetyl-GPC) by rabbit platelets. Washed platelets were incubated with alkyl[3H]acetyl-GPC ([3H]acetyl-PAF) or [3H]alkylacetyl-GPC ([3H]alkyl-PAF) and [Ca2+]i was subsequently elevated by the addition of the ionophore A23187 or thrombin. The catabolism of PAF-acether was studied by measuring the release of [3H]acetate or the formation of [3H]alkylacyl-GPC. The ionophore inhibited the release of [3H]acetate and the formation of [3H]alkylacyl-GPC with no accumulation of lyso-[3H]PAF, indicating that the deacetylation of PAF-acether was blocked. The effect of ionophore on the deacetylation of PAF-acether was parallel with the increase of [Ca2+]i and could be reversed by the addition of EGTA. In contrast with the prolonged inhibition evoked by ionophore, thrombin, which induced a transient elevation of [Ca2+]i, merely delayed the deacetylation of PAF-acether. Since intact platelets failed to convert exogenous lyso-PAF, the effect of Ca2+ on its acylation was investigated by using platelet homogenates. These experiments showed that the acylation of lyso-PAF was inhibited by the exogenously added Ca2+, with a maximum effect at 1 mM. When the formation of endogenous lyso-PAF from the labelled pool of alkylacyl-GPC was examined, a prolonged increase in the concentration of lyso-PAF with a parallel and equally prolonged decrease in the cellular level of alkylacyl-GPC were observed after the addition of ionophore to intact platelets. The addition of EGTA reversed the effect of ionophore, thus permitting reacylation of lyso-PAF. In contrast, only a transient change in the level of lyso-PAF and alkylacyl-GPC was evoked by the addition of thrombin. Therefore we conclude that the inhibitory effect of Ca2+ on the deacetylation-reacylation of PAF-acether may have an important role in the regulation of its biosynthesis.     

Metabolism of platelet activating factor (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) and 1-alkyl-2-acetyl-sn-glycerol by human endothelial cells

The metabolism of platelet activating factor (1-[1,2-3H]alkyl-2-acetyl-sn-glycero-3-phosphocholine) and 1-[1,2-3H]alkyl-2-acetyl-sn-glycerol was studied in cultures of human umbilical vein endothelial cells. Human endothelial cells deacetylated 1-[1,2-3H]alkyl-2-acetyl-sn-glycero-3-phosphocholine to the corresponding lyso compound (1-[1,2-3H]alkyl-2-lyso-sn-glycerol-3-phosphocholine) and a portion was converted to 1-[1,2-3H]alkyl-2-acyl(long-chain)-sn-glycero-3-phosphocholine. Lyso platelet activating factor (lyso-PAF) (1-[1,2-3H]alkyl-2-lyso-sn-glycero-3-phosphocholine) was detected in the media very early during the incubation and the amount remained higher than the level of the lyso product observed in the cells. Cellular levels of 1-[1,2-3H]alkyl-2-lyso-sn-glycero-3-phosphocholine were significantly higher than the acylated product (1-[1,2-3H]alkyl-2-acyl(long-chain)-sn-glycero-3-phosphocholine) at all times during the 60-min incubation period, which suggests that the ratio of acetylhydrolase to acyltransferase activities is greater in endothelial cells than in most other cells. When endothelial cells were incubated with 1-[1,2-3H]alkyl-2-acetyl-sn-glycerol, a known precursor of PAF, 1-[1,2-3H]alkyl-sn-glycerol was the major metabolite formed (greater than 95% of the 3H-labeled metabolites during 20- and 40-min incubations). At least a portion of the acetate was removed from 1-[1,2-3H]alkyl-2-acetyl-sn-glycerol by a hydrolytic factor released from the endothelial cells into the medium during the incubations. Only negligible amounts of the total cellular radioactivity (0.2%) was incorporated into platelet activating factor (1-[1,2-3H]alkyl-2-acetyl-sn-glycero-3-phosphocholine); therefore, it is unlikely that the previously observed hypotensive activity of 1-alkyl-2-acetyl-sn-glycerols can be explained on the basis of the conversion to platelet activating factor (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) by endothelial cells. Results of this investigation indicate that endothelial cells play an important role in PAF catabolism. Undoubtedly, the endothelium is important in the regulation of PAF levels in the vascular system.     

Conversion of alkylacetylglycerol to platelet-activating factor in HL-60 cells and subcellular localization of the mediator

A human promyelocytic leukemia (HL-60) cell line was used to investigate the conversion of 1-alkyl-2-acetyl-sn-glycerol (alkylacetyl-G) to platelet-activating factor (PAF; 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) by intact cells and in subcellular fractions in order to examine the fate of PAF synthesized de novo. Lipid extracts obtained from undifferentiated HL-60 cells incubated with [3H]alkylacetyl-G contained 2-4% of the label as [3H]PAF; several related metabolites were also detected. The yield of [3H]PAF could be dramatically increased by pretreating the cells with either oleic acid, an activator of CTP:phosphocholine cytidylyltransferase, or phenylmethylsulfonyl fluoride, an inhibitor of PAF acetylhydrolase. These results, together with a kinetic study of [3H]alkylacetyl-G metabolism, indicate the sequential participation of a cholinephosphotransferase for the conversion of [3H]-alkylacetyl-G to PAF and acetylhydrolase and transacylase activities in the remodeling pathway that metabolize the newly formed [3H]PAF to 1-[3H]alkyl-2-acyl(long chain)-sn-glycero-3-phosphocholine. The dithiothreitol-insensitive cholinephosphotransferase activity capable of converting alkylacetyl-G to PAF was localized in subcellular fractions that contain CDP-choline:1,2-dioleoyl-sn-glycerol cholinephosphotransferase (dithiothreitol-sensitive), as well as marker enzyme activities for the endoplasmic reticulum and Golgi membranes. Subcellular localization analyses also indicated that the majority of newly formed [3H]PAF and a large portion of its deacetylated metabolite were associated with the plasma membrane-containing fractions, whereas most of the 1-[3H]alkyl-2-acyl(long chain)-sn-glycero-3- phosphocholine was present in the intracellular organelles. Incubations of HL-60 cells with exogenous [3H]PAF produced a similar subcellular distribution of metabolites. Very little (less than 10%) of the [3H]PAF produced from [3H]alkylacetyl-G was released from intact cells under a variety of incubation conditions but 50% of the de novo-derived mediator was recovered in the medium of cells that were permeabilized with saponin. Our results indicate that PAF is rapidly translocated from its intracellular site of enzymatic synthesis to the plasma membrane where it is apparently sequestered in a pool that is not accessible to extracellular acceptors in contact with intact cells.     

Conversion of 1-O-[3H]alkyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine to lyso platelet-activating factor by the CoA-independent transacylase in membrane fractions of human neutrophils

The first step in the synthesis of platelet-activating factor (PAF) in stimulated neutrophils is generally accepted to be hydrolysis of 1-O-alkyl-2-acyl-sn-glycero-3-phosphorylcholine (1-O-alkyl-2-acyl-GPC), with 1-O-alkyl-2-arachidonoyl-GPC being the preferred precursor. Characterization of the enzymatic activity responsible for the hydrolysis of 1-O-alkyl-2-arachidonoyl-GPC has been hampered by lack of an active and reliable cell-free system for study. In the present studies, membrane preparations containing 1-O-[3H]alkyl-2-arachidonoyl-GPC were prepared from intact human neutrophils that had been labeled using 1-O-[3H]hexadecyl-2-lyso-GPC. When the labeled membrane preparations were incubated in the presence of unlabeled 1-O-alkyl-2-lyso-GPC (5 microM), rapid deacylation (up to 25% of the label in 10 min) of the 1-O-[3H]alkyl-2-arachidonoyl-GPC to 1-O-[3H]alkyl-2-lyso-GPC (lyso-PAF) was observed. The deacylation activity appeared to be the same in preparations from resting or stimulated cells. No requirement for Ca2+, various nucleotides, or protein kinase activation could be demonstrated. A number of observations indicated that [3H]lyso-PAF is formed in the system by the action of the CoA-independent transacylase present in the cells rather than by phospholipase A2. Both 1-O-alkyl-2-lyso-GPC and 1-acyl-2-lyso-GPC elicited deacylation of 1-O-[3H]alkyl-2-arachidonoyl-GPC, whereas neither 3-O-alkyl-2-lyso-GPC nor 1-O-alkyl-2-O-methyl-rac-glycero-3-phosphorylcholine, which should act as detergents but are not transacylase substrates, effected deacylation. The deacylation activity and CoA-independent transacylase activities were blocked in parallel by a number of inhibitors and by heat inactivation. In preparations containing 1-O-alkyl-2-[3H]arachidonoyl-GPC, no release of free [3H]arachidonic acid was observed. However, a shift of the [3H]arachidonate into exogenous 1-O-tetradecyl-2-lyso-GPC was observed in the system. These findings are consistent with the generation of [3H]lyso-PAF by the CoA-independent transacylase activity.     

Metabolism of platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) by human promyelocytic leukemic HL60 cells. Stimulated expression of phospholipase A2 and acetyltransferase requires differentiation

Human promyelocytic leukemia (HL60) cells can be induced to differentiate into mature granulocytes by exposure to dimethyl sulfoxide. The addition of N-formylMet-Leu-Phe or the Ca2+ ionophore A23187 to these differentiated cells generated 15-30 pmol of platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (alkylacetyl-GPC)/10(6) cells as quantified by platelet aggregation assays. Under identical conditions, uninduced cells produced little alkylacetyl-GPC. Upon the addition of ionophore A23187, differentiated cells, and not uninduced ones, released [14C]arachidonate from prelabeled phospholipids including ether-linked phosphatidylcholines, formed both 3H-labeled 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine (alkyllyso-GPC) and [3H]alkylacetyl-GPC from endogenous 3H-labeled 1-O-alkyl-2-(long chain) acyl-sn-glycero-3-phosphocholine (alkylacyl-GPC), and incorporated exogenously added [3H]acetate or [3H]alkyllyso-GPC into alkylacetyl-GPC. These results are suggestive that both phospholipase A2 and acetyltransferase activities are involved in alkylacetyl-GPC biosynthesis by HL60 cells and that these activities appear during differentiation. However, when measured in cell extracts, the activities of phospholipase A2 and acetyltransferase of uninduced cells were virtually indistinguishable from those of differentiated cells. Uninduced cells exhibited enhanced incorporation of [3H]alkyllyso-GPC or [3H]alkylacetyl-GPC into alkylacyl-GPC and of [14C]arachidonate and [14C]oleate into various phospholipids including phosphatidylcholine. However, such enhanced expression of acylation reactions could not account for the lack of accumulation of arachidonate or of alkylacetyl-GPC by uninduced cells. Furthermore, analyses of phospholipid classes by phosphorus determination showed no significant alterations in phospholipid composition of HL60 cells during differentiation. Together these data are suggestive that mechanisms regulating the activation of phospholipase A2 and acetyltransferase activities are defective in uninduced cells and that an increased concentration of cytosolic free Ca2+ alone is not a sufficient requirement for these mechanisms.     

Inactivation of platelet activating factor by rabbit platelets. Lyso-platelet activating factor as a key intermediate with phosphatidylcholine as the source of arachidonic acid in its conversion to a tetraenoic acylated product

[3H]PAF (platelet activating factor or 1-alkyl-2-acetyl-GPC) is converted to 1-alkyl-2-lyso-GPC and 1-alkyl-2-acyl-GPC by rabbit platelets (GPC is sn-glycero-3-phosphocholine). The deacetylation reaction does not involve the transfer of the acetate of PAF to any other lipid class and added exogenous lyso-PAF readily mixes with the cellular pool of the [3H]lyso-PAF intermediate formed from [3H]PAF. [3H]1-Alkyl-2-acyl-GPC produced during the inactivation of [3H]PAF contained primarily the tetraenoic acyl species (approximately 80% of the 3H in this fraction). The source of the arachidonic acid used for the reacylation of the lyso-PAF intermediate is the diacyl species, phosphatidylcholine.     

Function of the cloned putative neutral sphingomyelinase as lyso-platelet activating factor-phospholipase C

Sphingolipids such as ceramide and sphingosine have been regarded as novel signal mediators in cells. However, the mechanisms of generation of these lipids upon various stimulation remain to be elucidated. Neutral sphingomyelinase (N-SMase) is one of the key enzymes in the generation of ceramide, and recently the cloning of a putative N-SMase was reported. Because the function of the protein was unclear in the previous report, we investigated the role it plays in cells. N-SMase activity in cells overexpressing the protein with hexa-histidine tag was immunoprecipitated with anti-hexa-histidine antibody. The metabolism of ceramide and SM was not apparently affected in overexpressing cells. Radiolabeling experiments using [(3)H]palmitic acid or [(3)H]hexadecanol demonstrated an accumulation of 1-O-alkyl-sn-glycerol and a corresponding decrease of 1-alkyl-2-acyl-sn-glycero-3-phosphocholine in overexpressing cells. In vitro studies showed that both 1-acyl-2-lyso-sn-glycero-3-phosphocholine (lyso-PC) and 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine (lyso-platelet activating factor (lyso-PAF)) are good substrates of the protein. In further radiolabeling experiments, 1-acyl-lyso-PC was predominantly and equally metabolized into diacyl-PC in both vector and overexpressing cells. On the other hand, 1-O-alkyl-lyso-PC (lyso-PAF) was metabolized into both diradyl-PC and 1-O-alkyl-glycerol in overexpressing cells but only into diradyl-PC in vector cells. These results suggest that the protein acts as lyso-PAF-PLC rather than lyso-PC-PLC or N-SMase in cells.     

Lipoxygenase products of arachidonic acid modulate biosynthesis of platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) by human neutrophils via phospholipase A2

When human neutrophils, previously labeled in their phospholipids with [14C]arachidonate, were stimulated with the Ca2+-ionophore, A23187, plus Ca2+ in the presence of [3H]acetate, these cells released [14C]arachidonate from membrane phospholipids, produced 5-hydroxy-6,8,11,14-[14C]eicosatetraenoic acid (5-HETE) and 14C-labeled 5S,12R-dihydroxy-6-cis,8,10-trans, 14-cis-eicosatetraenoic acid ([14C]leukotriene B4), and incorporated [3H]acetate into platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine). Ionophore A23187-induced formation of these radiolabeled products was greatly augmented by submicromolar concentrations of exogenous 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE), 5-HETE, and leukotriene B4. In the absence of ionophore A23187, these arachidonic acid metabolites were virtually ineffective. Nordihydroguaiaretic acid (NDGA) and several other lipoxygenase/cyclooxygenase inhibitors (butylated hydroxyanisole, 3-amino-1-(3-trifluoromethylphenyl)-2-pyrazoline and 1-phenyl-2-pyrazolidinone) caused parallel inhibition of [14C]arachidonate release and [3H]PAF formation in a dose-dependent manner. Specific cyclooxygenase inhibitors, such as indomethacin and naproxen, did not inhibit but rather slightly augmented the formation of these products. Furthermore, addition of 5-HPETE, 5-HETE, or leukotriene B4 (but not 8-HETE or 15-HETE) to neutrophils caused substantial relief of NDGA inhibition of [3H]PAF formation and [14C]arachidonate release. As opposed to [3H]acetate incorporation into PAF, [3H]lyso-PAF incorporation into PAF by activated neutrophils was little affected by NDGA. In addition, NDGA had no effect on lyso-PAF:acetyl-CoA acetyltransferase as measured in neutrophil homogenate preparations. It is concluded that in activated human neutrophils 5-lipoxygenase products can modulate PAF formation by enhancing the expression of phospholipase A2.     

Metabolic conversion of platelet-activating factor into ethanolamine plasmalogen in an amnion-derived cell line.

Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, PAF) labeled with 3H in the alkyl side chain was taken up rapidly by amnion-derived WISH cells in culture. The radioactivity was found in a number of cellular metabolites, principally 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (alkyl-acyl-GPC) which was labeled at a rapid rate. No intracellular accumulation of lyso-PAF was detected. At longer time periods, a substantial proportion of the radioactivity was found in association with the phosphatidylethanolamine fraction extracted from the cells. This fraction contained a high proportion of the corresponding 1',2'-alkenyl derivative (plasmalogen), as judged by the formation of long-chain fatty aldehyde after exposure to acid. The magnitude of the conversion of PAF into ethanolamine plasmalogen is suggestive of a correlation between plasmalogen content and exposure to PAF in some tissues. The exact sequence of reactions leading from alkyl-acyl-GPC to the ethanolamine derivatives is yet to be established.