Acyl and plasmalogen analogs of platelet activating factor--new lipid cellular bioregulators]

Recent data concerning two structural platelet-activating factor (PAF) analogs-1-O-acyl-2-acetyl-sn-glycero-3-phosphocholine (acyl-PAF) and 1-O-alk-1'-enyl-2-acetyl-sn-glycero-3-phosphocholine (vinyl-PAF) identified in some cells and tissues are reviewed. Isolation, identification, biosynthesis, and metabolism of acyl-PAF and vinyl-PAF are considered. The activity of acyl-PAF and vinyl-PAF towards platelets, leukocytes, isolated myocardium, and ileum as well as its in vivo activity are discussed. The influence of acyl-PAF and vinyl-PAF on PAF platelet interaction, Ca2+ mobilization, and platelet adenylate cyclase activity is considered. It is concluded that similar to PAF, acyl-PAF and vinyl-PAF should be regarded as a family of PAF lipid bioregulators.     

Lycopene in association with alpha-tocopherol or tomato lipophilic extracts enhances acyl-platelet-activating factor biosynthesis in endothelial cells during oxidative stress

Lipophilic compounds contained in tomato can prevent cardiovascular diseases by modulating the atherogenic processes in vascular endothelium mediated by oxidized low-density lipoproteins (LDLs). We investigated the effects of lycopene on the metabolism of platelet-activating factor (PAF) and its much less biologically active acyl analog, acyl-PAF, known to prevent LDL oxidation. Lycopene, or lycopene in association with alpha-tocopherol, or whole tomato lipophilic extracts (containing more than 80% lycopene) were used in experiments in which endothelial cells (ECs) are known to synthesize PAF following H(2)O(2)-induced oxidative stress. The results indicated that in each case H(2)O(2)-stimulated PAF biosynthesis in ECs, which is catalyzed by acetyl-CoA acetyltransferase (AT), appeared strongly inhibited. However, acyl-PAF biosynthesis, which also occurs through the PAF-dependent transacetylase (TA), was significantly increased by lycopene only when it was in association with alpha-tocopherol or with the minor compounds present in the whole lipophilic tomato extract. These findings suggest that alpha-tocopherol or lipophilic compounds present in tomato juice potentiate the effects of lycopene on the modulation of PAF and acyl-PAF biosynthesis in ECs during oxidative stress.     

Modulation by flavonoids of PAF and related phospholipids in endothelial cells during oxidative stress

PAF-dependent transacetylase (TA) modifies the functions of platelet-activating factor (PAF), a potent inflammatory lipid, either by transferring the acetyl group from PAF to lysophospholipids (TAL activity), or to sphingosine (TAS activity) or by hydrolyzing PAF (acetylhydrolase activity). In stimulated endothelial cells (EC), TAL activity contributes to the synthesis of acyl-PAF, an acyl analog of PAF, that antagonizes PAF functions and is regulated by the cellular redox state. In this study, we investigated the possible involvement of TA in the flavonoid antioxidant mechanism(s) during oxidative stress in EC induced by hydrogen peroxide. The treatment of EC with H2O2 resulted in 4-fold increase of the acetyl-CoA acetyltransferase activity (AT), that is responsible for PAF biosynthesis, while the TAL activity increased only by 53%. However, the preincubation of H2O2-treated EC with the flavonoids hesperedin, naringin, and quercetin strongly inhibited AT activity and activated TAL by 290%, 340%, and 250%, respectively. The induction of TAL activity resulted in enhanced biosynthesis of 1-acyl-2-[3H]acetyl-PAF in intact EC and was related to the flavonoid structure. These findings suggest that TAL is involved in the flavonoid anti-inflammatory action by enhancing the production of acyl-PAF.     

Differential responsiveness of human neutrophils to the autocrine actions of 1-O-alkyl-homologs and 1-acyl analogs of platelet-activating factor.

The phlogistic actions of six molecular species of platelet-activating factor (PAF) (1-O-alkyl-PAF homologs, 16:0-, 18:0- and 18:1-alkyl-PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC) and their respective 1-acyl-PAF analog counterparts, 16:0-, 18:0- and 18:1-acyl-PAF, 1-acyl-2-acetyl-sn-glycero-3-phosphocholine (AGPC)) were assessed relative to five human neutrophilic polymorphonuclear leukocyte (PMN) functional responses: 1) lysosomal enzyme secretion; 2) specific desensitization to 16:0-AGEPC-induced lysosomal enzyme secretion; 3) O2- production; 4) chemotaxis; and 5) priming for enhanced O2- production. With respect to inducing lysozyme secretion, 18:0-AGEPC was 30- and 75-fold less potent than 16:0-AGEPC and 18:1-AGEPC, respectively, and was 25- and 40-fold less potent for inducing beta-glucuronidase secretion. 18:0-AGEPC was also 10-fold less active than 18:1- and 16:0-AGEPC for inducing O2- production. Thus, the rank order of potency of the alkyl-PAF homologs for inducing both lysosomal enzyme secretion and O2- production was 18:1- greater than or equal to 16:0- much greater than 18:0-AGEPC. In contrast, these three alkyl-PAF homologs had the same potency for desensitizing PMN to subsequent 16:0-AGEPC-induced lysosomal enzyme secretion and for priming PMN for augmented O2- production in response to FMLP or human recombinant C5a. Paradoxically, however, the rank order of potency of the alkyl-PAF homologs for effecting PMN chemotaxis was 18:0- greater than 18:1- much greater than 16:0-AGEPC. At concentrations as high as 1.0 microM, the acyl-PAF analogs did not initiate PMN lysosomal enzyme secretion, O2- production, or chemotaxis. However, the acyl-PAF analogs induced partial PMN desensitization to 16:0-AGEPC. A novel finding of potential (patho)-physiologic significance was the ability of acyl-PAF at nM concentrations to prime PMN for significantly enhanced O2- production after stimulation with FMLP or human recombinant C5a. The priming action of acyl-PAF was due to an increase in the rate as opposed to a prolongation of O2- production. The differing rank orders of potency of the alkyl-PAF homologs and acyl-PAF analogs for stimulating several physiologic responses of the same target cell, the human PMN, support the premise that there may be more than one PAF receptor subtype on the PMN and/or that differences in the biophysical properties of the various molecular species of PAF modulate their interaction with PAF receptor(s) linked to stimulus-response coupling.     

Regulation of the biosynthesis of acyl analogs of platelet-activating factor by purinergic agonist in endothlial cells

We have previously shown that platelet-activating factor (PAF)-dependent transacetylase (TA) contains three catalytic activities, namely PAF: lysophospholipid TA (TAL), PAF: sphingosine TA (TAs) and PAF acetylhydrolase. It serves as a modifier of PAF actions by producing different lipid signal molecules. The TAL activity is involved in the biosynthesis of acyl analogs of PAF (acyl-PAF, 1-acyl-2-acetyl-sn-glycero-3-phosphocholine, acylacetyl-GPC) in agonist-stimulated endothelial cells. In the present investigation, we have studied the mechanism(s) by which the TA activity is regulated in ATP-treated endothelial cells. We have demonstrated that ATP, and thiol-modifying agents with ATP, specifically regulate only the TAL part of the TA activities.     

PAF-Stimulated Protein Tyrosine Phosphorylation in Hippocampus: Involvement of NO Synthase

The effect of platelet-activating factor (PAF) on protein tyrosine phosphorylation was studied in rat hippocampal slices. PAF caused an increase in the tyrosine phosphorylation of two phosphoproteins, which we identified by immunoprecipitation assays as the focal adhesion kinase p125^FAK and crk-associated substrate p130^Cas. The PAF effect was time- and dose-dependent. In addition, the involvement of PAF receptor was demonstrated by using PCA-4248, a specific receptor antagonist. When NO synthase was inhibited by N^G-monomethyl-L-arginine (L-NMA), PAF-stimulated protein tyrosine phosphorylation was inhibited. In conclusion, our results indicate that PAF increased the tyrosine phosphorylation of both p125^FAK and p130^Cas proteins by the production of NO in hippocampus, suggesting that PAF may play a role in the functioning of this cerebral area.     

Effects of platelet-activating factor on single potassium channel currents in guinea pig ventricular myocytes

Platelet-activating factor (PAF) has been implicated as one of the mediators of cardiac anaphylaxis. This phospholipid has been shown to have numerous effects on a variety of tissues, including the heart. Among these effects are alterations in the resting potential and generation of arrhythmias at very low concentrations. This suggests that PAF may modulate the activity of the background, inwardly-rectifying potassium current (I_K1). Thus, the effects of PAF on I_K1 were examined at the single channel level. Ventricular cells were isolated from adult guinea pig hearts and single channel currents recorded from cell-attached patches. PAF had substantial effects on the single channel currents at sub-nanomolar concentrations (10^–11 to 10^–10 M). PAF initially caused flickering of the channels, followed by a gradual prolonged depression of channel activity. Since these potassium channels play a major role in determining the resting potential and excitability of the cardiac cell, the effects of PAF on I_K1 may play a major role in the deleterious electrophysiological actions of PAF on the heart.Abbreviations I_K1 Inwardly-rectifying background potassium current - Lyso-PAF Lyso-platelet-activating factor - PAF Platelet-activating factor     

Effect of platelet-activating factor (PAF) on sodium calcium exchange in cardiac sarcolemmal vesicles

Summary The effects of platelet-activating factor (PAF) on Na⁺-dependent calcium uptake in myocardial sarcolemmal vesicles were examined in order to clarify its mechanism of inotropic action on the heart. PAF (40 and 20 µM) significantly inhibited Na⁺-Ca²⁺ exchange by 61% and 37%, respectively. Both initial rate of exchange and maximal exchange were inhibited. The Km for the reaction was not altered but Vmax was lowered 55% by PAF. Lyso-PAF inhibited Na⁺-Ca²⁺ exchange to a similar degree as PAF. CV-3988, a specific PAF receptor antagonist, failed to diminish the inhibitory effect of PAF on Na⁺-Ca²⁺ exchange, suggesting that the effect of PAF on Na⁺-Ca ²⁺ exchange is not via a receptor mechanism. The passive permeability of sarcolemmal vesicles to Ca²⁺ was markedly elevated after PAF treatment. However, this effect could not account for the decrease in Na⁺-Ca²⁺ exchange. Interestingly, passive Ca²⁺ binding to cardiac sarcolemma was increased by 40 µM PAF. This study indicates that a depression of Na⁺-Ca²⁺ exchange probably does not play a role in the negative inotropic effect of PAF on the myocardium under physiological conditions. Its mechanism of action on Na⁺-Ca²⁺ exchange is discussed.     

Existence of lipid vesicles containing platelet-activating factor in endothelial cell lysate

Platelet aggregation activity due to platelet-activating factor (PAF) was detected at high molecular weight (HMW) and low molecular weight fractions after gel-filtration chromatography of cell lysate of endothelial cells. [³H]PAF added to the cell lysate was similarly distributed after chromatography. The radioactivity associated with HMW fraction was not reduced by digesting the lysate with trypsin, suggesting that PAF was not making complexes with proteins but was included in lipid vesicles in cell lysate. Further evidence showed that an unknown specific factor(s) was needed to form these PAF-containing lipid vesicles. Radioactivity was not found in HMW fraction when [³H]PAF was mixed with cell lysate of vascular smooth muscle cells. When monomeric PAF was added to endothelial cell lysate, the specific activity of aggregation decreased to the level exerted by endogenous PAF-containing lipid vesicles due to incorporation into lipid vesicles. PAF in the form of lipid vesicles was more stable in plasma than monomeric form.     

Presence of platelet-activating factor in squirrel monkey (Saimiri boliviensis) spermatozoa: seasonal differences

Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine) (PAF) is a potent signaling phospholipid which has pleiotropic biological properties in addition to platelet activation. PAF has been detected in the spermatozoa in a number of species. The concentration of PAF is inversely related to human spermatozoal quality. There are no reports on the presence of PAF in nonhuman primate spermatozoa. Therefore, the primary objective of this study was to determine if PAF is present in the spermatozoa from the squirrel monkey (which is a seasonal breeder). A second objective was to determine if PAF levels change from the breeding to the nonbreeding season. Endogenous lipids were extracted from mature Bolivian squirrel monkeys (Saimiri boliviensis) spermatozoa and assayed for the presence of PAF by [¹²⁵I] radioimmunoassay. PAF was detected in all samples assayed. PAF levels were significantly higher (P< 0.01) during the breeding season (mean: 3.58 ng/10⁶ spermatozoa) than the nonbreeding season (mean: 0.76 ng/10⁶ spermatozoa). The data demonstrate that PAF is present in squirrel monkey spermatozoa, with higher levels found during the breeding season. Additional studies are warranted to elucidate the role of PAF in spermatozoa function. Am. J. Primatol. 45:301–305, 1998. © 1998 Wiley-Liss, Inc.     

Autoradiographic localization of platelet-activating factor (PAF) binding sites in the rabbit endometrium during the peri-implantation period

Summary This communication describes the use of in-vivo and in-vitro autoradiography to map specific platelet-activating factor (PAF) receptors in the rabbit uterus. Specific [³H]PAF uptake was predominantly localized on epithelial, but not on stromal or myometrial cells. Very few silver grains were associated with the luminal epithelial cells in the uterus of the estrous rabbit, primarily because of the non-differentiated state of the epithelium. In the differentiated pregnant uterus, significantly more [³H]PAF was bound to the glandular epithelial cells, with the stromal cells binding consistently significantly less. The highest density of silver grains was observed at the implantation sites on day 7 of pregnancy. There was no apparent difference in [³H]PAF C18:0 uptake between the epithelial cells at the inter-implantation zone on day 7 and on day 6. Bound [³H]PAF was displaceable by lyso-PAF, U66985, CV3988, but not U66982, L652,731, SRI 63,441 or the inactive PAF isomer, oleoyl PAF. Bovine serum albumin (BSA) significantly inhibited tissue uptake of [³H]PAF C18:0. Intraluminally administered [³H]PAF C18:0 and intravenously injected [³H]methylcarbamyl-PAF, a non-metabolizable PAF analog, penetrated the implanted blastocyst and bound to the embryoblast. This event was reproducible in vitro with pre-implantation blastocysts from day-6 pregnant rabbits, which suggests that uterine-derived PAF may translocate into the blastocyst after attachment.     

Study on the metabolism of lyso-platelet activating factor (lyso-PAF) in human paranasal sinus mucosa. The cultured ciliated epithelium can convert lyso-PAF to PAF

The conversion of lyso-platelet activating factor (lyso-PAF) to PAF in cultured paranasal sinus mucosa obtained from normal human subjects was studied. The PAF concentration in the medium was determined after addition of lyso-PAF. PAF became detectable at 10 minutes after the addition of 10⁻⁸M lyso-PAF, and reached a maximum concentration (3.25×10⁻⁹M) at 20 minutes. The PAF level then gradually declined to become undetectable at 60 minutes after addition of lyso-PAF. Thus PAF is very unstable having a half-life calculated to be 12.8 minutes with an elimination constant of k=0.05377 minutes⁻¹. In contrast, lyso-PAF is known to be a stable metabolite of PAF as well as a precursor of PAF. The results obtained from this study suggest that the turnover of lyso-PAF to PAF may play a role in evoking prolonged inflammation in target organs or tissues.     

Enhancement of leukotriene D4-induced contraction of guinea-pig isolated trachea by platelet activating factor

The effect of platelet activating factor (PAF), a potent lipid mediator of inflammation, was examined in the induction of airway hyperreactivity to known mediators of anaphylaxis. Concentration-dependent contractions of the isolated guinea-pig trachea to PAF (10⁻⁷ − 10⁻⁵M) were produced and an EC₅₀ value was found to be 7.5 × 10⁻⁷M. Pretreatment for 30 min with a known PAF inhibitor, CV-3988 (10⁻⁵ or 10⁻⁴M), produced significant inhibition of PAF contractions; however, at 10⁻⁶M, CV-3988 had no effect. In the presence of meclofenamic acid (10⁻⁶M), the concentration-response curve to PAF was shifted significantly upward and to the left. This potentiation could be reversed by pretreating the tissues with the peptidoleukotriene antagonists, FPL 55712 or SK&F 102922 (10⁻⁵M). Pretreatment with PAF concentrations having essentially no intrinsic activity (10⁻⁸, 10⁻⁷) significantly enhanced the contraction of guinea-pig trachea to various concentrations of LTD₄ and to certain concentrations of a thromboxane mimic (U-46619). Pretreatment with lyso-PAF failed to potentiate the LTD₄ response, while pretreatment with CV-3988 reverse the potentiation by PAF of the lower concentrations of LTD₄. However, PAF failed to enhance contractions (with or without the presence of meclofenamic acid) to acetylcholine, histamine, PGD₂ or LTC₄ (in the presence of serine borate). These results indicate a possible role for PAF as a mediator of airway hyperreactivity.     

Production and Metabolism of Platelet-Activating Factor in the Normal and Ischemic Fetal Rat Brain

Abstract: Production and metabolism of platelet-activating factor (PAF) in the fetal rat brain under normal and under ischemic stress conditions were examined. Endogenous PAF levels, determined by a bioassay using PAF-stimulated platelet release of [³H]serotonin, averaged 2.32 ± 2.14 pg/mg in control brains and was reduced to 1.10 ± 1.06 pg/mg after 20 min of maternal-fetal blood flow occlusion. [³H]PAF administered intracranially into the fetuses in utero was removed in a biphasic, time-dependent manner: a rapid component with an estimated elimination rate constant of 0.067 min^?1 and t_1/2 of 10 min and a slower component with an elimination rate of 0.017 min^?1 and t_1/2 of 41 min. In fetal brains subjected to ischemia a delayed elimination of [³H]PAF was noticed in the slow component (t_1/2 = 59 min), indicating a possible difference between the clearance of exogenous and endogenous PAF. The disappearance of [³H]PAF was accompanied by an increase in the radioactivity associated with lyso-PAF that reached a plateau after 2.5 min, possibly indicating the degradation of the fast component. A steady increase in the alkyl-acyl-glycerophosphorylcholine radioactivity commenced after 5 min and continued up to 30 min. The endogenous production of PAF and the rapid degradation due to maternal-fetal blood flow occlusion indicate an additional target for therapeutic intervention in the pathology of intrauterine ischemia. Addition of the calcium ionophore A23187 stimulated in vitro formation of PAF and lyso-PAF from [³H]-choline-labeled fetal brain phospholipids, suggesting that intracellular calcium may play a major stimulatory role in PAF production. Degradation of polyphosphoinositides by a phospholipase C may constitute a major target for PAF generated either by decapitation or after blood flow occlusion, as evident from the protective effect of the in vivo administered BN52021 PAF antagonist.     

Saccharomyces cerevisiae Cells Produce Platelet-activating Factor in Response to Calcium Ionophore A23187

Synthesis of platelet-activating factor (PAF) was stimulated by the treatment of cells with the calcium ionophore A23187 in Saccharomyces cerevisiae. The amount of PAF production increased A23187-concentration- and time-dependently. The maximum PAF synthesis was observed when the yeast cells were treated with 2 μm of A23187 for 5 min, and the PAF amount decreased at the higher concentrations of A23187 and/or for the longer incubation time. Treatment of the cells with 1 mm of PMSF (phenylmethyl-sulfonyl fluoride), an inhibitor of PAF catabolism, resulted in transient accumulation of PAF; however, at more than 2 mm of PMSF, PAF production was reduced. Synthesized PAF was not released into the extracellular fraction and was found to be cell-associated. In the absence of A23187 or CaCl₂, and in the presence of EGTA, a Ca²⁺-chelator, no new synthesis of PAF was observed. These results suggested A23187-induced PAF production was Ca²⁺ dependent, and proceed via PAF remodeling pathway. PAF synthesis in response to A23187 was not detectable in the yeast cells in the logarithmic phase, but increased and reached maximum in the stationary phase of the cell growth. These findings suggest that PAF is related to yeast cell growth.     

Presence of Platelet-Activating Factor and Its Receptor in Baboon (Papio spp) Spermatozoa

Platelet-activating factor [1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine; PAF] is a novel potent signaling phospholipid which has unique pleiotropic biological properties in addition to platelet activation. PAF has been detected in the spermatozoa in several species. The concentration of PAF is inversely related to human spermatozoal quality. PAF is present in squirrel monkey (a seasonal breeder) spermatozoa and is significantly higher during the breeding season. PAFs mechanism of action is a receptor-mediated event. There is no report on the presence of PAF or the PAF-receptor in nonhuman Old World primate spermatozoa. Therefore, the primary objective of this study was to determine if PAF is present in the spermatozoa from baboons, which are year-round breeders. A secondary objective was to determine the presence and localization of the PAF-receptor in spermatozoa. We extracted endogenous lipids from mature hybrid baboon (Papio spp) epididymal spermatozoa and assayed them for the presence of PAF by [ ¹²⁵ I]-radioimmunoassay. We also exposed baboon spermatozoa to PAF-receptor antibody followed by FITC-conjugated antibody. PAF was in all samples assayed (mean: 2.29 (±0.63) pmol/10 ⁶ spermatozoa). Baboon spermatozoa possess PAF-receptors most prevalently along the neck and midpiece regions. The data demonstrates that PAF and its receptor are present in baboon spermatozoa. Additional studies will elucidate the role of PAF in spermatozoal function.     

Group A Streptococcus Secreted Esterase Hydrolyzes Platelet-Activating Factor to Impede Neutrophil Recruitment and Facilitate Innate Immune Evasion

The innate immune system is the first line of host defense against invading organisms. Thus, pathogens have developed virulence mechanisms to evade the innate immune system. Here, we report a novel means for inhibition of neutrophil recruitment by Group A Streptococcus (GAS). Deletion of the secreted esterase gene (designated sse) in M1T1 GAS strains with (MGAS5005) and without (MGAS2221) a null covS mutation enhances neutrophil ingress to infection sites in the skin of mice. In trans expression of SsE in MGAS2221 reduces neutrophil recruitment and enhances skin invasion. The sse deletion mutant of MGAS5005 (Δsse ^MGAS5005) is more efficiently cleared from skin than the parent strain. SsE hydrolyzes the sn-2 ester bond of platelet-activating factor (PAF), converting biologically active PAF into inactive lyso-PAF. K_M and k _cat of SsE for hydrolysis of 2-thio-PAF were similar to those of the human plasma PAF acetylhydrolase. Treatment of PAF with SsE abolishes the capacity of PAF to induce activation and chemotaxis of human neutrophils. More importantly, PAF receptor-deficient mice significantly reduce neutrophil infiltration to the site of Δsse ^MGAS5005 infection. These findings identify the first secreted PAF acetylhydrolase of bacterial pathogens and support a novel GAS evasion mechanism that reduces phagocyte recruitment to sites of infection by inactivating PAF, providing a new paradigm for bacterial evasion of neutrophil responses.     

Differential effect of platelet activating factor on 1-methyl-4-phenylpyridinium-induced cell death through regulation of apoptosis-related protein activation

Platelet activating factor (PAF) has been suggested to play a critical role in the pathogenesis of neurological disorders. We assessed the effect of PAF against the toxicity of 1-methyl-4-phenylpyridinium (MPP⁺), a parkinsonian toxin, in relation to apoptotic process. PAF exhibited differential effect against the MPP⁺ toxicity in differentiated PC12 cells depending on concentration. Treatment with 0.75 μM PAF significantly attenuated the MPP⁺-induced increase in Bax levels, decrease in Bid and Bcl-2 levels, and mitochondrial membrane potential loss that lead to the release of cytochrome c and subsequent caspase-3 activation. The inhibitory effect of PAF was not associated with nuclear factor-κB activation. In contrast, PAF at the concentrations greater than 2.5 μM exhibited a toxicity and additive effect on the MPP⁺ toxicity. The results show that PAF at low concentrations, which does not induce a significant toxicity, may prevent the MPP⁺ toxicity by suppressing the apoptosis-related protein activation and mitochondrial membrane permeability change that lead to the cytochrome c release and caspase-3 activation. The preventive effect seems to be associated with the inhibitory effect on the formation of reactive oxygen species and depletion of GSH. In contrast, PAF at higher concentrations may exhibit an additive toxic effect against the MPP⁺ toxicity by increasing apoptosis-related protein activation.     

The effects of platelet-activating factor on flow rate and eicosanoid release in the isolated perfused rat gastric vascular bed

In the isolated rat stomach perfused via the vasculature in situ under constant pressure bolus injections of platelet-activating factor (PAF, 3, 16, or 50 ng) induced dose-dependent, long-lasting reductions of flow rates and simultaneously significant increases in the release of cysteinyl-leukotrienes (cys-LT), thromboxane (TX) B₂ and 6-keto-prostaglandin (PG) F_1α. Reversed phase high pressure liquid chromatography demonstrated the release of a mixture of comparable amounts of LTC₄, LTD₄ and LTE₄ by PAF. Inhibition of cys-LT sythesis by the lipoxygenase inhibitors nordihydroguaiaretic acid (NDGA) and L-651, 896 did not significantly affect PAF-induced flow reduction indicating that endogenous cys-LT are of minor importance for the PAF effect on gastric vascular flow. This conclusion is supported by the fact that the cys-LT receptor antagonist FPL 55712 in a concentration (1 × 10⁻⁶ M) that completely antagonized gastric flow reduction by exogenous LTC₄ (1 × 10⁻⁷ M) had no effect on the PAF-induced reduction of flow. The cyclooxygenase inhibitor indomethacin aggravated the PAF-induced flow reduction suggesting that the endogenous vasodilator PGI₂ might act as a functional PAF antagonist in the rat gastric vascular bed. In contrast to FPL 55712 the PAF antagonist BN 52021 significantly and concentration-dependently antagonized the PAF effect on gastric vascular flow. The results demonstrate that PAF and LTC₄ induce flow reductions in the rat gastric vascular bed by activating different receptors and that endogenous eicosanoids released by PAF do not contribute significantly to the PAF effect on gastric vascular flow.     

Rapid Production of Platelet-activating Factor Is Induced by Protein Kinase Cα-mediated Phosphorylation of Lysophosphatidylcholine Acyltransferase 2 Protein

Platelet-activating factor (PAF), a potent proinflammatory lipid mediator, is synthesized rapidly in response to extracellular stimuli by the activation of acetyl-CoA:lyso-PAF acetyltransferase (lyso-PAFAT). We have reported previously that lyso-PAFAT activity is enhanced in three distinct ways in mouse macrophages: rapid activation (30 s) after PAF stimulation and minutes to hours after LPS stimulation. Lysophosphatidylcholine acyltransferase 2 (LPCAT2) was later identified as a Ca²⁺-dependent lyso-PAFAT. However, the mechanism of rapid lyso-PAFAT activation within 30 s has not been elucidated. Here we show a new signaling pathway for rapid biosynthesis of PAF that is mediated by phosphorylation of LPCAT2 at Ser-34. Stimulation by either PAF or ATP resulted in PKCα-mediated phosphorylation of LPCAT2 to enhance lyso-PAFAT activity and rapid PAF production. Biochemical analyses showed that the phosphorylation of Ser-34 resulted in augmentation of V_max with minimal K_m change. Our results offer an answer for the previously unknown mechanism of rapid PAF production.