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.     

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.     

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.     

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.     

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.     

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.     

Synthesis of 1-acyl-2-[3H]acetyl-SN-glycero-3-phosphocholine, a structural analog of platelet activating factor, by vascular endothelial cells

Human umbilical vein endothelial cells (HUVECS) were challenged with thrombin in the presence of [3H]acetate to stimulate the production of radiolabeled platelet activating factor (PAF, 1-O-alkyl-2-[3H]acetyl-sn-glycero-3-phosphocholine, 1-O-alkyl-2-[3H]acetyl-GPC). The 3H-product was isolated by thin-layer chromatography, and 1-radyl-2[3H],3- diacetylglycerols were prepared by phospholipase C digestion and subsequent acetylation at the sn-3 position. When the 1-radyl-2[3H],3-diacetylglycerols were analyzed by zonal thin-layer chromatography, 96-97% of the radiolabeled derivative migrated with 1-acyl-2,3-diacetylglycerol standard. Only minor amounts (3-4%) of 1-alkyl-2[3H],3-diacetylglycerol were observed, demonstrating that the predominant acetylated product synthesized by thrombin-stimulated HUVECS was 1-acyl-2-[3H]acetyl-GPC. This relative abundance of 1-acyl-2-[3H]-acetyl-GPC was not significantly affected by thrombin dose, incubation time, or cell passage, and was also observed in HUVECS challenged with ionophore A23187. In addition, the acetylated product from ionophore A23187- or bradykinin-stimulated bovine aortic endothelial cells contained 90% 1-acyl-2-[3H]acetyl-GPC, suggesting that the synthesis of the 1-acyl PAF analog is not unique to HUVECS. These findings demonstrate that PAF is a minor synthetic component of HUVECS and bovine aortic endothelial cells. In light of the integral role which the vascular endothelial cell plays in the regulation of thrombosis, these findings also suggest that the production of 1-acyl-2-acetyl-GPC may be biologically important.     

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)     

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.     

Calcium is necessary but not sufficient for the platelet-activating factor release in human neutrophils stimulated by physiological stimuli. Role of G-proteins

Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycerol-3-phosphocholine; PAF) enhances the release of newly synthesized PAF as measured by [3H]acetate incorporation into PAF in human neutrophils. The response was dose-dependent, rapid, transient, and inhibitable by the PAF antagonist BN-52021. The non-metabolizable bioactive PAF analogue (C-PAF) but not lyso-PAF enhances the release of newly synthesized PAF. Newly synthesized PAF was also released after stimulation of these cells with fMet-Leu-Phe. The human granulocyte-macrophage colony-stimulating factor potentiates the stimulated release of PAF. The intracellular calcium chelator BAPTA inhibits the rise of [Ca2+]i and the release of PAF but not the Na+/H+ antiport activity. PAF release, but not the rise in the intracellular concentration of free calcium, was inhibited in pertussis toxin-treated neutrophils stimulated with PAF. The release of PAF in pertussis toxin-treated cells was also inhibited in cells stimulated with fMet-Leu-Phe or opsonized zymosan. These results suggest that functional pertussis toxin-sensitive guanine nucleotide regulatory protein and/or one or more of the changes produced by phospholipase C activation are necessary for PAF release produced by physiological stimuli. It appears that PAF release requires a coordinated action of receptor-coupled G-proteins, calcium, and other parameters.     

An anti-inflammatory protein secreted from the rat seminal vesicle epithelium inhibits the synthesis of platelet-activating factor and the release of arachidonic acid and prostacyclin

Platelet-activating factor (PAF), a 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, is a mediator of inflammation and endotoxic shock produced by a variety of stimulated cells. Since the main biosynthetic pathway of PAF involves acetylation of 1-O-alkyl-sn-glycero-3-phosphocholine (lyso-PAF) generated from 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine by phospholipase A2, we suggest a general physiological role played by steroid-induced anti-(phospholipase A2) proteins in the modulation of PAF synthesis. The results of the present study support this hypothesis since an androgen-induced anti-inflammatory protein, SV-IV, secreted from rat seminal vesicles, inhibits PAF synthesis in stimulated polymorphonuclear neutrophils, macrophages and endothelial cells. SV-IV impairs PAF synthesis by inhibiting the activation of phospholipase A2, that also results in the inhibition of arachidonic acid and prostacyclin release, and of acetyl-CoA:lyso-PAF acetyltransferase.     

Biosynthesis of platelet-activating factor by two-cell mouse embryos.

Incubation of two-cell mouse embryos with a range of radiolabelled compounds resulted in the incorporation of label into platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) in the culture media. The demonstration that known precursors ([1-14C]hexadecanol, [1-3H]hexadecanol, 1-O-[alkyl-1'2'-3H]lyso-PAF, 1-O-[alkyl-1'2'-3H]acetyl-glycerol and [methyl-3H]choline chloride) were incorporated into PAF showed that embryo-derived PAF biosynthesis occurred via pathways present in other PAF-producing cells. The enzyme responsible for the formation of the ether linkage of the PAF molecule, alkyl-dihydroxyacetone-phosphate synthase, was present in the preimplantation embryo as [1-3H]hexadecanol was incorporated into PAF. Incorporation of label from alkylacetyl-glycerol and choline chloride into lyso-PAF was also observed, suggesting a role for lyso-PAF in the metabolism of embryo-derived PAF. Incubation of embryos with each of three [14C]carbohydrate energy substrates resulted in the incorporation of label into PAF in culture media, indicating that the composition of embryo culture media is important in the synthesis of PAF precursors. Incorporation of label from [2-14C]pyruvate was greatest and is consistent with the suggestion that pyruvate is the major energy source at the two-cell stage of development. L-[U-14C]Lactate was also incorporated into embryo-derived PAF, but the mean amount incorporated relative to the concentration of labelled substrate in the medium was 40 times less. The incorporation of D-[U-14C]glucose into PAF was 2405 times less than that from pyruvate, relative to the concentration in the medium.     

Binding of platelet activating factor by isolated membranes from U937 cells

Platelet activating factor (PAF), 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (1-O-alkyl-2-acetyl-GPC) has been known to have biological effect on cells. The mechanisms of the effect of the potent phospholipid on cells has not been established. We have used 1-O-[3H]alkyl-2-acetyl-GPC [( 3H]PAF) to study the interaction on the isolated membranes of U937 cells. The binding process was time, protein concentration, temperature dependent and reversible. The binding of [3H]PAF to the U937 cell membranes was slightly inhibited by the addition of PAF analogue, 3-O-Hexadecyl-2-acetyl-sn-glycerol-1-phosphorylcholine. U937 cell membranes showed high affinity binding sites for PAF with equilibrium dissociation constant (Kd) of 5 x 10(-9) M. The displacement of bound [3H]PAF with 500-fold excess of nonlabeled PAF was not altered suggesting that the bound [3H]PAF was not degraded during the binding. Binding of [3H]PAF on U937 cell membranes was inhibited by PAF antagonist, 59227RP. The kinetic of the inhibition by PAF antagonist is competitive suggesting that PAF and PAF antagonist bind at the same site.     

C20 polyunsaturated fatty acids and phorbol myristate acetate enhance agonist-stimulated synthesis of 1-radyl-2-acetyl-sn-glycero-3-phosphocholine in vascular endothelial cells

This study has investigated the effect of supplementation of vascular endothelial cells with arachidonate and other polyunsaturated fatty acids on the agonist-stimulated synthesis of platelet activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; 1-alkyl-2-acetyl-GPC). Incubation of calf pulmonary artery endothelial cells for 48 h in medium containing 40 microM arachidonate resulted in a 2-3-fold enhancement of [3H]acetate incorporation into 1-radyl-2[3H]acetyl-GPC in response to either bradykinin or calcium ionophore A23187. The effects of arachidonate supplementation were both dose- and time-dependent, requiring a minimum exogenous arachidonate concentration of 2.5 microM and an incubation time of 4-6 h. Eicosapentaenoate and docosahexaenoate also enhanced the synthesis of 1-radyl-2-[3H]acetyl-GPC, but were less potent than arachidonate; alpha-linolenate, linoleate and oleate were without effect. Although not effective as an agonist, phorbol myristate acetate potentiated A23187- and bradykinin-stimulated synthesis of 1-radyl-2-[3H]acetyl-GPC. The effects of arachidonate supplementation were synergistic with potentiation by phorbol myristate acetate. Sphingosine inhibited agonist-stimulated incorporation of [3H]acetate into 1-radyl-2-[3H]acetyl-GPC both in the presence and absence of PMA. Characterization of the radiolabeled material indicated that the primary product was the acyl analogue of PAF (1-acyl-2-acetyl-GPC) rather than PAF. The results from this study suggest that agonist-stimulated synthesis of 1-radyl-2-acetyl-GPC in vascular endothelial cells is modulated both by cellular fatty acyl composition and activation of protein kinase C. Enrichment of vascular endothelial cells with fatty acids, which are mobilized by agonist-stimulated phospholipase A2, may enhance subsequent deacylation of choline phospholipids and, thus, increase synthesis of both 1-acyl-2-acetyl-GPC and PAF.     

Phorbol diester enhances calcium ionophore A23187-induced [3H]acetate incorporation into platelet-activating factor in murine macrophages: predominant incorporation into 1-O-acyl-2-acetyl-sn-glycero-3-phosphocholine

Pretreatment of macrophages with 12-O-tetradecanoylphorbol-13-acetate (TPA) has been shown to enhance the release of arachidonic acid from cell phospholipids in response to agonist stimulation. This study describes the ability of TPA to also alter calcium ionophore A23187-induced incorporation of [3H]acetate into platelet activating factor (PAF). Cultured murine peritoneal macrophages were preincubated with [3H]acetate (25 muCi) and TPA (10 ng/ml) for 10 min, and subsequently incubated with 0.1 microM A23187 for 0.5-10 min. Buffer and cells were then extracted and PAF resolved by normal-phase HPLC. Sequential exposure to TPA and A23187 resulted in a greatly enhanced incorporation (11,861 dpm/10(6) cells) of [3H]acetate into PAF compared to TPA alone, which did not significantly influence [3H]acetate incorporation into PAF, and 0.1 microM A23187, which induced minimal incorporation (688 dpm/10(6) cells). Macrophage-produced [3H]PAF was resolved by HPLC, extracted, treated with phospholipase-C, and acetylated to facilitate quantitation of 1-O-alkyl-2-acetyl-GPC (PAF) from 1-O-acyl-2-acetyl-GPC (acylPAF). A23187 alone (1 microM) produced 72% 1-O-acyl-2-[3H]acetyl-GPC, and A23187 (0.1 microM) following TPA pretreatment produced 81% 1-O-acyl-2-[3H]acetyl-GPC. Less than 2% of the radioactivity of acylPAF was in the acyl moiety. These data support a role for protein kinase C in modulating agonist-induced PAF synthesis. The results also suggest that acetyltransferase of murine macrophages does not possess specificity for 1-O-alkyl-2-lyso-GPC, and that availability of specific species of lyso-phospholipid may determine the type of PAF produced.     

Translocation of exogenous platelet-activating factor and its lyso-compound through plasma membranes is a rate-limiting step for their metabolic conversions into alkylacylglycerophosphocholines in rabbit platelets and guinea-pig leukocytes

Platelets and leukocytes are known to degrade platelet-activating factor (PAF), a potential mediator of inflammation, to its lyso-derivative (lyso-PAF) and then convert this to 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholines. However, little is known about the mechanism of internalization of PAF and lyso-PAF, which is a prerequisite for their metabolism within the cells. In this work, the internalization of PAF and lyso-PAF by rabbit platelet and guinea-pig leukocyte plasma-membranes were examined by the washing method with bovine serum albumin. The rates of translocation of PAF and lyso-PAF across guinea-pig plasma membranes were significantly higher than those across rabbit platelets. In these cells, the translocation of PAF was found to be accelerated indirectly by activation of PAF receptors by a small portion of added PAF. Results suggest that a temperature-dependent diffusion process is involved in the internalization of these phospholipids. In both rabbit platelets and guinea-pig leukocytes, the translocation of PAF and lyso-PAF through the plasma membranes was shown to be rate-limiting for the metabolic conversion of these compounds to 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine.     

Stimulation of platelet-activating factor synthesis by a nonmetabolizable bioactive analog of platelet-activating factor and influence of arachidonic acid metabolites

Platelet-activating factor (PAF) is a potent neutrophil agonist operating through specific receptors located on the cell surface. Binding of PAF to its receptor may also stimulate further PAF synthesis, thus providing a means of amplifying the PAF signal for the cell of origin and/or other responsive cells. In this report we demonstrate that 1-O-alkyl-2-N-methylcarbamyl-sn-glycero-3-phosphocholine (C-PAF), a nonmetabolizable bioactive analog of PAF, stimulates human neutrophils to synthesize PAF, as detected by [3H]acetate incorporation into PAF. This approach allowed us to conclude that [3H]acetate-labeled PAF was formed from endogenous precursor rather than mere turnover of the stimulatory dose of PAF. PAF's ability to initiate further PAF synthesis was confirmed by measuring the PAF-stimulated conversion of 1-O-[3H]alkyl-2-acylglycerophosphocholine to 1-O-[3H]alkyl-2-acetylglycerophosphocholine by prelabeled human neutrophils and by determining the molecular species of 1-O-alkyl-2-[3H]acetylglycerophosphocholine produced by cells stimulated with a single molecular species of PAF (C15:0). Degradation of exogenously added [3H]PAF was not inhibited by C-PAF/5-hydroxyeicosatetraenoic acid treatment. Thus, inhibition of PAF degradation was ruled out as the mechanism accounting for the appearance of labeled PAF in the stimulated cells. Synthesis of PAF in response to C-PAF was not dependent on cytochalasin B pretreatment but was dramatically potentiated by 5-hydroxyeicosatetraenoic acid, which alone was without effect. Additionally, we have demonstrated that another major arachidonate metabolite of neutrophils, leukotriene B4, stimulates PAF production. Thus, at least three products of activated neutrophils, including PAF itself, can promote PAF synthesis by these cells. This positive feedback effect may amplify autacoid production and the final cellular response.     

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.     

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.     

Rabbit blastocysts accumulate platelet-activating factor (PAF) and lyso-PAF in vitro.

Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine; PAF) is a very potent phospholipid, which has been demonstrated to stimulate smooth muscle and change vascular permeability. PAF has been detected in the rabbit preimplantation uterine endometrium and has been demonstrated to bind specifically to rabbit uterine membranes. To evaluate the possible role of PAF in maternal-embryonic chemical communication, we report here that rabbit blastocysts can accumulate [3H]PAF from their environment. Blastocysts were able to accumulate [3H]PAF as time-, buffer-, age-, and concentration-dependent functions. The accumulation was inhibited by some PAF receptor antagonists, such as U66985, as well as by unlabeled PAF and lyso-PAF, indicating that the accumulation process may be receptor mediated. The data support the current model of PAF as a paracrine factor in preimplantation stages of reproduction.