Platelet-activating factor (PAF)-dependent transacetylase and its relationship with PAF acetylhydrolases

Platelet-activating factor (PAF)-dependent transacetylase (TA) is an enzyme that transfers an acetyl group from PAF to acceptor lipids such as lysophospholipids and sphingosine. This enzyme is distributed in membrane and cytosol of the cells. We previously revealed that TA purified from rat kidney membrane showed an amino acid sequence similarity to that of bovine PAF-acetylhydrolase (AH) (II). In the present study, we purified TA from the rat kidney cytosol and analyzed its amino acid sequence. The amino acid sequence of the cytosolic TA is similar to that of bovine PAF-AH (II) and membrane TA. To clarify the relationship between TA and PAF-AH (II), we isolated cDNA of rat PAF-AH (II). The predicted amino acid sequence of rat PAF-AH (II) from isolated cDNA included all the sequences found in TAs purified from the membrane and cytosolic TAs. In addition, monoclonal antibody to recombinant PAF-AH (II) cross-reacted with both cytosolic and membrane TAs. Consistent with sequence identity, recombinant PAF-AH (II) showed TA activity, whereas recombinant PAF-AH Ib, which is a different subtype of intracellular PAF-AHs, did not possess TA activity. Analysis of a series of site-directed mutant PAF-AH (II) proteins showed that TA activity was decreased, whereas PAF-AH activity was not affected in C120S and G2A mutant proteins. Thus, Cys(120) and Gly(2) are implicated in the catalysis of TA reaction in this enzyme. Furthermore, the transfer of acetate from PAF to endogenous acceptor lipids was significantly increased in a time-dependent manner in CHO-K1 cells transfected with PAF-AH (II) gene. These results demonstrate that PAF-AH (II) can function, as a TA in intact cells, and PAF-AH (II) and TA are the same enzyme.     

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

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

Release, purification, and characterization of platelet-activating factor (PAF)

Platelet-activating factor (PAF) is a phospholipid mediator, released by basophils, macrophages and neutrophils under immunological and non immunological stimuli. It aggregates platelets and liberates their vasoactive contents. We studied the "spontaneous" release of PAF from hog blood leukocytes : optimal conditions were 22 degrees C, pH 9.5 in BSA and Ca2+-containing Tyrode's. This release was inhibited by the Ca2+-chelating agent, EDTA, and by the phospholipase A2 inhibitor, bromophenacyl bromide. Disruption of the cells did not yield PAF, indicating that it is not a "preformed" mediator. A preparative procedure for the extraction and purification of bulk quantities of PAF was developed. Purification was performed by silicic acid columns followed by high pressure liquid chromatography. The active fraction was eluted between sphingomyelin and lysophosphatidylcholine. The PAF purest fractions were still contaminated with these phospholipids as shown by thin layer chromatography and chemical ionization mass spectrometry. PAF activity was not affected by treatment with diazomethane, acetylation or hydrogenation. Our results combined with those obtained from our previous studies of the PAF structure using specific phospholipases indicate that PAF is a glycero-phospholipid devoid of ester function at position 1. This allowed us to establish precise criteria to distinguish PAF from other aggregating agents.     

Immunofluorescent localization of platelet-activating factor (PAF) in the rat

Summary An immunofluorescent staining method for detecting platelet-activating factor (PAF) is described. This method employs a polyclonal anti-PAF rabbit antibody. When rat brain, heart, lung, liver or kidney tissue was stained using this method, the heart, lung and kidney exhibited PAF-specific staining. Analysis of the amount of PAF in different organs, either by immunofluorescence or by bioassay, showed that kidney tissue contains the greatest amount of PAF.     

Existence of endogenous inhibitors of platelet-activating factor (PAF) with PAF in rat uterus

Two kinds of phospholipids in normal rat uterus were found to inhibit the aggregation of washed rabbit platelets induced by 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (alkylacetyl-GPC) and were named Inhibitor I and Inhibitor II and identified by mass spectrometry. Inhibitor I was a mixture of 1-acyl (16:0, 18:0, 18:1, 18:2, and 20:4)-2-lyso-sn-glycero-3-phosphocholine (acyllyso-GPC) and 1-alkyl (16:0, 18:0, and 18:1)-2-lyso-sn-glycero-3-phosphocholine (alkyllyso-GPC). 16:0 acyllyso-GPC was the most inhibitory, followed by 18:1, 18:2, 20:4, and 18:0 acyllyso-GPCs and 16:0 alkyllyso-GPC. Their IC50 values were in the range of 1-4 X 10(-5) M against the platelet aggregation induced by 1 X 10(-10) M 16:0 alkylacetyl-GPC, indicating that they were about 100 times weaker inhibitors than CV-3988. Inhibitor II was a mixture of N-acyl sphing-4-enyl phosphocholine (18:1/18:0, 18:1/20:0, 18:1/24:0, and 18:1/24:2). The most inhibitory of these components were 18:1/20:0 and 18:1/24:0, followed by 18:1/24:2 and 18:1/18:0, and their IC50 values were in the range of 4-5 X 10(-5) M against platelet aggregation induced by the alkylacetyl-GPC. Quantitatively, about 10(5) times higher concentrations of these inhibitors should be necessary to inhibit platelet aggregation induced by 1 X 10(-10) M 16:0 alkylacetyl-GPC. In fact, the contents of Inhibitors I and II, respectively, were approximately 10(5) times (4.7 X 10(-2) and 7.1 X 10(-2) mol/mol lipid-phosphorus of the original uterine phospholipids) than that of 16:0 alkylacetyl-GPC (1.4 X 10(-6) mol/mol lipid-phosphorus). The role of alkylacetyl-GPC in normal rat uterus is uncertain, but it coexists in situ with two kinds of endogenous inhibitors, choline containing lysoglycerophospholipids and sphingophospholipids.     

Occurrence of platelet-activating factor (PAF) in normal rat stomach and alteration of PAF level by water immersion stress

We detected platelet-activating substance in gastrointestinal areas, which was confirmed to be platelet-activating factor (PAF) on the basis of the following findings: 1) it comigrated with authentic PAF on thin-layer chromatography; 2) it did not aggregate PAF-desensitized platelets; and 3) its activity was completely antagonized by the receptor antagonists CV3988 and L-652,731. The level of PAF was determined with a bioassay method based on the release of [3H]serotonin from washed rabbit platelets. In the normal rat stomach, the level of PAF was high in the antrum (940 +/- 200 nmol PAF/mol phosphorus of original phospholipids), especially in the antral mucosa (1801 +/- 426 nmol/mol phosphorus of original phospholipids). The stomach PAF level was significantly altered by water immersion stress. Stress for a period of 1 h was associated with a decrease in the antral PAF level to 39 +/- 7% of that of untreated controls. This low PAF level persisted during stress. On the other hand, in the corpus, stress for periods of 1 and 3 h was associated with decreases in the PAF content, and further stress (7 h) resulted in restoration of the PAF level to normal. Furthermore, 7 h of stress was associated with distinct hemorrhagic lesions, which were prevented by CV3988 infused i.v. before the stress. This is the first report of an association between a decrease of the endogenous PAF level in animal tissues and tissue damage.     

Purification and characterization of a novel neurotensin-degrading peptidase from rat brain synaptic membranes

A peptidase that cleaved neurotensin at the Pro10-Tyr11 peptide bond, leading to the formation of neurotensin-(1-10) and neurotensin-(11-13), was purified nearly to homogeneity from rat brain synaptic membranes. The enzyme appeared to be monomeric with a molecular weight of about 70,000-75,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high pressure liquid chromatography filtration. Isoelectrofocusing indicated a pI of 5.9-6. The purified peptidase could be classified as a neutral metallopeptidase with respect to its sensitivity to pH and metal chelators. Thiol-blocking agents and acidic and serine protease inhibitors had no effect. Studies with specific peptidase inhibitors clearly indicated that the purified enzyme was distinct from enzymes capable of cleaving neurotensin at the Pro10-Tyr11 bond such as proline endopeptidase and endopeptidase 24-11. The enzyme was also distinct from other neurotensin-degrading peptidases such as angiotensin-converting enzyme and a recently purified rat brain soluble metalloendopeptidase. The peptidase displayed a high affinity for neurotensin (Km = 2.6 microM). Studies on its specificity revealed that neurotensin-(9-13) was the shortest neurotensin partial sequence that was able to fully inhibit [3H]neurotensin degradation. Shortening the C-terminal end of the neurotensin molecule as well as substitutions in positions 8, 9, and 11 by D-amino acids strongly decreased the inhibitory potency of neurotensin. Among 20 natural peptides, only angiotensin I and the neurotensin-related peptides (xenopsin and neuromedin N) were found as potent as unlabeled neurotensin.     

Regulation of platelet-activating factor (PAF) activity in human diseases by phospholipase A2 inhibitors, PAF acetylhydrolases, PAF receptor antagonists and free radical scavengers.

The aim of this review is to present recent findings indicating the likely involvement of platelet-activating factor (PAF) in human diseases, and possible ways of alleviating its harmful effects. PAF is a potent proinflammatory mediator and promotes adhesive interactions between leukocytes and endothelial cells, leading to transendothelial migration of leukocytes, by a process of juxtacrine intercellular signalling. This process leads to activation of leukocytes and the release of reactive oxygen radicals, lipid mediators, cytokines and enzymes. These reaction products subsequently contribute to the pathological features of various inflammatory diseases. The reactive oxygen radicals cause low density lipoprotein (LDL) oxidation which mediates the development of atherosclerosis. Oxidized LDL may damage cellular and subcellular membranes, leading to tissue injury and cell death. Among the therapeutic approaches considered are agents that inhibit/degrade proinflammatory mediators and thereby have anti-inflammatory and/or anti-atherogenic potential. These include inhibitors of phospholipase A2 activity, PAF-acetylhydrolases, PAF antagonists and free radical scavengers/antioxidants, the latter protecting against oxidized LDL-induced cytotoxicity.     

Role of platelet-activating factor (PAF) in the initiation of the decidual reaction in the rat

Injection of PAF into the left uterine horn induced a dose-dependent decidua-like reaction in the pseudopregnant rat. This reaction was maximal when PAF was injected at Day 5 of pseudopregnancy and was blocked by the specific PAF antagonist, BN 52021. BN 52021 did not interfere with the decidual reaction induced by prostaglandin E-2 or insertion of a cotton thread in the uterine horn. In contrast, a decidua-like reaction was not evoked by the inactive lyso-PAF, demonstrating the specificity of the action of PAF. The decidua-like reaction induced by PAF involves the generation of cyclooxygenase metabolites of arachidonic acid since it was inhibited by indomethacin. The histological alterations induced by PAF were similar to those observed after embryo implantation, strengthening the postulate for a role of the autacoid in the early stages of pregnancy.     

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.     

Purification and characterization of galactocerebroside sulfotransferase from rat kidney

Galactocerebroside sulfotransferase (EC 2.8.2.11) was purified to apparent homogeneity from rat kidneys. The purified protein is stable at -20 degrees C, and has an estimated molecular weight of 64,000 and a pI of 5.1. In contrast to other known sulfotransferases, the enzyme appears not to require divalent metal ions for activity. The Km for the donor, 3'-phosphoadenosine 5'-phosphosulfate, is 5.2 microM. Structural studies on this "active" sulfate donor show the requirement of a phosphate group at the 3' position of the ribose moiety. Modification of the amino group at either the 6 or 8 position on the purine ring renders the corresponding compounds poor substrates. Both galactosylceramide and lactosylceramide are effective acceptors for this enzyme, while galactosylsphingosine and galactosylglycerolipids are sulfated only poorly, suggesting that the in vivo sulfation of these glycolipids is carried out by different sulfotransferases. The active site of the enzyme contains arginine residues which appear to be important in binding the sulfate donor. The enzyme protein is hydrophobic and binds 0.17 mg [3H]Triton X-100/mg protein. The purified enzyme contains bound lipids, consisting primarily of cholesterol and phosphatidylcholine. The lipid environment affects the activity of the enzyme which, in turn, regulates the sulfation of glycolipids.     

Activity of platelet-activating factor (PAF) acetylhydrolase in plasma from patients with ischemic cerebrovascular disease

Platelet-activating factor (PAF) is metabolized by a specific enzyme, PAF acetylhydrolase, which may play an important role in the manifestation of the biological activities of PAF in vivo. The activity of PAF acetylhydrolase in plasma of patients with ischemic stroke was higher than that in healthy controls. The incidence of irreversible platelet aggregation in response to PAF, as well as to ADP, was found to be higher in patients than in controls. The patients whose platelets responded with irreversible aggregation to PAF displayed a higher activity of plasma PAF acetylhydrolase than those with only reversible aggregation. In controls, PAF acetylhydrolase activity correlated positively, although weakly, with LDL-cholesterol, which may reflect the major role of LDL in carrying this enzyme. However, since there was no significant difference in plasma levels of lipids and apoproteins between patients and controls (except for apo B) and there was no significant relationship between the enzyme activity and the levels of other lipids and apoproteins, it is unlikely that increased plasma level of PAF acetylhydrolase activity in stroke patients is accounted for by an abnormality of lipoprotein metabolism. Platelet hyperfunction may be associated with augmented generation of PAF, which, in turn, may bring about the induction of the inactivating enzyme, PAF acetylhydrolase.     

Simple and rapid measurement of platelet-activating factor (PAF) in whole blood.

A simple assay of platelet-activating factor (PAF) in whole blood was developed, employing acetone extraction and thin layer chromatography (TLC) purification of blood sample. The activity of acetylhydrolase present in blood sample was almost completely suppressed by ice-cold acetone extraction, and other inhibitory substances interfering the activity of PAF were effectively removed from the acetone extract by TLC. Then, the treated samples were subjected to a conventional PAF bioassay using rabbit platelets. The recovery rate of PAF by the above procedure was constant and feasible (46-48%). The lower limit of the present assay was estimated to be 1.0 x 10(-10) M. Employing the present method, it was able to determine the amount of PAF in blood (1.2-6.0 x 10(-10) M) of 6 out of 14 septic patients, while no significant PAF activity was detected in the samples from 6 healthy subjects. These results indicate a potential application of the present method in the clinical assay of PAF in blood.     

Generation and release of platelet-activating factor (PAF) from enriched preparations of rabbit basophils; failure of human basophils to release PAF

Rabbit mononuclear cells containing up to 20% basophils and uncontaminated by neutrophils release PAF when stimulated with goat antiserum to rabbit IgE. The amount of PAF detected was a function of basophil concentration but decreased on a per cell basis at high basophil or high total cell concentrations. Calcium ionophore A23187, but not protein A, C5a, or FMLP, initiated rabbit basophil degranulation and PAF release. By contrast, extensive studies using a variety of human leukocyte preparations failed to demonstrate the release of significant levels of PAF from human basophils by IgE-dependent or -independent mechanisms. These results suggest that cells other than the peripheral blood basophil (e.g., the neutrophil) may act as the primary site of PAF production in man.     

Modulation of platelet-activating factor (PAF) synthesis and release from human polymorphonuclear leukocytes (PMN): role of extracellular Ca2+

Extracellular Ca2+ regulated the synthesis and release of platelet-activating factor (PAF) from human polymorphonuclear leukocytes (PMN) stimulated with N'-formyl-methionyl-leucyl-phenylalanine (FMLP) in the presence of cytochalasin B. Maximum PAF synthesis and release required the presence of 0.14 mM Ca2+ whereas 1.4 mM Ca2+ was necessary for maximum lysosomal enzyme secretion. The synthesis of PAF occurred within 2.5 min after PMN stimulation in the presence of 1.4 mM Ca2+; however, PAF release did not occur until 5 min after stimulation. Peak PAF release occurred by 7.5 min but accounted for only 30-40% of the total amount of PAF synthesized, the remainder being retained on or within the PMN. Stimulation of PMN in the presence of 0.01 M EDTA or EGTA decreased PAF synthesis and release by greater than 95%. In the absence of extracellular Ca2+, stimulated PMN synthesized PAF in amounts that were 10-30% of maximum, but there was no release of the newly synthesized PAF. At Ca2+ concentrations greater than 0.01 mM, there was a dose-dependent (up to 0.14 mM) increase in PAF synthesis that was associated with the initiation and concomitant increase in the amount of PAF released. These data suggest the presence of a PAF synthesis-release coupling mechanism in which the extracellular Ca2+-dependent release of PAF stimulates additional PAF synthesis.     

Roles of cytosolic phospholipase A(2) and platelet-activating factor receptor in the Ca-induced biosynthesis of PAF

Casein-elicited peritoneal exudate cells (PEC), mainly consisted of neutrophils, were collected from platelet-activating factor receptor-knock-out (PAFR-KO), cytosolic phospholipase A(2) knock-out (cPLA(2)-KO), and wild-type (WT) mice. After stimulation of PEC with calcium ionophore A 23187, PAF levels were measured by radio-ligand binding assay using receptor-rich membrane fraction prepared from the PAF receptor transgenic mice. We found that the level of PAF production by PEC was not different between WT and PAFR-KO mice. On the other hand, cPLA(2)-KO mice were deficient in the PAF production. These results provide the direct evidence while cPLA(2) is essential in the production of PAF, PAF receptor deficiency has little effect on the PAF production.