The animal ether phospholipid platelet-activating factor stimulates acidification of the incubation medium by cultured soybean cells

Addition of the animal ether phospholipid platelet-activating factor, 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine, (PAF) stimulates medium acidification in cultured soybean (Glycine max L.) cells. The pH of the medium after 8–10 hours is on the average one pH unit lower than in controls. With fusicoccin an average pH difference of 1.7 units is reached. Phospholipids, glycerol, 1-oleyl-2-acetyl-sn-glycerol, 1-0-hexadecyl-sn-glycerol, and triolein at the same concentrations as PAF had no stimulatory effect on medium acidification. The detergents CHAPS and deoxycholate lead to alkalinization of the medium whereas lysophosphatidylcholine (LPC), a detergent with structural similarity to PAF, shows no effect.Abbreviations CHAPS (3-((3-cholamylopropyl) dimethylamino)-1-propanesulfonate) - DOC deoxycholic acid - FC fusicoccin - LPC lysophosphatidylcholine - OAG 1-oleyl-2-acetyl-sn-glycerol - PAF platelet-activating factor = 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine - IAA indole-3-acetic acid     

Comparison of 1-O-alkyl-, 1-O-alk-1'-enyl-, and 1-O-acyl-2-acetyl-sn-glycero-3-phosphoethanolamines and -3-phosphocholines as agonists of the platelet-activating factor family

Four naturally occurring platelet-activating factor (PAF) analogs, 1-alk-1'-enyl-2-acetyl-sn-glycero-3-phosphocholine, 1-hexade-canoyl-2-acetyl-sn-glycero-3-phosphocholine, 1-octadecanoyl-2-acetyl-sn-glycero-3-phosphocholine, and 1-alkyl-2-acetyl-sn-glycero-3-phosphoethanolamine, stimulated human neutrophils (PMN) to mobilize Ca²⁺, degranulate, and produce Superoxide anion. They were, respectively, 5-, 300-, 500-, and 4000-fold weaker than PAF in each assay; inhibited PMN-binding of [³H]PAF at concentrations paralleling their biological potencies; and showed sensitivity to the inhibitory effects of PAF antagonists. PAF and the analogs, moreover, desensitized PMN responses to each other but not to leukotriene B₄ and actually increased (or primed) PMN responses to N-formyl-MET-LEU-PHE. Finally, 5-hydroxyicosatetraenoate-enhanced PMN responses to PAF and the analogs without enhancing the actions of other stimuli. It stereospecifically raised each analog's potency by as much as 100-fold and converted a fifth natural analog, 1-alk-1'-enyl-2-acetyl-sn-glycero-3-phosphoethanolamine from inactive to a weak stimulator of PMN. PAF and its analogs thus represent a structurally diverse family of cell-derived phospholipids which can activate, prime, and desensitize neutrophils by using a common, apparently PAF receptor-dependent mechanism.     

Continuous binding of the PAF molecule to its receptor is necessary for the long-term aggregation of platelets

Human and rabbit platelets fully aggregated byplatelet-activating factor (PAF) underwent slow disaggregation but wererapidly disaggregated by the PAF receptor antagonists WEB-2086,Y-24180, SM-12502, and CV-3988. Whereas the1-alkyl-2-[³H]acetyl-sn-glycero-3-phosphocholine([³H]acetyl-PAF)specifically bound to platelet receptors underwent slow and spontaneousdissociation, it dissociated promptly from its receptor when WEB-2086was added, in parallel with platelet disaggregation and disappearanceof P-selectin on the cell surface. Extracellular[³H]acetyl-PAF wasrapidly deacetylated by normal rabbit platelets; some of the[³H]acetyl-PAF wasbound to the cells and a very small amount of [³H]acetate wasdetected in the cells. In contrast, when1-[³H]alkyl-2-acetyl-sn-glycero-3-phosphocholinewas added to the platelets, the radioactivity was rapidly incorporatedinto the 1-alkyl-2-acyl-sn-glycero-3-phosphocholinefraction. These results indicate that1) continuous binding of PAF to itsreceptor is necessary for prolonged platelet aggregation, which may bemediated through an unknown signaling system for a long-term cellresponse rather than a transient signaling system, and2) most of the[³H]acetyl-PAF boundto platelets is metabolized extracellularly by ecto-type PAFacetylhydrolase, with the lyso-PAF generated being incorporated rapidlyinto the cells and converted to1-alkyl-2-acyl-sn-glycero-3-phosphocholine.

     

Platelet activating factor-acetylhydrolase in insects: Identification and partial characterization of a 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine acetylhydrolase in a cell-free system of Heliothis

A specific acetylhydrolase that inactivates platelet activating factor (PAF; 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine), a potent cellular mediator in mammalian cells, by removal of the sn-2 acetyl moiety, has been found in the cytosolic fraction of several postembryonic developmental stages and specific tissues of the corn earworm, Heliothis zea (Boddie). Effects of magnesium, calcium, EGTA, deoxycholate, dithiothreitol, diisopropylfluorophosphate, egg phosphatidylcholine, and an acylacetyl-glycerophosphocholine show that hydrolysis of the acetate moiety is due to a specific acetylhydrolase for PAF. The activity does not appear to be due to a typical cellular phospholipase A₂ that utilizes phospholipid substrates with a long-chain acyl group at position sn-2 of glycerol. Specific activities and properties of the acetylhydrolase from this insect match closely with those described from tissues of vertebrate animals.     

A plant protein kinase and plant microsomal H+ transport are stimulated by the ether phospholipid platelet-activating factor

ATP-dependent H⁺ transport in microsomes from zucchini hypocotyls is stimulated by the ether lipid 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor = PAF) known as a hormone-like substance from mammals. The stimulation can only be observed when soluble cytosolic proteins are present. A soluble protein mediating the PAF-dependent H⁺ transport and a PAF-stimulated protein kinase are coeluted by DEAE-Sephacel chromatography. Stimulation of phosphorylation by PAF of a 55 kDa polypeptide without Ca²⁺ and, additionally, of a 35 kDa polypeptide in the presence of Ca²⁺ is observed in zucchini microsomal membranes. This is evidence for a novel phospholipid-stimulated protein kinase in plants. Phosphorylation of regulatory proteins may be involved in the stimulation of in vitro H⁺ transport by PAF.Abbreviations BTP 1,3-bis (tris(hydroxymethyl)-methylamino) propane - DTT dithiotreitol - EGTA ethylene glycol-bis (ß-aminoethyl ether)-N,N,N,N,-tetraacetic acid - Mes 2-(N-morpholino) ethanesulfonic acid - PAF platelet-activating factor = 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine - SDS sodium dodecylsulfate - TCA trichloroacetic acid     

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

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

Metabolism of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine by cell cultures

The metabolism of 1-alkyl-2-acetyl-$\text{sn}$-glycero-3-phosphocholine (platelet-activating factor) was studied using various cultured cell lines. All incubations were done in the presence of bovine serum albumin and serum-free media, since albumin eliminates the adsorption of 1-alkyl-2-acetyl-$\text{sn}$-glycero-3-phosphocholine to cultureware and serum enzymes interfere. Human leukemia (HL-60) cells, MDCK canine kidney cells, and transformed and nontransformed clones of mouse C3H1OT1/2 cells display varying rates of uptake, degradation, and capacities for reacylation of 1-alkyl-2-acetyl-$\text{sn}$-glycero-3-phosphocholine. HL-60 cells displayed the highest uptake rate (24.6 pmol/mg cell protein/15 min). Whereas C3H10T1/2 cells in culture showed uptake rates comparable to other cells tested, they displayed a relative metabolic inertness towards 1-alkyl-2-acetyl-$\text{sn}$-glycero-3-phosphocholine.     

Inactivation of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine by a plasma acetylhydrolase: Higher activities in hypertensive rats

We have partially characterized the properties of a specific acetylhydrolase in plasma from spontaneous hypertensive rats. This enzyme inactivates 1-alkyl-2-acetyl-$\text{sn}$-glycero-3-phosphocholine (a lipid involved in platelet aggregating, hypotensive, and allergic responses) by removal of the acetate group. The extent of acetate hydrolysis was linear with both time and protein concentration, and the enzyme had an apparent K_m of 2.5 μM and a V_max of 2.6 nmol/min/mg protein. As with an intracellular acetylhydrolase previously characterized by us, the plasma activity was not affected by addition of phosphatidylcholine, EDTA, or Ca²⁺. However, in contrast to the acetylhydrolase activity in the rat kidney soluble fraction, the plasma activity was associated with a higher molecular weight protein resolved on a Sepharose 6B column and the plasma acetylhydrolase was not inhibited by treatment with trypsin, pronase, or subtilisin. We also compared the acetylhydrolase activity in plasma of age-matched spontaneous hypertensive rats and their normotensive controls, and found approximately 20% higher levels of activity in plasma from the hypertensive animals ($\text{P}$ <0.01).     

Activities of enzymes that metabolize platelet-activating factor (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) in neutrophils and eosinophils from humans and the effect of a calcium ionophore

Enzymatic systems in human blood cells are described for the activation and inactivation of a biologically active phospholipid (1-alkyl-2-acetyl-$\text{sn}$-glycero-3-phosphocholine) with hypotensive, platelet-aggregating, and inflammatory properties. The results document the presence of alkyldihydroxyacetone-phosphate synthase (forms the $\text{O}$-alkyl linkage in lipids), 1-alkyl-2-lyso-$\text{sn}$-glycero-3-phosphocholine:acetyl-CoA acetyltransferase (produces the biologically active molecule), and 1-alkyl-2-acetyl-$\text{sn}$-glycero-3-phosphocholine: acetylhydrolase (destroys the biological activity) in human neutrophils and eosinophils. Both the acetyltransferase and acetylhydrolase activities are increased severalfold after treatment of normal neutrophils with ionophore A23187; however, alkyldihydroxyacetone-phosphate synthase activity is not influenced by the ionophore. Eosinophils isolated from patients with eosinophilia have significantly greater activities of all the enzymes studied than the eosinophils isolated from normal individuals. Our results indicate the acetyltransferase responsible for 1-alkyl-2-acetyl-$\text{sn}$-glycero-3-phosphocholine synthesis may serve an important role in human blood cells that release this biologically active phospholipid. Moreover, the acetyltransferase activity was found to be dramatically influenced by calcium flux.     

In vivo metabolism of a new class of biologically active phospholipids: 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine,a platelet activating-hypotensive phospholipid

This report describes the in vivo metabolism of a new class of naturally occurring biologically active phospholipids (1-alkyl-2-acetyl-$\text{sn}$-glycero-3-phosphocholines) that can cause hypotension and platelet aggregation. After intravenous injection in male rats, the acetylated ether phospholipid (1-[1′,2′-³H]alkyl) is rapidly cleared ($\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{?30}\text{s}$) from blood and its metabolites are found in a variety of tissues. The tissues containing the highest levels of radioactivity are lung, liver, spleen, and kidney. Chromatographic results showed that a considerable portion of the active lipid is not readily catabolized in some of the major tissues examined; however, inactive metabolites were also found, mainly 1-alkyl-2-lyso-$\text{sn}$-glycero-3-phosphocholine and 1-alkyl-2-acyl-$\text{sn}$-glycero-3-phosphocholine; the latter has a long chain fatty acid at the $\text{sn}$-2 position instead of the acetate. The findings are consistent with our earlier data that show these same tissues have the most active enzyme systems for metabolizing 1-alkyl-2-acetyl-$\text{sn}$-glycero-3-phosphocholine.     

Maintenance of respiratory control by beef heart mitochondria incubated at 25 degrees C: response to protective agents and to protective agents and to prior stress.

Lysophospholipase D (EC 3.1.4.-) activity was demonstrated in rat kidneys, intestines, lungs, testes, and liver. The liver enzyme was studied in greatest detail and its labeled products were identified by chemical and Chromatographic techniques. This enzyme hydrolyzes 1-[1-¹⁴C]hexadecyl-sn-glycero-3-phosphoethanolamine and 1-[1-¹⁴C]hexadecyl-sn-glycero-3-phosphocholine to yield 1-[1-¹⁴C]hexadecyl-sn-glycero-3-phosphate; the initial product is subsequently dephosphorylated by a phosphohydrolase in microsomes to form 1-[1-¹⁴C]hexadecyl-sn-glycerol. The possibility that phospholipase C and a phosphotransferase were responsible for the formation of 1-[1-¹⁴C]hexadecyl-sn-glycero-3-phosphate was ruled out. Neither 1-[1-¹⁴C]hexadecyl-2-acyl-sn-glycero-3-phosphoethanolamine nor 1-[1-¹⁴C]hexadecyl-2-acyl-sn-glycero-3-phosphocholine was hydrolyzed. The enzyme requires Mg²⁺, is inhibited by Ca²⁺, and is stimulated by high salt concentrations; it is localized in the microsomal fraction and has a pH optimum between 7.0 and 7.6. Inhibition by sulfhydryl reagents and protection by glutathione and dithiothreitol suggest that a sulfhydryl group is required for activity. The enzyme is inhibited by detergents and by organic solvent extraction. It appears to be tightly bound to the microsomes, since repeated freeze-thawing or sonication did not release the activity, and trypsin digestion (either in the presence or in the absence of 0.04% deoxycholate) did not destroy the activity. Lysophospholipase D was previously known to occur only in brain (R. L. Wykle and J. M. Schremmer, 1974, J. Biol. Chem., 249, 1742–1746).     

Lipid Acetylation Reactions and the Metabolism of Platelet-Activating Factor

In this review properties of lipid acetyltransferase enzymes are outlined. The three activities of interest are lyso PAF acetyltransferase (acetyl CoA: 1-alkyl-sn-glycero-3-phosphocholine acetyltransferase), AGP acetyltransferase (acetyl CoA: 1-alkyl sn-glycero-3-phosphate acetyltransferase) and a transacetylase activity that can transfer acetyl groups from PAF to lipid acceptors in the formation of 1-alkenyl-2-acetyl-sn-glycero-3-phosphoethanolamine and N-acetyl sphingosine (C₂ ceramide). This review focuses on the role of acetyltransferases and transacetylases within the metabolism of platelet-activating factor and specifically addresses characteristics of the enzymes, including subcellular localization, substrate selectivity, and enzymatic regulation     

Metabolism of hepatic haem and `green pigments'' in rats given 2-allyl-2-isopropylacetamide and ferric citrate. A new model for hepatic haem turnover

The acyl specificities of several acyltransferases located in the microsomal fraction of lactating rat mammary gland have been investigated using palmitate and oleate as substrates along with CoA, ATP and Mg²⁺, bovine serum albumin and NaF. With either sn-glycerol 3-phosphate or dihydroxyacetone phosphate (plus NADPH) as acyl acceptor, phosphatidic acid containing palmitate preferentially esterified at position-2 and oleate at position-1 was the major product. Dihydroxyacetone phosphate and sn-glycerol 3-phosphate competitively inhibited each other's acylations, suggesting that a single enzyme might be responsible for both esterifications and oleate was the preferred substrate for the formation of acyldihydroxyacetone phosphate. The specificities of the acyl-CoA–1-monoacyl-sn-glycerol 3-phosphate and the acyl-CoA–2-monoacyl-sn-glycerol 3-phosphate acyltransferases were also studied. The specificities observed combined with the relative velocities of these reactions suggest that phosphatidic acid is formed in the mammary gland with the first acylation occurring at position-1 favouring oleate followed by the second acylation at position-2 favouring palmitate. This is consistent with the unusual structure found in the triacylglycerols of rat milk. When a mouse liver microsomal fraction was used the opposite specificities were observed consistent with the structure of the triacylglycerols of mouse liver. The microsomal acylation of the monoacyl-sn-glycerol 3-phosphocholines was also investigated. Although no marked acyl specificity could be detected when the 2-monoacyl-sn-glycerol 3-phosphocholine was used as the acyl acceptor, both oleate and linoleate were esterified in preference to palmitate to the 1-monoacyl-sn-glycerol 3-phosphocholine.     

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

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

Quantification of distinct molecular species of the 2-lyso metabolite of platelet-activating factor by gas chromatography—negative-ion chemical ionization mass spectrometry

The biological activity of platelet-activating factor (PAF) is comprised by a few molecular species of phosphatidylcholine which contain a fatty alcohol connected by an ether linkage to the sn-1 position of the glycerol backbone and an acetate ester at the sn-2 position. The various molecular species of PAF differ in chain length and degree of unsaturation in the fatty alcohol residue side-chain. PAF is rapidly hydrolyzed to lyso-PAF by an acetylhydrolase enzyme which is quite active in a number of cells that synthesize PAF. We describe a method for quantitation of lyso-PAF which involves conversion to its propionate derivative in the presence of an internal standard (deuterium-labelled PAF), digestion to the diglyceride with Bacillus cereus phospholipase C, conversion to the pentafluorobenzoate derivative and capillary column gas chromatographic—negative-ion methane chemical ionization mass spectrometric analysis. Distinct molecular species of lyso-PAF can be individually quantitated at levels of 1 ng or less. These methods are applied to the demonstration of lyso-PAF accumulation in renal tissue from transplanted allografts undergoing acute rejection, in renal tissue from kidneys subjected to cold storage and autotransplantation, and in intestinal mucosa subjected to warm ischemia and reperfusion.     

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.     

Intracellular erythrocyte platelet-activating factor acetylhydrolase I inactivates aspirin in blood

Aspirin (acetylsalicylic acid) prophylaxis suppresses major adverse cardiovascular events, but its rapid turnover limits inhibition of platelet cyclooxygenase activity and thrombosis. Despite its importance, the identity of the enzyme(s) that hydrolyzes the acetyl residue of circulating aspirin, which must be an existing enzyme, remains unknown. We find that circulating aspirin was extensively hydrolyzed within erythrocytes, and chromatography indicated these cells contained a single hydrolytic activity. Purification by over 1400-fold and sequencing identified the PAFAH1B2 and PAFAH1B3 subunits of type I platelet-activating factor (PAF) acetylhydrolase, a phospholipase A(2) with selectivity for acetyl residues of PAF, as a candidate for aspirin acetylhydrolase. Western blotting showed that catalytic PAFAH1B2 and PAFAH1B3 subunits of the type I enzyme co-migrated with purified erythrocyte aspirin hydrolytic activity. Recombinant PAFAH1B2, but not its family member plasma PAF acetylhydrolase, hydrolyzed aspirin, and PAF competitively inhibited aspirin hydrolysis by purified or recombinant erythrocyte enzymes. Aspirin was hydrolyzed by HEK cells transfected with PAFAH1B2 or PAFAH1B3, and the competitive type I PAF acetylhydrolase inhibitor NaF reduced erythrocyte hydrolysis of aspirin. Exposing aspirin to erythrocytes blocked its ability to inhibit thromboxane A(2) synthesis and platelet aggregation. Not all individuals or populations are equally protected by aspirin prophylaxis, the phenomenon of aspirin resistance, and erythrocyte hydrolysis of aspirin varied 3-fold among individuals, which correlated with PAFAH1B2 and not PAFAH1B3. We conclude that intracellular type I PAF acetylhydrolase is the major aspirin hydrolase of human blood.     

Studies on ether-phospholipids of vascular smooth muscle cells. Identification of a rapid Ca2+-dependent hydrolysis of alkyl-phosphatidylethanolamine promoted by endothelin-1

We have investigated the metabolism of 1-O-[³H]octadecyl-sn-glycero-3-phosphocholine ([³H]lyso PAF) and [³H]myristic acid in secondary cultures of aortic smooth muscle cells (SMC) to characterize the origin of second messengers generated upon stimulation with endothelin-1 (ET-1). When cells were labelled with [³H]lyso PAF, we observed a transfer of the label from phosphatidylcholine (PC) to phosphatidylethanolamine (PE). In contrast, incubation with [³H]myristate labelled mainly PC. Both precursors were incorporated into all PC and PE subclasses. However, [³H]lyso PAF labelled mainly alkyl-subclasses while [³H]myristate was associated with diacyl-subclasses. Using these specific labelling procedures, we have shown that ET-1 induced a strong hydrolysis of PE. This hydrolysis was specific for alkyl-PE with a maximum after 5 s of stimulation. We have also observed an extracellular Ca²⁺-dependent increase in diglyceride (DG), phosphatidic acid (PA) and mainly triglyceride (TG) concomitant to alkyl-PE hydrolysis. Thus, alkyl-DG generated from alkyl-PE appears to be a major product in ET-1 stimulation of SMC. These results suggest a new level of complexity in the signal transduction cascade involving a specificity for phospholipid subclasses.     

Effect of platelet activating factor on leukocytes II. Enhancement of eosinophil chemotactic factor and β-glucuronidase release

Synthetic 1-O-alkyl-2-O-acetyl-sn-glyceryl-3-phosphorylcholine (PAF) and 1-O-alkyl-sn-glyceryl-3-phosphorylcholine (lyso-PAF) have previously been shown to induce chemotaxis and chemokinesis of human neutrophils. We present here data showing that these agents are inactive by themselves, but that they enhance neutrophil secretion once it has been initiated by a calcium ionosphore or by zymosan. Two substances, the lipid eosinophil chemotactic factor (ECF) and the lysosomal enzyme β-glucuronidase, are used as markers for neutrophil release. PAF augments secretion of both substances in a dose-dependent fashion, with lyso-PAF being less potent. The kinetics of enhancement are very rapid (<2 min) and are not reversible by washing of the cells. A pyrazoline derivative that inhibits arachidonate cyclo-oxygenation and lipoxygenation, reduces the enhancing effect of PAF and lyso-PAF. PAF, and less so lyso-PAF, are thus potentially important modulators of neutrophil secretion during inflammatory processes.     

Possible influence of lysophospholipase on the production of 1-acyl-2-acetylglycerophosphocholine in macrophages.

The rate of production of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF) and 1-acyl-2-acetyl-sn-glycero-3-phosphocholine (acylPAF) was measured in macrophages following the incorporation of [3H]acetate. Upon activation by A23187, guinea pig alveolar macrophages incorporated [3H]acetate into PAF, but a little radioactivity was found in acylPAF. However, labeling of acylPAF and PAF with [3H]acetate was greatly enhanced in A23187-stimulated alveolar macrophages that had been pretreated with phenylmethanesulphonyl fluoride (PMSF). [3H]PAF was predominantly converted to 1-[3H]alkyl-2-acyl glycerophosphocholine, but [14C]acylPAF rapidly hydrolyzed to 14C-labeled free fatty acid by the incubation with lysates prepared from macrophages. The deacetylation of [14C]acylPAF and [3H]PAF by acetylhydrolase and also the hydrolysis of [14C]lysoPC by lysophospholipase were strongly inhibited in macrophages that had been pretreated with PMSF, while PMSF failed to inhibit the activities of acetyltransferase and acyltransferase. The relative proportions of PAF and acylPAF were quite different in different types of cells. In contrast to alveolar macrophages, peritoneal macrophages, neutrophils and spleen cells from guinea pigs incorporated 2-4 times more [3H]acetate into acylPAF than into PAF. The presence of high levels of acylPAF in peritoneal macrophages was confirmed by GLC-MS analysis. The activities of lysophospholipase, acetylhydrolase and acetyltransferase were measured in alveolar and peritoneal macrophages to determine whether the preferential formation of acylPAF as compared to PAF in peritoneal macrophages was due to differences in these activities between alveolar and peritoneal macrophages. The activity of acetylhydrolase of peritoneal macrophages was almost the same as that in alveolar macrophages. The activity of acetyltransferase in peritoneal macrophages was about half of that in alveolar macrophages. However, the activity of lysophospholipase in peritoneal macrophages was one-sixth of that in alveolar macrophages. These results suggest that lysophospholipase is one of the primary factors involved in the control of the production of acylPAF in activated cells, and that it acts by modulating the availability of lysoPC for the synthesis of acylPAF. Furthermore, high levels of activity of lysophospholipase allow the preferential formation of PAF, via the rapid hydrolysis of lysoPC which would act as a competitive inhibitor of the incorporation of acetate into lysoPAF.