Lyso Platelet-activating Factor Research Articles

Accumulation of alkyl-lysophosphatidylcholines in Niemann-Pick disease type C1

Lysosomal function is impaired in Niemann-Pick disease type C1 (NPC1), a rare and inherited neurodegenerative disorder, resulting in late endosomal/lysosomal accumulation of unesterified cholesterol. The precise pathogenic mechanism of NPC1 remains incompletely understood. In this study, we employed metabolomics to uncover secondary accumulated substances in NPC1. Our findings unveiled a substantial elevation in the levels of three alkyl-lysophosphatidylcholine [alkyl-LPC, also known as lyso-platelet activating factor (PAF)] species in NPC1 compared to controls across various tissues, including brain tissue from individuals with NPC1, liver, spleen, cerebrum, cerebellum, and brain stem from NPC1 mice, as well as in both brain and liver tissue from NPC1 cats. The three elevated alkyl-LPC species were as follows: LPC O-16:0, LPC O-18:1, and LPC O-18:0. However, the levels of PAF 16:0, PAF 18:1, and PAF 18:0 were not altered in NPC1. In the NPC1 feline model, the brain and liver alkyl-LPC levels were reduced following 2-hydroxypropyl-β-cyclodextrin (HPβCD) treatment, suggesting that alkyl-LPCs are secondary storage metabolites in NPC1 disease. Unexpectedly, cerebrospinal fluid (CSF) levels of LPC O-16:0 and LPC O-18:1 were decreased in individuals with NPC1 compared to age-appropriate comparison samples, and their levels were increased in 80% of participants 2 years after intrathecal HPβCD treatment. The fold increases in CSF LPC O-16:0 and LPC O-18:1 levels were more pronounced in responders compared to nonresponders. This observation suggests that LPC O-16:0 and LPC O-18:1 may serve as potential markers for monitoring treatment response in NPC1 patients.

Bioactive Lipid Mediators Predict Platelet Function in Transfusion Recipients: A Phase 1 Randomized Clinical Trial in Healthy Humans

Background: Platelets are stored at room temperature for 5-7 days (RSP) and are transfused to patients who are bleeding or at risk of bleeding. Due to frequent and severe shortages, the FDA recently approved cold-stored platelets in plasma (CSP) for up to 14 days despite a lack of post-transfusion data. It is also unclear which donors are best suited to provide platelets for either RSP or CSP and which in vitro markers accurately predict post-transfusion RSP or CSP platelet function. Objective: To evaluate the post-transfusion function and predictors of post-transfusion function of platelets stored for the maximum approved storage times (7-day RSP, 14-day CSP) in healthy volunteers on acetylsalicylic acid (ASA). Methods: We conducted a randomized cross-over study in ten healthy humans. Subjects on ASA were randomized to receive either autologous RSP or CSP first. We obtained blood from recipients before and after transfusion for platelet function testing and samples from the storage bag before and after storage for lipid metabolite analysis. The first round was followed by a second round with transfusion of the alternative product and the same testing sequence. Results: RSP reversed platelet inhibition significantly better in αIIbβ3 integrin activation-dependent assays. In contrast, CSP led to significantly more thrombin generation in recipients. Overall, RSP contained more pro-inflammatory lipid mediators while CSP had more anti-inflammatory lipid mediators. In particular, LPC species including LPC-O species (formerly known as Lyso-platelet activating factor [LPAF]), LPE, and LPI were significantly more abundant in RSP than in CSP, while LPS was more abundant in CSP than in RSP. Similarly, the relative increase of LPC, LPE, and LPI, and linoleic acid-dependent oxylipins, such as diHOMEs and HODEs were higher in RSP recipients than in CSP recipients. Linoleic acid-dependent oxylipins correlated positively with platelet aggregation in units and recipients of RSP and CSP. Polyunsaturated fatty acids (PUFAs) showed a negative correlation with platelet aggregation in both RSP and CSP units and recipients. Most importantly, LPC-O (LPAF) species showed a strong negative correlation with endogenous thrombin generation potential in CSP units after collection (pre-storage), post-storage, and in transfusion recipients. Conclusion: Results from our trial provide the first efficacy data of autologous and extended-stored CSP in plasma. We report the first correlational data between unit and recipient lipid mediators and post-transfusion platelet function. Taken together, we show that the main mechanism of post-transfusion CSP function appears to be based on procoagulant properties. In the future, testing platelet donors for LPC-O (LPAF) species could identify donors whose platelets are better suited for cold storage than room-temperature storage.

Cytoprotective Effects of Lysophospholipids from Sea Cucumber Holothuria atra.

Lysophospholipids are important signaling molecules in animals and metazoan cells. They are widely distributed among marine invertebrates, where their physiological roles are unknown. Sea cucumbers produce unique lysophospholipids. In this study, two lysophospholipids were detected in Holothuria atra for the first time, lyso-platelet activating factor and lysophosphatidylcholine, with nuclear magnetic resonance and liquid chromatography–time-of-flight mass spectrometric analyses. The lipid fraction of H. atra contained lyso-platelet activating factor and lysophosphatidylcholine, and inhibited H2O2-induced apoptosis in the macrophage cell line J774A.1. The antioxidant activity of the lysophospholipid-containing lipid fraction of H. atra was confirmed with the oxygen radical absorbance capacity method. Our results suggest that the lysophospholipids from H. atra are potential therapeutic agents for the inflammation induced by oxidative stress.

Interaction Of Platelet Activating Factor Acetyl Hydrolase (Paf Ah) Enzyme In Gln281 To Arg281 Mutation Toward Paf And Its Molecular Dynamic

Platelet Activating Factor Acetyl Hydrolase (PAF AH) or LpPLA2 is key enzyme in myocardial infarction catalyzes the sn-2 acetyl group of Platelet Activating Factor (PAF) into lyso PAF and acetate as non-potent inflammatory molecules. PAF AH plays a critical role in arterial plaque development of Coronary Artery Disease (CAD). A crystal structure of PAF AH complexes with other ligand and effects of amino acid alteration to protein plasma consequence have also been reported. Here we report on the result of molecular docking and Molecular dynamic (MD) simulation carried out for PAF AH wild type (WT)/PAF and mutant Q281R/PAF complexes. Docking result shown that amino acid residues on active site of Q281 PAF AH mutant have not recognized on PAF AH. Eelectrostatics and hydrophobic bonds significantly reduced in Q281R than wild type. In the 7500 ps MD simulation Q281R showed less dynamics than WT but enzymatic machinary of mutant Q281R was not interrupted during MD simulation as well as PAF AH wildtype. These findings clearly indicated the importance effect of mutant Q281R in PAF AH recognition to its substrate

Alkylglycerol monooxygenase

Alkylglycerol monooxygenase (E.C. 1.14.16.5), also called glyceryl ether monooxygenase, is a tetrahydrobiopterin-dependent enzyme. It is the only enzyme known to cleave the ether bond of alkylglycerols and lyso-alkylglycerol phospholipids, including lyso-platelet activating factor. Although it has been first described already in 1964, it was not possible so far to purify the protein. It took until 2010 to assign a sequence to this labile integral membrane enzyme by bioinformatic selection of candidate genes, recombinant expression of these, and sensitive monitoring of the enzymatic activity by a fluorescence-based assay. The sequence shows no significant similarity with the other known tetrahydrobiopterin-dependent enzymes but contains the fatty acid hydroxylase protein motif signature. Proteins containing this signature are all labile and catalyze reactions similar to the alkylglycerol monooxygenase reaction. They are thought to use a di-iron centre for catalysis. Site directed mutagenesis of alkylglycerol monooxygenase defined a region of the active site and a conserved glutamate residue important for tetrahydrobiopterin interaction. Current research now focuses on defining a physiological role of this enzyme which occurs not only in mammals but also in commonly used model organisms such as zebrafish and the nematode Caenorhabditis elegans. © 2013 IUBMB Life 65(4):366–372, 2013.

Enzymatic activity of the human 1-acylglycerol-3-phosphate-O-acyltransferase isoform 11: upregulated in breast and cervical cancers

The conversion of lysophosphatidic acid (LPA) to phosphatidic acid is carried out by the microsomal enzymes 1-acylglycerol-3-phosphate-O-acyltransferases (AGPATs). These enzymes are specific for acylating LPA at the sn-2 (carbon 2) position on the glycerol backbone and are important, because they provide substrates for the synthesis of phospholipids and triglycerides. At least, mutations in one isoform, AGPAT2, cause near complete loss of adipose tissue in humans. We cloned a cDNA predicted to be an AGPAT isoform, AGPAT11. This cDNA has been recently identified also as lysophosphatidylcholine acyltransferase 2 (LPCAT2) and lyso platelet-activating factor acetyltransferase. When AGPAT11/LPCAT2/lyso platelet-activating factor acetyltransferase cDNA was expressed in CHO and HeLa cells, the protein product localized to the endoplasmic reticulum. In vitro enzymatic activity using lysates of Human Embryonic Kidney-293 cells infected with recombinant AGPAT11/LPCAT2/lyso platelet-activating factor-acetyltransferase cDNA adenovirus show that the protein has an AGPAT activity but lacks glycerol-3-phosphate acyltransferase enzymatic activity. The AGPAT11 efficiently uses C18:1 LPA as acyl acceptor and C18:1 fatty acid as an acyl donor. Thus, it has similar substrate specificities for LPA and acyl-CoA as shown for AGPAT9 and 10. Expression of AGPAT11 mRNA was significantly upregulated in human breast, cervical, and colorectal cancer tissues, indicating its adjuvant role in the progression of these cancers. Our enzymatic assays strongly suggest that the cDNA previously identified as LPCAT2/lyso platelet-activating factor-acetyltransferase cDNA has AGPAT activity and thus we prefer to identify this clone as AGPAT11 as well.

LPT1 Encodes a Membrane-bound O-Acyltransferase Involved in the Acylation of Lysophospholipids in the Yeast Saccharomyces cerevisiae

Phospholipids are major components of cellular membranes that participate in a range of cellular processes. Phosphatidic acid (PA) is a key molecule in the phospholipid biosynthetic pathway. In Saccharomyces cerevisiae, SLC1 has been identified as the gene encoding lysophosphatidic acid acyltransferase, which catalyzes PA synthesis. However, despite the importance of PA, disruption of SLC1 does not affect cell viability (Nagiec, M. M., Wells, G. B., Lester, R. L., and Dickson, R. C. (1993) J. Biol. Chem. 268, 22156-22163). We originally aimed to identify the acetyl-CoA:lyso platelet-activating factor acetyltransferase (lysoPAF AT) gene in yeast. Screening of a complete set of yeast deletion clones (4741 homozygous diploid clones) revealed a single mutant strain, YOR175c, with a defect in lysoPAF AT activity. YOR175c has been predicted to be a member of the membrane-bound O-acyltransferase superfamily, and we designated the gene LPT1. An Lpt1-green fluorescent protein fusion protein localized at the endoplasmic reticulum. Other than lysoPAF AT activity, Lpt1 catalyzed acyltransferase activity with a wide variety of lysophospholipids as acceptors, including lysophosphatidic acid, lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylglycerol, lysophosphatidylinositol, and lysophosphatidylserine. A liquid chromatography-mass spectrometry analysis indicated that lysophosphatidylcholine and lysophosphatidylethanolamine accumulated in the Deltalpt1 mutant strain. Although the Deltalpt1 mutant strain did not show other detectable defects, the Deltalpt1 Deltaslc1 double mutant strain had a synthetic lethal phenotype. These results indicate that, in concert with Slc1, Lpt1 plays a central role in PA biosynthesis, which is essential for cell viability.

Movement of monoglyceride derived from hydrolysis of fluorescence-labeled lyso platelet-activating factor by lysophospholipase C through plasma membranes of porcine kidney epithelial cell line LLC-PK1

To investigate the mechanisms of the release of lyso platelet-activating factor (PAF), an alkyl ether-linked lysophosphatidylcholine, from the kidney epithelial cell line LLC-PK1, the cell monolayer was incubated with a fluorescence-labeled lysoPAF analog, Bodipy-lysoPAF, on either the basolateral or apical side. The fluorescent lipids in the culture media mixed with or without bovine serum albumin at a final concentration of 2% were analyzed by thin layer chromatography. In both cases, two major bands, assignable to Bodipy-lysoPAF and Bodipy-monoglyceride (MG), were detected in the culture medium to which Bodipy-lysoPAF had been added, whereas the culture medium at the opposite side exhibited only the major band of Bodipy-MG. Our results suggest that lysoPAF was degraded by high ecto-lysophospholipase C activity. The possible physiological significance of this metabolic pathway is discussed.

Protective Effect of the Inhibition of PAF Remodeling and Adhesion Molecule on the Oxidative Stress of the Lungs of Rats Given Endotoxin Intratracheally

Backgraound : There have been many reports on the pathogenesis of sepsis-induced acute respiratory distress syndrome(ARDS) but, the precise mechanism has not been elucidated. This study examined the protective effect of an inhibition of platelet activating factor(PAF) remodeling and the adhesion molecule on the oxidative stress of the lungs in rats with an endotoxin induced acute lung injury(ALI). Methods : ALI was induced in Sprague-Dawley rats by instilling an E-coli endotoxin into the trachea. Ketotifen and fucoidan were used respectively to inhibit PAF remodeling and adhesion molecule. The lung leak index, lung myeloperoxidase(MPO) activity, bronchoalveolar lavage(BAL) fluid neutrophil count and lyso PAF acetyltransferase activity(AT), were measured and an ultrastructural study and cytochemical electron microscopy were performed. Results : The lung leak index, lung MPO activity, BAL fluid neutrophil count and lyso PAF AT activity was higher in the endotoxin-treated rats. In addition, severe destruction of the pulmonary architecture and increased hydrogen peroxide production were identified. These changes were reversed by ketotifen. However, fucoidan did not appear to have any protective effects. Conclusion : The inhibition of PAF remodeling appeared to be effective in decreasing the endotoxin-induced ALI. In addition, this effect might be derived from the inhibition of neutrophilic oxidative stress. However, the inhibition of the adhesion molecules by fucoidan appeared to be ineffective in decreasing the endotoxin-induced ALI.

CDNA Cloning and Expression of Biologically Active Platelet Activating Factor-Acetylhydrolase (PAF-AH) from Bovine Mammary Gland

Platelet activating factor (PAF)-acetylhydrolase (PAF-AH) is an enzyme that hydrolyzes the acetyl ester at the sn-2 position of PAF, and converts it to the inactive metabolite, lyso PAF. This enzyme is distributed widely in the intracellular as well as the extracellular matrix and is believed to be a defense mechanism that protects the host against the toxic effects of PAF and other biologically active oxidized phospholipids. Purification and expression of cDNA cloning of the intracellular and extracellular types of PAF-AH from several sources from different species have been reported. In this study, the cDNA for PAF-AH was cloned by reverse transcription (RT)-PCR from total RNA of bovine mammary gland. The complete amino acid sequences from the cDNA contains 444 amino acids and was identical to that of the PAF-AH isolated from the bovine spleen cDNA library except for two mismatches of amino acid residues (Thr-247 to Met and Ile-431 to Thr). Recombinant PAF-AH was expressed in HEK 293 cells, which exhibited enzyme activity in the in vitro assay system. Furthermore, recombinant bovine PAF-AH was identified by western blot using human plasma PAF-AH antibody as a monomeric polypeptide with a molecular weight of approximately 43 kDa. This protein can be applied to in vivo models to test its protective role against the deleterious PAF actions.

Chromogenic Assay for the Activity of Sphingomyelinase from Bacillus cereus and Its Application to the Enzymatic Hydrolysis of Lysophospholipids

We developed a convenient chromogenic assay method for the activity of sphingomyelinase (SMase) from Bacillus cereus. SMase reaction was quenched by Zn(2+), and the released phosphocholine was converted into a choline by the action of alkaline phosphatase. After that, the choline was converted into a chromogenic dye by the actions of choline oxidase and peroxidase in the presence of EDTA to trap the added Zn(2+) which could interfere with the choline oxidase/peroxidase reactions. Triton X-100 also was added to the reaction mixture, in order to remove turbidity generated from ceramide which had been produced by the SMase reaction. To test a large number of samples in a short period of time, this assay was performed using 96-well microtiter plates. This method proved to be applicable not only to the measurement of the hydrolysis of sphingomyelin but also to those of lysophosphatidylcholine (lysoPC) and lyso platelet-activating factor by B. cereus SMase. Using this method, the kinetic parameters (K(m) and k(cat)) for B. cereus SMase toward various types of substrates were then determined, and the effect of Triton X-100 on the hydrolysis of lysoPC was examined.

JulyNonenzymatic synthesis of glycerolipids catalyzed by imidazole

Imidazole catalyzed acylations of lysolipids by acyl-CoAs in water at room temperature and at a pH close to neutrality. In the presence of oleoyl-CoA and either lysophosphatidylcholine, 1-palmitoyl-sn-glycero-3-phosphocholine (LPC); lysophosphatidylglycerol, monoacyl-sn-glycero-3-phosphoglycerol; lysophosphatidyl acid, 1-oleoyl-sn-glycero-3-phosphate; lysophosphatidylserine, monoacyl-sn-glycero-3-phosphoserin; or lysophosphatidylethanolamine, monoacyl-sn-glycero-3-phosphoethanolamine, the corresponding phospholipids were synthesized. Similarly, the use of lyso-platelet activating factor, an ether analog of LPC, yielded the formation of 1-O-alkyl-2-oleoyl-sn-glycero-3-phosphocholine. In the presence of LPC, an imidazole-catalyzed synthesis of phosphatidylcholine (PC) occurred when medium, long, and very long chain acyl-CoAs were added. With hydroxyacyl-CoA, a similar PC synthesis was obtained.The process described in the present paper appears to offer several potential applications of interest for the synthesis of glycerophospholipids and triglycerides with labeled and/or an unusual or fragile fatty acid, or when suitable acyltransferases have not yet been described in the literature and/or are not commercially available. The method described is very safe and simple since lipids can be synthesized in tubes containing 0.7% imidazole in water, and left for a few hours at room temperature on the bench.

Plasmodium falciparum phospholipase C hydrolyzing sphingomyelin and lysocholinephospholipids is a possible target for malaria chemotherapy.

Sphingomyelinase (SMase) is one of the principal enzymes in sphingomyelin (SM) metabolism. Here, we identified a Plasmodium falciparum gene (PfNSM) encoding a 46-kD protein, the amino acid sequence of which is ∼25% identical to that of bacteria SMases. Biochemical analyses of the recombinant protein GST-PfNSM, a fusion protein of the PfNSM product with glutathione-S-transferase, reveal that this enzyme retained similar characteristics in various aspects to SMase detected in P. falciparum–infected erythrocytes and isolated parasites. In addition, the recombinant protein retains hydrolyzing activity not only of SM but also of lysocholinephospholipids (LCPL) including lysophosphatidylcholine and lysoplatelet-activating factor, indicating that PfNSM encodes SM/LCPL-phospholipase C (PLC). Scyphostatin inhibited SM/LCPL-PLC activities of the PfNSM product as well as the intraerythrocytic proliferation of P. falciparum in a dose-dependent manner with ID50 values for SM/LCPL-PLC activities and the parasite growth at 3–5 μM and ∼7 μM, respectively. Morphological analysis demonstrated most severe impairment in the intraerythrocytic development with the addition of scyphostatin at trophozoite stage than at ring or schizont stages, suggesting its effect specifically on the stage progression from trophozoite to schizont, coinciding with the active transcription of PfNSM gene.

Platelet activating factor degradation in tear fluid from guinea pigs with allergic conjunctivitis.

The purpose of this study was to investigate the role of platelet-activating factor (PAF) and PAF acetylhydrolase (AH) in conjunctiva. The influence of PAF on conjunctival vascular permeability and the presence of PAF or its metabolites in tears from guinea pigs with allergic conjunctivitis were investigated. We instilled PAF to the eyes of guinea pigs and evaluated vascular permeability. Tear samples were collected from passively sensitized guinea pigs, and the concentration of PAF and its metabolites determined by liquid chromatography-tandem mass spectrometry. Exogenous PAF degradation in tear samples was evaluated with or without diisopropyl fluorophosphate (DFP). Topically applied PAF increased vascular permeability in conjunctiva. In the tear samples from guinea pigs with allergic conjunctivitis, PAF could not be detected. However, 40 +/- 6 ng/ml of lyso-platelet activating factor (lyso-PAF) and 230 +/- 50 ng/ml of 1-alkyl-2-acyl-sn-glycero-3-phosphocholine were detected at 10 min after challenge. Exogenous PAF was rapidly degraded in the tear samples from guinea pigs with allergic conjunctivitis, but not from normal guinea pigs. This PAF degradation was inhibited by DFP. These results suggest that PAF in the tear fluid is quickly hydrolyzed to lyso-PAF by PAF AH, which may be released or activated in allergic conjunctivitis.

Phospholipase A 2 sécrétée de type IIA et syndrome inflammatoire

Phospholipase A2 are enzymes that hydrolyse the sn-2 position of glycerophospholipids, and play a pivotal role in the generation of lipid inflammatory mediators. The type IIA secretory phospholipase A2 (sPLA2) is a distal effector of inflammation, produced by a number of cells challenged with inflammatory stimuli such as tumor necrosis factor or interleukin-1. Hydrolysis of the glycerophospholipids generates arachidonic acid, which is implicated in eicosanoid synthesis, and some important lipid mediators such as lyso-platelet activating factor, lysophosphatidic acid (LPA), or lysophosphatidylcholine (LPC). High levels of sPLA2 are found in the plasma of patients suffering from sepsis, septic shock, adult respiratory distress syndrome and multiple organ failure. The levels are correlated to the intensity of the systemic inflammatory response and possibly to the outcome. This review presents important results concerning the pathophysiological implications of sPLA2 in inflammatory disorders. We also present the role of LPA and LPC. The utility of the determination of sPLA2 activity in clinical practice is discussed.

Cloning and expression of rat neutral sphingomyelinase: enzymological characterization and identification of essential histidine residues

Using cross-species sequence homology, we cloned a cDNA for rat neutral sphingomyelinase (nSMase) composed of 422 amino acids that shares 87.6 and 79.0% identity with the mouse and human forms respectively. The rat nSMase expressed in Escherichia coli catalyzed sphingomyelin hydrolysis at neutral pH in a Mg 2+-dependent manner, and required Triton X-100, dithiothreitol, and KCl for its full activity. The cloned rat enzyme shares conserved sequences with nSMases from both eukaryotes and prokaryotes. Introduction of single mutations into either of the histidine residues at positions 136 and 272, putative active sites, entirely abolished the activity, supporting a common mechanism for the nSMase family independent of the species. However, mutation in histidine 151, conserved only in eukaryotes, also abolished the activity, suggesting eukaryote-specific control of nSMase linked to this histidine 151. This enzyme also catalyzed the hydrolysis of lyso-platelet activating factor to yield 1-alkylglycerol at a rate that is slightly lower than that with sphingomyelin.

Effect of the inhibition of PLA2 and PAF on the neutrophilic respiratory burst and apoptosis

Background Since the exact pathogenesis of sepsis-induced ARDS has not been elucidated, the mechanisms of enhanced neutrophilic respiratory burst were probed in endotoxin primed neutrophils associated with the roles of phospholipase A2 (PLA2), platelet activating factor (PAF) and apoptosis. Methods In isolated fresh human neutrophils, effects of the inhibition of PLA2 and PAF on the apoptosis were examined by the method of Annexin-FITC/dual PI flow cytometry. The roles of PLA2 and PAF on the neutrophilic respiratory burst were also examined by measuring oxidant generation in cytochrome-c reduction assay. Activities of the PLA2 and lysoPAF acetyltransferase (lysoPAF AT) of the neutrophils were determined to understand the effect of endotoxin on these enzymatic activities which may be related to the neutrophilic respiratory burst and apoptosis. In addition, the role roles of PLA2 and PAF in neutrophilic adhesion to bovine endothelial cells were examined in vitro by neutrophil adhesion assay. To investigate the effect of oxidants on pulmonary surfactant, cytochemical ultrastructural microscopy was performed. To inhibit PLA2 and PAF, non-specific PLA2 inhibitor mepacrine (100 nM) and WEB 2086 (100 nM) or ketotifen fumarate (10 kg/ml) were used respectively in all in vitro experimental sets. WEB 2086 is PAF receptor antagonist, and ketotifen fumarate is a lyso PAF AT inhibitor. Results The mapacrine treatment, provided after the endotoxin (ETX) treatment, resulted in increased apoptosis of neutrophils (p<0.001) while treatments of WEB 2086 and ketotifen did not. The inhibition of PLA2 and PAF decreased (p<0.001) production of oxidants from PMA-stimulated neutrophils. While endotoxin increased the PLA2 activity of neutrophils (p<0.01), mepacrine supressed (p<0.001) the activity, provided after treatment of ETX. The lyso PAF actyltransferase activity (lyso PAF AT) increased(p<0.01) after treatment of ETX. In contrast, mepacrine, WEB 2086 and ketotifen showed a tendency of decreasing the activity after treatment of ETX. The Treatment of ETX increased (p<0.001) neutrophil adhesion to endothelial cells, which was reversed by inhibition of PLA2 and PAF (p<0.001). The binding of oxidants to pulmonary surfactant was identified histologically. Conclusions The enhanced neutrophilic respiratory burst by ETX plays a pivotal role in the pathogenesis of ARDS in term of oxidayive oxidative stress. Increased production of oxidants form neutrophils is mediated by the activations of PLA2 and lyso PAF AT.

The regulation of CoA-independent transacylation reactions in neuronal nuclei by lysophospholipid, free fatty acid, and lysophospholipase: the control of nuclear lyso platelet-activating factor metabolism.

CoA-independent transacylase activities generating alkylacylglycerophosphocholine (AAGPC) from alkylglycerophosphocholine (1-alkyl GPC) were considerably enriched in neuronal nuclei isolated from rabbit cerebral cortex. Specific nuclear transacylation activities were 13 times the corresponding microsomal values. Several lysophospholipids, notably 1-acyl glycerophosphocholine (1-acyl GPC), 1-alkenyl GPC and 1-alkenyl GPE (1-alkenyl glycerophosphoethanolamine) inhibited the transacylation of 1-alkyl GPC. The inhibitory effects of 1-acyl GPC were seen in the presence of MAFP (methyl arachidonoylfluorophosphonate) or free oleate, compounds that inhibit neuronal nuclear lysophospholipase. When neuronal nuclei were preincubated with 1-alkyl GPC, the radioactive AAGPC product served as donor in transacylation reactions, to generate 1-alkyl GPC. In these nuclear reactions, 1-palmitoyl GPE and 1-palmitoyl GPC appeared to be poor acceptor substrates, when compared with corresponding 1-alkyl and 1-alkenyl analogues. The presence of free oleate or MAFP in the reactions containing 1-acyl GPC boosted the release of 1-alkyl GPC from AAGPC. These observations are of particular relevance to brain ischemia in which lysophospholipid, free fatty acid, and platelet-activating factor (PAF) levels rise dramatically. PAF can be made by the nuclear acetylation of 1-alkyl GPC, which is formed by nuclear transacylation mechanisms. Yet transacylase also removes 1-alkyl GPC, and thus this enzyme activity can regulate 1-alkyl GPC availability. Our observations indicate that lysophospholipids promote the formation of 1-alkyl GPC from nuclear AAGPC via transacylation, while free fatty acid likely prolongs the lifetime of 1-acyl lysophospholipids substrates by lysophospholipase inhibition. Similarly, once 1-alkyl GPC is formed, other lysophospholipids effectively compete with this 1-alkyl analogue and reduce its conversion back to AAGPC by transacylation. Free oleate, in this case, sustains 1-acyl lysophospholipid inhibitors of 1-alkyl GPC transacylation. Thus the cycle of transacylation may favour 1-alkyl GPC formation during ischemia, increasing levels of 1-alkyl GPC for nuclear acetylation reactions and PAF formation. The nuclear generation of PAF is of considerable importance as PAF can play regulatory roles in transcription events associated with inflammation.

MgATP has different inhibitory effects on the use of 1-acyl-lysophosphatidylcholine and lyso platelet-activating factor acceptors by neuronal nuclear acetyltransferase activities

The inhibitory effects of MgATP on neuronal nuclear acetyltransferase activities were studied using lyso platelet-activating factor (lyso-PAF, 1-alkyl- sn-glycero-3-phosphocholine) and lysophosphatidylcholine (lyso-PC, 1-acyl- sn-glycero-3-phosphocholine). The nuclear (N 1) acetylation of lyso-PC was more profoundly inhibited by MgATP. MgATP did not alter the apparent K m for acetyl-CoA in either acetylation reaction. The inhibitory effects of MgATP were not seen for other nucleotides or MgAMP-PCP. Kinase inhibitors such as staurosporine (1 μM), chelerythrine, and R59022 (diglyceride kinase inhibitor I) did not block the MgATP inhibition of either acetylation. However, the addition of phospholipids to the assays indicated a selective inhibitory effect for PIP (25–50 μM) in the nuclear acetylation of lyso-PAF. When N 1 was incubated with [γ- 33P]ATP, phosphatidic acid and PIP were the principal radioactive lipid products. While the extent of MgATP inhibition of lyso-PAF acetylation was similar at different concentrations of lyso-PAF, increasing lyso-PC concentrations greatly decreased the MgATP inhibition seen in lyso-PC acetylations. Nuclear envelopes prepared in the presence of PMSF, and fraction N 1 exposed to PMSF, did not show the inhibitory effect of MgATP on lyso-PC acetylation. PMSF (an inhibitor of certain phospholipase and lysophospholipase activities) did not reduce the MgATP inhibition of lyso-PAF acetylation. Arachidonoyl trifluoromethylketone, an inhibitor of cytosolic phospholipases A 2 and of lysophospholipase activity associated with cPLA 2, also blocked the inhibitory effect of MgATP on lyso-PC acetylation. Using radioactive lyso-PC substrate, fraction N 1 produced labeled free fatty acid and phosphatidylcholine. In the presence of acetyl-CoA, the production of radioactive phosphatidylcholine increased almost 6-fold when MgATP was also included in these incubations. In the presence of MgATP and acetyl-CoA, PMSF reduced the levels of radioactive free fatty acid and phosphatidylcholine derived from lyso-PC, while Triacsin C, an inhibitor of acyl CoA synthetase, decreased phosphatidylcholine labeling. These findings suggest that MgATP inhibition of lyso-PC acetylation results from a loss of lyso-PC substrate that is largely mediated by nuclear lysophospholipase, acyl-CoA synthetase and lyso-PC acylation. Thus the neuronal nuclear production of Acyl PAF may be regulated by paths that compete for the lyso-PC substrate. In contrast, the acetylation of lyso-PAF is inhibited by PIP, a product of nuclear PI kinase reactions.

Production, metabolism and effect of platelet-activating factor on the growth of the human K562 erythroid cell line

The human immature K562 erythroid cell line was studied for its capacity to produce and to metabolize the phospholipid molecule platelet-activating factor (PAF). K562 cells produced PAF under calcium ionophore stimulation. Lyso PAF and acetyl-CoA (the acetate donor molecule for the acetylation of lyso PAF into PAF) had no effect on the amounts of PAF produced by ionophore-stimulated cells. The metabolism of PAF and lyso PAF by K562 cells was compared to that of freshly-isolated human bone marrow erythroblasts and blood erythrocytes. K562 cells rapidly metabolized [ 3 H ]PAF and [ 3 H ]lyso PAF with 1-alkyl analogue of phosphatidylcholine as the major metabolic product. In contrast, blood erythrocytes did not. PAF acetylhydrolase activity levels in K562 cells and bone marrow erythroblasts were similar and higher than in blood erythrocytes. PAF (1–100 nM) stimulated [ 3 H ]thymidine incorporation in K562 cells grown in low serum concentration, a non-metabolizable PAF agonist being more potent than PAF to stimulate thymidine incorporation. PAF receptor mRNA was detected in K562 cells by polymerase chain reaction on reverse transcripts. The present study demonstrates that K562 cells produce and metabolize PAF and underlines the putative role of erythroid precursors in the modulation of bone marrow PAF concentrations. The effect of PAF on the growth of K562 cells might be mediated through PAF receptors suggesting a potential role of PAF on the proliferation and functions of human erythroid marrow precursors.