Ten-Ching Lee

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

Abstract Enzymatic systems in human blood cells are described for the activation and inactivation of a biologically active phospholipid (1-alkyl-2-acetyl- sn -glycero-3-phosphocholine) with hypotensive, platelet-aggregating, and inflammatory properties. The results document the presence of alkyldihydroxyacetone-phosphate synthase (forms the O -alkyl linkage in lipids), 1-alkyl-2-lyso- sn -glycero-3-phosphocholine:acetyl-CoA acetyltransferase (produces the biologically active molecule), and 1-alkyl-2-acetyl- 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- 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.

Regulation of platelet activating factor synthesis: Modulation of 1-alkyl-2-lyso-sn-glycero-3-phosphocholine:Acetyl-CoA acetyltransferase by phosphorylation and dephosphorylation in rat spleen microsomes

1-Alkyl-2-lyso-sn-glycero-3-phosphocholine:acetyl-CoA acetyltransferase plays an important regulatory role in the biosynthesis of platelet activating factor, a potent bioactive mediator. We tested the hypothesis that the activity of acetyltransferase may be modulated by enzymatic phosphorylation and dephosphorylation. The results showed that acetyltransferase activity in rat spleens was 2- to 3-fold higher in microsomes isolated in the presence of F-than in those isolated in the presence of Cl-. The microsomal acetyltransferase could be activated by preincubation of microsomes, isolated in the presence of Cl-, with ATP, Mg2+, and the soluble fraction from rat spleen. Addition of phosphatidylserine, diacylglycerols, plus Ca2+ further enhanced the activity. The increase in the activity of acetyltransferase was abolished by treatment of the activated microsomes with alkaline phosphatase. Conversely, the activity of acetyltransferase can be reactivated in the alkaline phosphatase-treated microsomes with incubation conditions that favor phosphorylation. Therefore, our findings suggest that acetyltransferase activity is regulated by reversible activation/inactivation through phosphorylation/dephosphorylation.

1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor) stimulates calcium influx in rabbit platelets

Summary 1-Alkyl-2-acetyl- sn -glycero-3-phosphocholine (platelet-activating factor) induces an influx of Ca 2+ in rabbit platelets that is time-, temperature-, and concentration-dependent. The ionophoretic activity of 1-alkyl-2-acetyl- sn -glycero-3-phosphocholine is much higher (>2-fold at 10 −10 M) than 3-hexadecyl-2-acetyl- sn -glycero-l-phosphocholine, 1-hexadecyl-2-lyso- sn -glycero-3-phosphocholine, 1-acyl-2-acetyl- sn -glycero-3-phosphocholine, or 1-hexadecanoyl-2-lyso- sn -glycero-3-phosphocholine.

Substrate specificity in the biocleavage of the O-alkyl bond: 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (a hypotensive and platelet-activating lipid) and its metabolites☆

Abstract 1-Alkyl-2-acetyl- sn -glycero-3-phosphocholine possesses both hypotensive and platelet-activating properties. Our data show that after removal of the acetyl group at the sn -2 position by an acetyl hydrolase, the product, 1-alkyl-2-lyso- sn -glycero-3-phosphocholine, can be cleaved by a microsomal tetrahydropteridine-dependent alkyl monooxygenase in the liver and spleen of rats. Results obtained for the tetrahydropteridine requirement, tissue distribution, responses to thermal inactivation and catalase, and substrate inhibition of the enzyme indicate that the ether linkage of 1-alkyl-2-lyso- sn -glycero-3-phosphocholine is hydrolyzed by a monooxygenase that appears to be identical to the one responsible for cleavage of the O -alkyl moiety of alkylglycerols.

Phase transitions of alkyl ether analogs of phosphatidylcholine.

The phase-transition temperatures of aqueous dispersions of diester, monoether and diether analogs of phosphatidylcholine were determinmed using transparinaric acid as a fluorescent probe. The diether analog of phosphatidylcholine has a higher phase-transition temperature, whilst the monoether analog has a lower phase-transition temperature than their diester counterpart.

Stimulation of calcium uptake by 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor) in rabbit platelets: possible involvement of the lipoxygenase pathway.

1-Alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor) induces an increase of Ca2+ uptake in rabbit platelets. This process depends upon the extracellular concentration of Ca2+ with the maximum stimulation occurring at 1-3 mM; uptake under these conditions is blocked by verapamil, a calcium-entry blocker. Increase of calcium uptake by the bioactive phospholipid was independent of ADP-induced platelet responses and of metabolites of arachidonic acid metabolism formed through the cyclooxygenase pathway. However, mepacrine, p-bromophenacyl bromide, eicosatetraynoic acid, and nordihydroguaiaretic acid significantly or totally inhibited the stimulation of Ca2+ uptake by 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine. When arachidonic acid was given sufficient time to be metabolized to other products by the platelets, stimulation of Ca2+ uptake also occurred. Arachidonic acid and platelet-activating factor did not produce an additive or synergistic effect. Our data suggest that a metabolite(s) generated from arachidonic acid through the lipoxygenase pathway may be the mediator(s) responsible for the action of platelet-activating factor in the induction of increased Ca2+ uptake in rabbit platelets.

Quantitative analysis of ether-linked lipids as alkyl- and alk-1-enyl-glycerol benzoates by high-performance liquid chromatography

Methodology for the quantitative and qualitative analyses of alkyl- and alk-l-enyl-glycerols derived by Vitride reduction of ether-linked glycerolipids in the presence of an internal standard was developed. The procedure involved preparation of benzoate derivatives that were subsequently analyzed by high-performance liquid chromatography with detection at 228 nm. Separation of the glycerol ether dibenzoates on a C18 reverse-phase column allowed for the simultaneous quantitation and the determination of chain length of both alkyl- and alk-l-enyl-glycerols in a single chromatographic run of less than 15 min. The method was accurate (less than 10% error), reproducible, and sensitive (less than 1 microgram per component). Application of the method to the analysis of phospholipids from L-M cells grown in the presence and absence of elaidic acid demonstrated that the cells incorporated a portion of the trans acid supplement (presumably via the fatty alcohol) into the side chains of both alkyl- and alk-l-enyl-glycerol-containing phosphatides.

Ether-Linked Glycerolipids and Their Bioactive Species:Enzymes and Metabolic Regulation

The pioneering studies of those scientists who first discovered (1915–1924) and ultimately identified the precise nature of the chemical structure of the alkyl and alk-l-enyl (plasmalogens) linkages in glycerolipids are described in exquisite detail in a review of the history (the years 1915–1960) of ether lipids by Debuch (1972)It is noteworthy that the chemical nature of the alkyl linkage in glycerolipids was firmly established 29 years before the precise chemical structure of the alk-l-enyl linkage in plasmalogens became known.

Membrane lipid modification and stearoyl-coenzyme A desaturase activity in L-M cells.

Abstract The stearoyl-coenzyme A desaturase system of L-M cells, grown as monolayers, was examined in microsomal membranes that contained 8.2% phosphatidylisopropylethanolamine, an unnatural phospholipid analog. Desaturation of both [1- 14 C]stearic acid by whole cells and [1- 14 C]stearoyl-coenzyme A by cell-free homogenates, or microsomes, was decreased to about 40% of control levels in cells that had been grown for 24 h in the presence of 10 m m N -isopropylethanolamine. No decrease in microsomal NADH- or NADPH-dependent cytochrome c reductase activities or the level of cytochrome b 5 was found in the L-M cells that had been treated for 24 h with N -isopropylethanolamine. Although amino acid transport into L-M cells was not affected by treatment with N -isopropylethanolamine, protein synthesis was decreased by about 30%. These results indicate that the decrease in stearoyl-coenzyme A desaturation in the modified membranes is specifically associated with the terminal oxidase activity (cyanide-sensitive factor) of the desaturase enzyme complex.

Metabolism of platelet activating factor (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) and 1-alkyl-2-acetyl-sn-glycerol by human endothelial cells

The metabolism of platelet activating factor (1-[1,2-3H]alkyl-2-acetyl-sn-glycero-3-phosphocholine) and 1-[1,2-3H]alkyl-2-acetyl-sn-glycerol was studied in cultures of human umbilical vein endothelial cells. Human endothelial cells deacetylated 1-[1,2-3H]alkyl-2-acetyl-sn-glycero-3-phosphocholine to the corresponding lyso compound (1-[1,2-3H]alkyl-2-lyso-sn-glycerol-3-phosphocholine) and a portion was converted to 1-[1,2-3H]alkyl-2-acyl(long-chain)-sn-glycero-3-phosphocholine. Lyso platelet activating factor (lyso-PAF) (1-[1,2-3H]alkyl-2-lyso-sn-glycero-3-phosphocholine) was detected in the media very early during the incubation and the amount remained higher than the level of the lyso product observed in the cells. Cellular levels of 1-[1,2-3H]alkyl-2-lyso-sn-glycero-3-phosphocholine were significantly higher than the acylated product (1-[1,2-3H]alkyl-2-acyl(long-chain)-sn-glycero-3-phosphocholine) at all times during the 60-min incubation period, which suggests that the ratio of acetylhydrolase to acyltransferase activities is greater in endothelial cells than in most other cells. When endothelial cells were incubated with 1-[1,2-3H]alkyl-2-acetyl-sn-glycerol, a known precursor of PAF, 1-[1,2-3H]alkyl-sn-glycerol was the major metabolite formed (greater than 95% of the 3H-labeled metabolites during 20- and 40-min incubations). At least a portion of the acetate was removed from 1-[1,2-3H]alkyl-2-acetyl-sn-glycerol by a hydrolytic factor released from the endothelial cells into the medium during the incubations. Only negligible amounts of the total cellular radioactivity (0.2%) was incorporated into platelet activating factor (1-[1,2-3H]alkyl-2-acetyl-sn-glycero-3-phosphocholine); therefore, it is unlikely that the previously observed hypotensive activity of 1-alkyl-2-acetyl-sn-glycerols can be explained on the basis of the conversion to platelet activating factor (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) by endothelial cells. Results of this investigation indicate that endothelial cells play an important role in PAF catabolism. Undoubtedly, the endothelium is important in the regulation of PAF levels in the vascular system.