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.     

The final step in the de novo biosynthesis of platelet-activating factor. Properties of a unique CDP-choline:1-alkyl-2-acetyl-sn-glycerol choline-phosphotransferase in microsomes from the renal inner medulla of rats

Final steps in the synthesis of platelet activating factor (PAF) occur via two enzymatic reactions: the acetylation of 1-alkyl-2-lyso-sn-glycero-3-phosphocholine by a specific acetyltransferase or the transfer of the phosphocholine base group from CDP-choline to 1-alkyl-2-acetyl-sn-glycerol by a dithiothreitol (DTT)-insensitive cholinephosphotransferase. Our studies demonstrate that rat kidney inner medulla microsomes synthesize PAF primarily via the DTT-insensitive cholinephosphotransferase since the specific activity of this enzyme is greater than 100-fold higher than the acetyltransferase. The two cholinephosphotransferases that catalyze the biosynthesis of phosphatidylcholine and PAF have similar Mg2+ or Mn2+ requirements and are inhibited by Ca2+. Also topographic experiments indicated that both activities are located on the cytoplasmic face of microsomal vesicles. PAF synthesis was slightly stimulated by 10 mM DTT, whereas the enzymatic synthesis of phosphatidylcholine was inhibited greater than 95% under the same conditions. The concept of two separate enzymes for PAF and phosphatidylcholine synthesis is further substantiated by the differences in the two microsomal cholinephosphotransferase activities with respect to pH optima, substrate specificities, and their sensitivities to temperature, deoxycholate, or ethanol. Study of the substrate specificities of the DTT-insensitive cholinephosphotransferase showed that the enzyme prefers a lipid substrate with 16:0 or 18:1 sn-1-alkyl chains. Short chain esters at the sn-2 position (acetate or propionate) are utilized by the DTT-insensitive cholinephosphotransferase, but analogs with acetamide or methoxy substituents at the sn-2 position are not substrates. Also, CDP-choline is the preferred water-soluble substrate when compared to CDP-ethanolamine. Utilization of endogenous neutral lipids as a substrate by the DTT-insensitive cholinephosphotransferase demonstrated that sufficient levels of alkylacetylglycerols are normally present in rat kidney microsomes to permit the synthesis of physiological quantities of PAF. These data suggest the renal DTT-insensitive cholinephosphotransferase could be a potentially important enzyme in the regulation of systemic blood pressure.     

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.     

Development of a novel scintillation proximity radioimmunoassay for platelet-activating factor measurement: comparison with bioassay and GC/MS techniques

A novel, facile and sensitive scintillation proximity radioimmunoassay (SPRIA) for quantitation of PAF has been developed. No separation of antibody bound [3H]PAF from free [3H]PAF is required as the assay employs protein A - coated fluomicrospheres (beads containing scintillant). The assay system was suitable for the quantitation of 0.03 to 2 pmol of 1-hexadecyl-2-acetyl-sn-glycero-3- phosphocholine. The cross-reactivity was high with 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine but was very low with PAF analogs such as 1-alkyl- and 1-acyl-2-lyso-sn-glycero-3-phosphocholine, 1-acyl-2-acetyl-sn-glycero-3-phosphocholine, and 1-alk-1'-enyl-2-acetyl-sn-glycero-3-phosphocholine. The specificity of SPRIA was higher than that of bioassay (platelet degranulation assay). PAF receptor antagonists (L-652,731, WEB2086, and FR900452) at up to 10 nmol per tube had no affect on the SPRIA. These observations indicate that the specificity of the PAF antibody is quite different from that of the platelet receptor. The values obtained using SPRIA for the measurement of PAF produced in polymorphonuclear leukocytes with stimuli are comparable to those obtained by SIM/GC/MS analysis.     

A comparison of the reversibility of phosphoethanolamine transferase and phosphocholine transferase in rat brain microsomes

The reversibility of phosphoethanolamine transferase (EC 2.7.8.1) in rat brain is demonstrated in this paper. Microsomal ethanolamine glycerophospholipids were prelabeled with an intracerebral injection of [3H]ethanolamine 4 h before killing young rats. Labeled CDPethanolamine was produced by incubation of the microsomes with CMP, although to a lesser extent than for the previously observed release of CDPcholine. Ethanolamine and choline glycerophospholipids were labeled with [2-3H]glycerol by incubation with primary cultures of rat brain. Microsomes from rat brains, with diisopropyl phosphofluoridate for inhibition of lipases, were incubated with the labeled glycerophospholipids separately, and labeled diacylglycerols were produced. The kinetic parameters of phosphoethanolamine transferase and phosphocholine transferase (EC 2.7.8.2) were compared by incubating rat brain microsomes with [3H]CMP. Inclusion of AMP in the reaction mixture was necessary in order to inhibit the hydrolysis of CMP by an enzyme with the properties of 5'-nucleotidase (EC 3.1.3.5). For phosphoethanolamine transferase and phosphocholine transferase respectively, the Km values for CMP were 40 and 125 microM and the V values were 2.3 and 21.6 nmol/h per mg protein. The reversibility of both enzymes permits the interconversion of the diacylglycerol moieties of choline and ethanolamine glycerophospholipids. During brain ischemia, a principal pathway for degradation of ethanolamine glycerophospholipids may be by reversal of phosphoethanolamine transferase followed by hydrolysis of diacylglycerols by the lipase.     

Evidence that increasing the cellular content of eicosapentaenoic acid does not reduce the biosynthesis of platelet-activating factor

Our study has examined platelet-activating factor (PAF) biosynthesis in neutrophils from individuals on a fish oil-enriched diet and in mast cells enriched with eicosapentaenoic acid (EPA) in vitro. Neutrophils isolated from males who were fed fish oil supplement (EPA; 2.8 g/day) for 5 wk contained large quantities of eicosapentaenoate in phosphatidylcholine (PC) and phosphatidylethanolamine and less in phosphatidylinositol. The ratio arachidonate/eicosapentaenoate in PC and phosphatidylethanolamine decreased from greater than 10 before the enriched diet to approximately 3 after the diet. The putative precursor of PAF, 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (1-O-alkyl-2-acyl-GPC) contained the bulk of eicosapentaenoate in PC subclasses with smaller quantities found in 1-acyl and 1-alk-1'-enyl linked species. Ionophore A23187-stimulated neutrophils produced similar quantities of PAF before and after enriched diet. Neutrophils during normal diet acylated 1-O-alkyl-2-lyso-GPC only with arachidonate whereas neutrophils from individuals on enriched diet transferred both arachidonate and eicosapentaenoate into exogenously-provided 1-O-alkyl-2-lyso-GPC. This allowed for the labeling of neutrophils with 1-O-[3H]-alkyl-2-arachidonoyl-GPC (before diet) as well as neutrophils with 1-O-[3H]-alkyl-2-eicosapentaenoyl-GPC and 1-O-[3H]-alkyl-2-arachidonoyl-GPC (after diet). Neutrophils after diet converted similar quantities of these labeled precursors to labeled PAF upon stimulation as those before the diet. Analysis of the nature of the long chain acyl residue remaining in the sn-2 position of 1-alkyl-2-acyl-GPC after cell stimulation indicated that arachidonate and eicosapentaenoate were both released from 1-O-alkyl-2-acyl-GPC at comparable rates. Finally, in vitro supplementation of murine mast cells (PT-18) with arachidonic acid or EPA caused a marked increase in the amount of PAF produced by the cell without having any effect on histamine release. Data from these experiments suggest that EPA is incorporated into a PAF precursor pool. However, this appears not to inhibit PAF production because phospholipase A2 can use eicosapentaenoate- as well as arachidonate-containing phospholipids in the initial step of PAF biosynthesis.     

ENZYMATIC ACYLATION OF 1-ALKYL-, 1-ALKENYL-AND 1-ACYL GLYCERO-3-PHOSPHORYLETHANOLAMINE IN DEVELOPING RAT BRAIN

Abstract— Rat brain particulate fractions were shown to acylate [³²P]1-alkyl- sn -glycero-3-phosphorylethanolamine (GPE). While the main product is 1-alkyl-2-acyl GPE, about 12 per cent of the radioactivity was also found in 1-alkenyl-2-acyl GPE. The acyl transferase activity was completely dependent on added ATP and CoA and it was localized mainly in the microsomal fraction. A comparative study of acyl transferase activities to 1-alkyl-, 1-alkenyl-, and 1-acyl GPE by crude mitochondrial fraction and microsomes of 10, 16 and 22-day-old rat brains showed a progressive increase in activity with development. In the 22-day-old rat brain the order of activity towards the three substrates is as follows: 1-acyl GPE ± 1-alkenyl GPE ± 1-alkyl GPE with a crude mitochondrial fraction and 1-acyl GPE ± 1-alkyl GPE ± 1-alkenyl GPE with microsomes.     

Metabolism and function of platelet-activating factor in fetal rabbit lung development

Previously, platelet-activating factor (PAF, PAF-acether, 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) had been identified in association with a lamellar-body-enriched fraction of human amniotic fluid obtained from women in labor. In consideration of the fact that fetal lung is the source of lamellar bodies, we have investigated the capacity of the developing lung to synthesize PAF. The specific activity of the PAF biosynthetic enzyme, 1-alkyl-2-lyso-sn-glycero-3-phosphocholine: acetyl-CoA acetyltransferase, increased from 116 pmol/min per mg protein in day 21 fetal rabbit lung to 332 pmol/min per mg protein by day 31. Although this enzymatic activity in fetal kidney also increased, it never reached the level found in lung. In contrast, the actyltransferase activity decreased by 80% in fetal liver between days 21 and 31. The acetyltransferase activity in lung was primarily localized in the microsomal fraction (105 000 X g pellet); however a significant proportion of the activity was found in the 18 000 X g pellet. The specific activity of acetyltransferase in adult alveolar type II rat pneumonocytes was significantly higher than that of adult rat lung or rat alveolar macrophages, suggesting that type II cells make a significant contribution to the actyltransferase activity of lung tissue. PAF acetylhydrolase remained relatively constant throughout the gestation in all tissues. The concentration of PAF in the fetal lung increased by 3-fold from 12 to 35 fmol/mg protein, between day 21 and day 31 of development. The concentrations of the PAF precursors, 2-lyso-PAF (1-alkyl-2-lyso-sn-glycero-3-phosphocholine) and the 2-acyl derivative, were several orders of magnitude higher than the PAF concentration. The pulmonary glycogen content decreased from 163 at day 21 to 35 micrograms/mg protein at day 31 of gestation. We suggest that the increase in PAF concentration may participate in the regulation of glycogen breakdown in fetal lung as it does in perfused rat liver (Shukla, S.D., Buxton, D.B., Olson, M.S. and Hanahan, D.J. (1983) J. Biol. Chem. 258, 10212-10214). The formation of PAF in the developing lung and its secretion, in association with lamellar bodies, into amniotic fluid is discussed in relation to parturition.     

Acyl specificity of hamster heart CDP-choline 1,2-diacylglycerol phosphocholine transferase in phosphatidylcholine biosynthesis

The acyl specificity of 1,2-diacylglycerol: CDP-choline phosphocholine transferase (EC 2.7.8.2) for the formation of phosphatidylcholine with the appropriate acyl groups in hamster heart was investigated. Enzyme activity was determined in the microsomal fraction with 1,2-diacylglycerols of known acyl content. Maximum enzyme activity was obtained with diacylglycerol containing a monoenoic acyl group at the C-2 position of the glycerol moiety, regardless of the acyl group at the C-1 position. The specificity of the enzymes was also investigated by perfusing the isolated hamster heart with labelled glycerol. Comparison of the molecular species of the labelled diacylglycerols and phosphatidylcholine subsequent to perfusion revealed that the specificity of phosphocholine transferase was not limited to the monoenoic species of diacylglycerol. The difference in specificity observed between the in vitro assay and the perfusion study may partly be attributed to the presence of detergent in the enzyme assay mixture (to facilitate solubility of diacylglycerol). It is concluded that in the hamster heart, phosphocholine transferase has only limited ability to select the appropriate acyl groups for phosphatidylcholine biosynthesis. It appears that the majority of the newly formed phosphatidylcholine in the heart via the CDP-choline pathway is subsequently resynthesized by deacylation-reacylation process.     

Regulation of the synthesis of platelet-activating factor and its inactive storage precursor (1-alkyl-2-acyl-sn-glycero-3-phosphocholine) from 1-alkyl-2-acetyl-sn-glycerol by rabbit platelets

We have established previously that 1-alkyl-2-acetyl-sn-glycerol (alkylacetyl-G) can be converted into at least six metabolites by rabbit platelets, including alkylacetyl-sn-(glycero-3-phosphocholine) (-GPC), i.e. platelet-activating factor (PAF) and 1-alkyl-2-acyl-sn- (alkylacyl)-GPC. Since part of the biological functions of alkylacetyl-G can be explained by its metabolic conversion to PAF and also to alkylacyl-GPC as an inactive storage precursor of PAF, the present study focused on the regulation of the synthesis of PAF and alkylacyl-GPC from alkylacetyl-G. Our results document the presence of a specific dithiothreitol (DTT)-insensitive cholinephosphotransferase in saponin-permeabilized rabbit platelets and show that DTT potentiates the production of PAF from alkylacetyl-G but inhibits the formation of phosphatidylcholine from diolein. We also demonstrated that the availability of CDP-choline controls the generation of PAF from alkylacetyl-G. Furthermore, when CTP: phosphocholine cytidylyltransferase is activated to produce more CDP-choline through the translocation of this enzyme from the cytosol to membranes by incubating the rabbit platelets with 0.2 mM sodium oleate, the production of PAF from alkylacetyl-G is increased 5-fold. More importantly, our experiments reveal the presence of two metabolic pathways that are responsible for the synthesis of alkylacyl-GPC from alkylacetyl-G, with each producing a unique molecular species composition of the stored PAF precursor, alkylacyl-GPC. The latter is enriched in polyunsaturates (70.7-78.5% 20:4) when formed through the remodeling pathway of PAF cycle via alkylacetyl-G (DTT-insensitive cholinephosphotransferase)----alkylacetyl-GPC----alkyllyso-GPC---- alkylacyl-GPC . Alkylacyl-GPC containing saturated species (71.8% 16:0) is generated by the retroconversion/de novo pathway according to the reaction scheme of alkylacetyl-G----alkyl-G----alkyllyso-glycero-3-phosphate (-GP)----alkylacyl-GP----alkylacyl-G (DTT-sensitive cholinephosphotransferase)----alkylacyl-GPC. Inactivation of PAF through the remodeling/PAF cycle can generate alkylacyl-GPC at both low (1.75 x 10(-7) M) and high (10(-6) M) concentrations of PAF whereas the conversion of alkylacetyl-G to alkylacyl-GPC via PAF through the remodeling pathway only occurs at a low concentration (1.75 x 10(-7) M). At a high concentration (10(-6) M), alkylacetyl-G is converted to alkylacyl-GPC via the retroconversion/de novo route. These data suggest that the formation of PAF by the DTT-insensitive cholinephosphotransferase activity limits the amounts of alkylacyl-GPC produced from alkylacetyl-G through this remodeling pathway (PAF cycle).(ABSTRACT TRUNCATED AT 400 WORDS)     

A new de novo pathway for the formation of 1-alkyl-2-acetyl-sn-glycerols, precursors of platelet activating factor. Biochemical characterization of 1-alkyl-2-lyso-sn-glycero-3-P:acetyl-CoA acetyltransferase in rat spleen

1-Alkyl-2-acetyl-sn-glycero-3-phosphocholine (alkylacetyl-GPC, platelet activating factor (PAF] can be biosynthesized either by acetylation of alkyllyso-GPC through a remodeling pathway or by the transfer of phosphocholine to alkylacetyl-sn-glycerol (alkylacetyl-G) via a putative de novo pathway involving a dithiothreitol-insensitive cholinephosphotransferase. However, the relevance of the de novo pathway in the biosynthesis of PAF depends on the existence of enzymes that can directly synthesize alkylacetyl-G from 1-alkyl-2-lyso-sn-glycero-3-P (alkyllyso-GP) or some other source. In this study, we demonstrated that microsomal preparations of rat spleen can synthesize alkylacetyl-GP by an alkyllyso-GP:acetyl-CoA acetyltransferase and that this intermediate is subsequently dephosphorylated by an alkylacetyl-GP phosphohydrolase to generate alkylacetyl-G. The properties of alkyllyso-GP:acetyl-CoA acetyltransferase were characterized under conditions where the contaminating activity of alkylacetyl-GP phosphohydrolase was minimal; this was accomplished by inhibiting the phosphohydrolase with the addition of sodium vanadate and sodium fluoride to the assay mixtures and incubating at relatively low temperatures (23 degrees C). Alkyllyso-GP:acetyl-CoA acetyltransferase had a pH optimum of 8.4 at 23 degrees C and was located in the microsomal fraction. The apparent Km for acetyl-CoA under these conditions was 226 microM and the optimal concentration of alkyllyso-GP ranged between 16 and 25 microM. Based on pH optima, substrate inhibition studies, and sensitivities to preincubation temperatures of the microsomes, it appears that alkyllyso-GP:acetyl-CoA acetyltransferase differs from the acetyltransferase responsible for the transfer of acetate from acetyl-CoA to alkyllyso-GPC to form PAF. A variety of tissues had high activities of alkyllyso-GP:acetyl-CoA acetyltransferase, which indicates that this pathway is operational in many cell types. Our results document the existence of a complete de novo biosynthetic pathway for the assembly of PAF, and this route could be responsible for maintaining physiological levels of platelet activating factor for normal cell function.     

Vertebrate class distribution of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine acetylhydrolase in serum

Serum from numerous mammals and lower vertebrates contains an enzyme activity that is specific for the hydrolysis of the acetate moiety of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF, platelet activating factor). Acetylhydrolase (EC 3.1.1.47, 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine acetylhydrolase) was found in all mammalian sera with activity ranging from 11 (fetal calf) to 178 (rabbit) pmol acetate liberated/microliter serum/min. The enzyme is not present in avian serum but is a constituent of reptiles and bony fishes.     

A rapid method for the determination of CTP and phosphocholine in rat liver and baby hamster kidney 21 cells

A rapid and sensitive assay for CTP and phosphocholine was required for us to determine the concentration of these compounds in tissues and cell cultures. Such a procedure was devised with CTP:phosphocholine cytidylyltransferase, an enzyme which is highly specific for CTP and phosphocholine. The 0--22% ammonium sulfate precipitate of a cytosolic extract from rat liver was used as the source of the enzyme. The amount of CTP in an extract was estimated by the conversion of [3H]phosphocholine to 3H-labelled CDP-choline. Similarly, the concentration of phosphocholine was estimated by the formation of 3H-labelled CDP-choline from 3H-labelled CTP. The conversion of CTP and phosphocholine to CDP-choline was 90% when inorganic pyrophosphatase was added to the incubations. The formation of CDP-choline was linear between 1 and 10 nmol of CTP or phosphocholine. The concentration of CTP was determined in rat liver (62 nmol/g wet weight) and baby hamster kidney 21 (BHK-21) cells (161 nmol/g wet weight). The concentration of phosphocholine in rat liver was 1.16 mumol/g wet weight whereas in BHK-21 cells it was much less (69 nmol/g wet weight). By this procedure, it may be possible to establish the importance of CTP and phosphocholine in the control of phosphatidylcholine biosynthesis.     

Effect of platelet activating factor on the motility and acrosome reaction of buffalo (Bubalus bubalis ) spermatozoa

Platelet activating factor (PAF; 1-0-alkyl-2 acetyl-sn-glycerol-3 phosphocholine) has been shown to have a wide range of biological activities. In this study, PAF was used to induce acrosome reactions in fresh as well as frozen-thawed buffalo spermatozoa at different incubation periods and PAF levels. As the period of incubation increased, there was a gradual decrease in motility and increase in acrosome reaction in both fresh and frozen-thawed spermatozoa. With increasing PAF levels, the motility of fresh spermatozoa decreased and acrosome reaction increased whereas in frozen-thawed semen, motility remained almost constant, and the increase in acrosome reaction was not pronounced. Differences in motility and acrosome reaction among different bulls, types of semen, periods of incubation and PAF levels were significant (P < 0.01). A PAF level of 100 microM and an incubation period of 15 min were found to be optimum for inducing acrosome reaction in buffalo spermatozoa, since at this combination acrosome reaction increased significantly (P < 0.01) over that of the control without much loss of motility.     

Stimulation of platelet-activating factor synthesis in human endothelial cells by activation of the de novo pathway. Phorbol 12-myristate 13-acetate activates 1-alkyl-2-lyso-sn-glycero-3-phosphate:acetyl-CoA acetyltransferase and dithiothreitol-insensitive 1-alkyl-2-acetyl-sn-glycerol:CDP-choline cholinephosphotransferase.

Human umbilical vein endothelial cells (HUVEC) produce platelet-activating factor (PAF) by a remodeling pathway involving a phospholipase A2 followed by an acetyl-CoA-dependent acetyltransferase which acetylates a lyso-PAF intermediate to form PAF and is stimulated by a variety of agents that generate inflammatory and allergic responses. A second route for PAF synthesis in mammalian tissues is a de novo pathway, which requires the participation of three enzymes: 1-alkyl-2-lyso-sn-glycero-3-phosphate (alkyllyso-GP): acetyl-CoA acetyltransferase, 1-alkyl-2-acetyl-sn-glycero-3-phosphate phosphohydrolase, and dithiothreitol (DDT)-insensitive 1-alkyl-2-acetyl-sn-glycerol (alkylacetyl-G):CDP-cholinecholinephosphotransferase. In the present study we show that protein kinase C activation by phorbol 12-myristate 13-acetate (PMA) induces PAF production in HUVEC by an increase of both alkyllyso-GP:acetyl-CoA acetyltransferase and DTT-insensitive alkylacetyl-G:CDP-choline choline-phosphotransferase. PAF synthesis, labeled precursors [( 3H]acetate and [methyl-3H]choline) incorporation, and both enzyme activities of the de novo pathway increase concomitantly in response to different doses of PMA. PMA does not activate the enzymes of the remodeling pathway. We conclude that both remodeling and the de novo pathway for PAF synthesis are present in HUVEC and might be alternatively activated depending on the conditions of cell stimulation.     

A new class of antihypertensive neutral lipid: 1-alkyl-2-acetyl-sn-glycerols, a precursor of platelet activating factor

A new type of neutral lipid is described that possesses hypotensive activity in genetic hypertensive (SHR) and normotensive (WKY) rats. 1-Alkyl-2-acetyl-sn-glycerols and 1-alkyl-2-propionyl-sn-glycerols are both equally effective in eliciting the hypotensive response. Requirement for the 1-alkyl and 2-acetyl or 2-propionyl structure of the active isomer was documented by the negative responses obtained with closely related neutral lipid analogs (1-alkyl-2-acyl-, 1-alkyl-3-acetyl-, 1-acyl-2-acetyl-, 1-alkyl-2,3-diacetyl-, and 1-alkyl-glycerols). Although less potent than PAF (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine), the 1-alkyl-2-acetyl-sn-glycerols produce a response of significantly longer duration and may have fewer immediate side effects than PAF. The mechanism for the biological activity is unknown; however, we have demonstrated previously that the enzymatic synthesis of 1-alkyl-2-acetyl-sn-glycerols to PAF occurs via a specific cholinephosphotransferase and therefore the observed blood pressure response might be due to the conversion of the neutral lipid precursor to PAF in vivo.     

Platelet-activating factor in normal rat uterus

Platelet-activating factor (PAF) was found in normal rat uterus and identified as 1-0-hexadecyl/octadecenyl-2-acetyl-sn-glycero-3-phosphocholine. PAF was purified by several successive chromatographic procedures. It showed platelet aggregating activity, which was inhibited by CV 3988, and had no effect on platelets desensitized with 1-0-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine. The tert-butyldimethyl-silylderivative of 1-0-alkyl-2-acetyl-sn-glycerol, which was obtained by hydrolysis of uterine PAF with phospholipase C, was analyzed by gas chromatography-mass spectrometry. One rat uterus contained approximately 21.3 ng of 1-0-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine. This is the first report of the occurrence of a significant amount of PAF in a normal animal tissue.     

Increased biosynthesis of platelet-activating factor in activated human eosinophils

1-Alkyl-2-lyso-sn-glycero-3-phosphocholine:acetyl-CoA acetyltransferase catalyzes the conversion of biologically inactive lysophospholipid to bioactive platelet-activating factor (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine, PAF) by an acetylation reaction. The activity of this enzyme in eosinophils isolated from patients with eosinophilia is stimulated (up to 4-fold) in a dose-, time-, and Ca2+/Mg2+-dependent manner after exposure to the eosinophil chemotactic factor of anaphylaxis (ECF-A), C5a, formyl-methionylleucylphenylalanine (fMLP), or ionophore A23187. The three naturally occurring chemotactic factors (ECF-A, C5a, and fMLP) cause a rapid and transient increase of enzyme activity, with a maximum at 1 or 3 min, whereas ionophore A23187 maintains an elevated level for up to 15 min. The activity of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine acetylhydrolase, an enzyme that catalyzes the breakdown of PAF to lyso-PAF, is not affected by C5a, fMLP, or ionophore A23187. The presence of PAF in eosinophils was established by demonstrating the lipid nature of the compound, the RF value being identical with that of synthetic 1-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine on thin layer chromatograms, and by its ability to induce serotonin release from rabbit platelets. Furthermore, ECF-A, C5a, fMLP, and ionophore A23187 all induce the secretion of PAF from eosinophils. These findings suggest that the generation and release of PAF could be a consequence of eosinophil chemotactic activation and may thus function in inflammatory and allergic reactions in which eosinophils participate.     

Biosynthesis of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet activating factor and a hypotensive lipid) by cholinephosphotransferase in various rat tissues

The unique alkyl phospholipid, 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine, has been reported to exhibit powerful antihypertensive activity (Blank, M.L., Snyder, F., Byers, L.W., Brooks, B. and Muirhead, E.E. (1979) Biochem. Biophys. Res. Commun. 90, 1194-1200) and appears to be an extremely potent platelet-activating factor (Demopoulos, C.A., Pinckard, R.N. and Hanahan, D.J. (1979) J. Biol. Chem. 254, 9355-9358). In the present study, microsomal preparations from several rat tissues were found to catalyze the synthesis of 1-alkyl-1-acetyl-sn-glycero-3-phosphocholine by 1-alkyl-2-acetyl-sn-glycerol:CDPcholine cholinephosphotransferase reaction. Optimal conditions to measure enzyme activity were established. A subcellular survey of this cholinephosphotransferase activity showed that the enzyme was of microsomal origin. Enzyme activity was found in microsomes from several tissues; however, spleen has the highest activity of the tissues examined. Three different species of 1-alkyl-2-acetyl-sn-glycerol were all found to be substrates. The 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine synthesized in the microsomes could be hydrolyzed by adding the 100,000 x g supernatant fraction to the incubation medium. The optimum pH for formation of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine was 8.0, which was different from the pH optimum of 8.5 observed for the long-chain diacylglycerol cholinephosphotransferases. Activity of cholinephosphotransferase towards 1-alkyl-2-acetyl-sn-glycerol was slightly enhanced and stabilized by dithiothreitol, whereas the activity towards a diacylglycerol was inhibited by dithiothreitol. The possible involvement of two different enzymes in the conversion of 1-alkyl-2-acetyl-sn-glycerol and diacylglycerol to their respective phospholipid products is discussed.     

Enzymic synthesis of 1-alkyl-2-acyl-sn-glycero-3-phosphorylethanolamines by the CDP-ethanolamine: 1-radyl-2-acyl-sn-glycerol ethanolaminephosphotransferase from microsomal fraction of rat brain

The incorporation of radioactivity from cytidine-5'-phosphate-[(32)P]phosphorylethanolamine into 1-alkyl-2-acyl-sn-glycero-3-phosphorylethanolamines and 1,2-diacyl-sn-glycero-3-phosphorylethanolamines was stimulated more than fourfold by 1-alkyl-2-acyl-sn-glycerols and 1,2-diacyl-sn-glycerols, respectively, with an ethanolaminephosphotransferase (EC 2.7.8.1) present in the microsomal fraction from brains of mature rats. The K(m) values, 0.28 mm for CDP-ethanolamine and 1.9 mm for 1-alkyl-2-acyl-sn-glycerols, were similar to those obtained by other investigators with other 1-radyl-2-acyl-sn-glycerols. The formation of 1,2-diacyl-sn-glycero-3-phosphorylethanolamines from endogenous 1,2-diacyl-sn-glycerols was inhibited by 1-alkyl-2-acyl-sn-glycerols. These properties indicate that the ethanolaminephosphotransferase lacks specificity for the type of group at the 1-position of the lipid substrate. The synthesis of 1-alkyl-2-acyl-sn-glycero-3-phosphorylethanolamines from 1-alkyl-2-acyl-sn-glycerols and CDP-ethanolamine by an enzyme from rat brain supports the inclusion of this reaction in the metabolic pathway for the synthesis of 1-alk-1'-enyl-2-acyl-sn-glycero-3-phosphorylethanolamines.