PAF Biosynthesis Research Articles

Computational design and engineering of an Escherichia coli strain producing the nonstandard amino acid para-aminophenylalanine

SummaryIntroducing heterologous pathways into host cells constitutes a promising strategy for synthesizing nonstandard amino acids (nsAAs) to enable the production of proteins with expanded chemistries. However, this strategy has proven challenging, as the expression of heterologous pathways can disrupt cellular homeostasis of the host cell. Here, we sought to optimize the heterologous production of the nsAA para-aminophenylalanine (pAF) in Escherichia coli. First, we incorporated a heterologous pAF biosynthesis pathway into a genome-scale model of E. coli metabolism and computationally identified metabolic interventions in the host’s native metabolism to improve pAF production. Next, we explored different approaches of imposing these flux interventions experimentally and found that the upregulation of flux in the chorismate biosynthesis pathway through the elimination of feedback inhibition mechanisms could significantly raise pAF titers (∼20-fold) while maintaining a reasonable pAF production-growth rate trade-off. Overall, this study provides a promising strategy for the biosynthesis of nsAAs in engineered cells.

Associations between red blood cells fatty acids, desaturases indices and metabolism of platelet activating factor in healthy volunteers

IntroductionPlatelet-activating-factor is an inflammatory lipid mediator. Key enzymes of its biosynthesis are CDP-choline:1-alkyl-2-acetyl-sn-glycerol-cholinephosphotransferase (PAF-CPT) and acetyl-CoA:lyso-PAF-acetyltransferases (Lyso-PAF-AT) while PAF-AH/Lp-PLA2 degrade PAF. The interplay between PAF and fatty acids metabolism was explored. Material and methodsIn a healthy population, PAF levels, its metabolic enzymes activity and RBC fatty acids were measured while desaturases indices (D) were estimated. A principal component analysis was also applied to assess patterns of RBC fatty acids. ResultsSFA were related to increased PAF biosynthesis and decreased Lp-PLA2 only in women. MUFA were inversely associated with PAF biosynthesis and positively with Lp-PLA2. Omega-6 fatty acids were positively correlated only with PAF-CPT while no significant correlations were observed with n3 fatty acids. D6 index was positively related with PAF biosynthetic enzymes and inversely with Lp-PLA2 while D9 correlated positively with Lp-PLA2. The pattern of high MUFA and low n6 was associated with reduced PAF biosynthesis and/or increased catabolism in both sexes. ConclusionThe role of fatty acids in amplifying or reducing inflammation seems to be also reflected in PAF metabolism.

Tricarboxylic Acid Cycle Activity and Remodeling of Glycerophosphocholine Lipids Support Cytokine Induction in Response to Fungal Patterns

Increased glycolysis parallels immune cell activation, but the role of pyruvate remains largely unexplored. We found that stimulation of dendritic cells with the fungal surrogate zymosan causes decreases of pyruvate, citrate, itaconate, and α-ketoglutarate, while increasing oxaloacetate, succinate, lactate, oxygen consumption, and pyruvate dehydrogenase activity. Expression of IL10 and IL23A (the gene encoding the p19 chain of IL-23) depended on pyruvate dehydrogenase activity. Mechanistically, pyruvate reinforced histone H3 acetylation, and acetate rescued the effect of mitochondrial pyruvate carrier inhibition, most likely because it is a substrate of the acetyl-CoA producing enzyme ACSS2. Mice lacking the receptor of the lipid mediator platelet-activating factor (PAF; 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine) showed reduced production of IL-10 and IL-23 that is explained by the requirement of acetyl-CoA for PAF biosynthesis and its ensuing autocrine function. Acetyl-CoA therefore intertwines fatty acid remodeling of glycerophospholipids and energetic metabolism during cytokine induction.

Docosahexaenoic Acid Inhibits Proliferation of EoL-1 Leukemia Cells and Induces Cell Cycle Arrest and Cell Differentiation.

Τhe effect of docosahexaenoic acid (DHA, an omega-3 polyunsaturated fatty acid) upon the proliferation of EoL-1 (Eosinophilic leukemia) cell line was assessed, while additional cellular events during the antiproliferative action were recorded. DHA inhibited EoL-1 cells growth dose-dependently by inducing growth arrest at G0/1 phase of the cell cycle. After DHA addition to the cells, the expression of MYC oncogene was decreased, PTAFR-mRNA overexpression was observed which was used as a marker of differentiation, and PLA2G4A-mRNA increase was recorded. The enzymatic activities of phospholipase A2 (PLA2), a group of hydrolytic enzymes, whose action precedes and leads to PAF biosynthesis through the remodeling pathway, as well as platelet activating factor acetylhydrolase (PAFAH) which hydrolyses and deactivates PAF, were also measured. DHA had an effect on the levels of both the intracellular and secreted activities of PLA2 and PAFAH. The inflammatory cytokines IL-6 and TNF-α were also detected in high levels. In conclusion, DHA-induced EoL-1 cells differentiation was correlated with downregulation of MYC oncogene, overexpression of PTAFR and PLA2G4A-mRNAs, increase of the inflammatory cytokines production, and alteration of the enzymatic activities that regulate PAF levels. DHA is a natural substance and the understanding of its action on EoL-1 cells on molecular level could be useful in further investigation as a future therapeutic tool against F/P + hypereosinophilic syndrome.

Tricarboxylic Acid Cycle Activity and Glycerophosphocholine Lipid Recycling Support Dendritic Cell Cytokine Induction in Response to Fungal Patterns

Increased glycolysis parallels immune cell activation, but the role of pyruvate flux remains largely unexplored. We show that stimulation of dendritic cells with the fungal surrogate zymosan induces a drop of pyruvate, citrate, itaconate, and α-ketoglutarate, as well as an increase in oxaloacetate, succinate, lactate, oxygen consumption, and activation of pyruvate dehydrogenase activity. The expression of IL10 and IL23A (the gene encoding the p19 chain of IL-23) depended on mitochondrial activity. Pyruvate underpinned histone H3 acetylation and acetate rescued the effect of mitochondrial pyruvate carrier inhibition, most likely because of compensation by the acetyl-CoA producing enzyme ACSS2. Mice lacking the receptor of the lipid mediator PAF (1-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine), showed a reduced production of IL-10 and IL-23 that is explained by the requirement of acetyl-CoA for PAF biosynthesis and the ensuing autocrine function of PAF. Acetyl-CoA is the nexus in the intertwining of phospholipid recycling and energetic metabolism involved in cytokine induction.

Nuclear localization of platelet-activating factor receptor controls retinal neovascularization.

Platelet-activating factor (PAF) is a pleiotropic phospholipid with proinflammatory, procoagulant and angiogenic actions on the vasculature. We and others have reported the presence of PAF receptor (Ptafr) at intracellular sites such as the nucleus. However, mechanisms of localization and physiologic functions of intracellular Ptafr remain poorly understood. We hereby identify the importance of C-terminal motif of the receptor and uncover novel roles of Rab11a GTPase and importin-5 in nuclear translocation of Ptafr in primary human retinal microvascular endothelial cells. Nuclear localization of Ptafr is independent of exogenous PAF stimulation as well as intracellular PAF biosynthesis. Moreover, nuclear Ptafr is responsible for the upregulation of unique set of growth factors, including vascular endothelial growth factor, in vitro and ex vivo. We further corroborate the intracrine PAF signaling, resulting in angiogenesis in vivo, using Ptafr antagonists with distinct plasma membrane permeability. Collectively, our findings show that nuclear Ptafr translocates in an agonist-independent manner, and distinctive functions of Ptafr based on its cellular localization point to another dimension needed for pharmacologic selectivity of drugs.

Antiobese function of platelet‐activating factor: increased adiposity in platelet‐activating factor receptor‐deficient mice with age

Platelet-activating factor receptor (PAFR)-deficient mice developed a more severe obese state characterized by higher body mass (~25%) and epididymal fat mass (~55%) with age than that of wild-type (WT) littermates. PAFR-deficient mice did not show changes in the expression of critical genes involved in anabolic and catabolic metabolism in adipose, liver, and muscle tissues between 6 and 36 wk. However, a 38-81% reduction in β3/β1-adrenergic receptor (AR) and uncoupling protein 1 (UCP1) mRNA and protein levels was observed in the interscapular brown adipose tissue (BAT) of PAFR-deficient mice. Whereas a single injection of the β3-adrenergic agonist, CL-316,243 (25 μg/kg) increased temperatures in the brown fat and rectums of WT mice, this increase in temperature was markedly suppressed in PAFR-deficient mice. Acetyl-CoA:lyso-platelet-activating factor (PAF) acetyltransferase, which is involved in PAF biosynthesis, and the PAF receptor were predominantly localized in BAT macrophages, whereas brown adipocytes possessed the enzyme and functional PAF receptors. The stimulation of brown adipocytes by PAF induced the expression of β3-AR mRNA and protein (1.5- and 1.9-fold, respectively), but not that of UCP1. These results indicate that obesity in PAFR-deficient mice resulted from impaired BAT activity and suggest that the antiobese function of PAF occurs through β3-AR/UCP1 expression in BAT.

Fish polar lipids retard atherosclerosis in rabbits by down-regulating PAF biosynthesis and up-regulating PAF catabolism

BackgroundPlatelet activating factor (PAF) has been proposed as a key factor and initial trigger in atherosclerosis. Recently, a modulation of PAF metabolism by bioactive food constituents has been suggested. In this study we investigated the effect of fish polar lipid consumption on PAF metabolism.ResultsThe specific activities of four PAF metabolic enzymes; in leukocytes, platelets and plasma, and PAF concentration; either in blood cells or plasma were determined. Samples were acquired at the beginning and at the end of a previously conducted study in male New Zealand white rabbits that were fed for 45 days with atherogenic diet supplemented (group-B, n = 6) or not (group-A, n = 6) with gilthead sea bream (Sparus aurata) polar lipids.The specific activity of PAF-Acetylhydrolase (PAF-AH); a catabolic enzyme of PAF, was decreased in rabbits' platelets of both A and B groups and in rabbits' leukocytes of group A (p < 0.05). On the other hand the specific activity of Lipoprotein-associated Phospholipase A2 (Lp-PLA2); the catabolic enzyme of PAF in plasma was increased in both A and B groups in both leukocytes and platelets (p < 0.05). PAF-cholinephosphotransferase (PAF-CPT); a biosynthetic enzyme of PAF showed increased specific activity only in rabbits' leukocytes of group A (p < 0.05). Neither of the two groups showed any change in Lyso-PAF-acetyltransferase (Lyso-PAF-AT) specific activity (p > 0.05). Free and bound PAF levels increased in group A while decreased in group B (p < 0.05).ConclusionsGilthead sea bream (Sparus aurata) polar lipids modulate PAF metabolism upon atherosclerotic conditions in rabbits leading to lower PAF levels and activity in blood of rabbits with reduced early atherosclerotic lesions compared to control group.

Tea Polyphenols Inhibit Acetyl-CoA:1-Alkyl-sn-Glycero-3-Phosphocholine Acetyltransferase (a Key Enzyme in Platelet-Activating Factor Biosynthesis) and Platelet-Activating Factor-Induced Platelet Aggregation

Background: Tea extracts have antiallergic and anti-inflammatory actions in rats and mice. However the mechanism through which tea polyphenols act in vivo are still incompletely understood. We found inhibitory effects of black tea extracts on an fMLP-induced aggregating response in a rabbit platelet-polymorphonuclear leukocyte (PMN) system. Method: To elucidate whether 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF) production in PMNs and/or PAF-stimulated platelet activation were inhibited, the effects of tea polyphenols were investigated on the enzyme activity of acetyl-CoA:1-alkyl-sn-glycero-3-phosphocholine acetyltransferase (EC 2.3.1.67), PAF biosynthesis in A23187-activated rabbit PMNs, and rabbit platelet aggregation. By comparing the inhibitory effects of 31 galloyl esters and gallic acid, the structure-inhibitory activity relationship was characterized. Results: Theaflavin and its galloyl esters and pentagalloyl glucose were found to be potent inhibitors of the acetyltransferase (IC₅₀ = 28–58 µM) and the PAF biosynthesis as well as (–)-epicatechin-3-O-gallate (IC₅₀ = 72 ± 13 µM) and (–)-epigallocatechin-3-O-gallate (IC₅₀ = 46 ± 6 µM). On the other hand, flavan-3-ols without galloyl group at C-3 and gallic acid did not show significant enzyme inhibition. In addition, theaflavin and its galloyl esters (IC₅₀ = 32–77 µM) and geranyl gallate, farnesyl gallate and geranylgeranyl gallate (IC₅₀ = 6.4–7.6 µM) were found to be potent inhibitors of PAF- and TPA-induced rabbit platelet aggregation but not A23187-induced aggregation. Conclusions: Theaflavin and its galloyl esters in black tea extract, and isoprenyl gallates were potent inhibitors of PAF synthesis and platelet aggregation and these activities may be relevant to the claimed therapeutic effects of tea extracts.

Application of a TCA-precipitation method for the determination of 1-alkyl-sn-glycero-3-phosphate:Acetyl-CoA acetyltransferase in human renal tissue

The activity of 1-alkyl-sn-glycero-3-phosphate:Acetyl-CoA acetyltransferase, which catalyses the first step of the de novo biosynthesis of PAF, was determined and characterised in cortical and medullary human renal tissues. A novel thin-layer chromatographic system as well as a trichloroacetic acid precipitation method, were utilised in order to determine the enzyme’s activity. The acetyltransferase activity was associated with the membranous fractions of the renal tissue, it showed an optimum pH of 8.4 and it had a bell-shaped dependence on BSA concentration. One or more disulphide bonds were necessary for the action of acetyltransferase while the enzyme seemed to be independent from divalent cations. Two assay products were extracted from the incubation mixture namely alkylacetylphosphatidic acid, produced by the acetylating action of the acetyltransferase on alkyllyso-phosphatidic acid and alkylacetyl-glycerol, which is produced by the action of a phosphohydrolase on alkylacetylphosphatidic acid. The presence of NaF in the assay mixture resulted to a decreased degradation of alkylacetylphosphatidic acid, as well as to an increased overall product formation. Cortical and medullary acetyltransferases share similar biochemical properties and there is no statistical difference between the two activities.

Application of a TCA-precipitation method for the determination of 1-alkyl-sn-glycero-3-phosphate:Acetyl-CoA acetyltransferase in human renal tissue

The activity of 1-alkyl-sn-glycero-3-phosphate:Acetyl-CoA acetyltransferase, which catalyses the first step of the de novo biosynthesis of PAF, was determined and characterised in cortical and medullary human renal tissues. A novel thin-layer chromatographic system as well as a trichloroacetic acid precipitation method, were utilised in order to determine the enzyme’s activity. The acetyltransferase activity was associated with the membranous fractions of the renal tissue, it showed an optimum pH of 8.4 and it had a bell-shaped dependence on BSA concentration. One or more disulphide bonds were necessary for the action of acetyltransferase while the enzyme seemed to be independent from divalent cations. Two assay products were extracted from the incubation mixture namely alkylacetylphosphatidic acid, produced by the acetylating action of the acetyltransferase on alkyllyso-phosphatidic acid and alkylacetyl-glycerol, which is produced by the action of a phosphohydrolase on alkylacetylphosphatidic acid. The presence of NaF in the assay mixture resulted to a decreased degradation of alkylacetylphosphatidic acid, as well as to an increased overall product formation. Cortical and medullary acetyltransferases share similar biochemical properties and there is no statistical difference between the two activities.

Regulation of platelet-activating factor synthesis in human monocytes by dipalmitoyl phosphatidylcholine.

Platelet-activating factor (PAF) has a major role in inflammatory responses within the lung. This study investigates the effect of pulmonary surfactant on the synthesis of PAF in human monocytic cells. The pulmonary surfactant preparation Curosurf significantly inhibited lipopolysaccharide (LPS)-stimulated PAF biosynthesis (P<0.01) in a human monocytic cell line, Mono mac-6 (MM6), as determined by (3)H PAF scintillation-proximity assay. The inhibitory properties of surfactant were determined to be associated, at least in part, with the 1,2-dipalmitoyl phosphatidylcholine (DPPC) component of surfactant. DPPC alone also inhibited LPS-stimulated PAF biosynthesis in human peripheral blood monocytes. DPPC treatment did not affect LPS-stimulated phospholipase A(2) activity in MM6 cell lysates. However, DPPC significantly inhibited LPS-stimulated coenzyme A (CoA)-independent transacylase and acetyl CoA:lyso-PAF acetyltransferase activity. DPPC treatment of MM6 cells decreased plasma membrane fluidity as demonstrated by electron paramagnetic resonance spectroscopy coupled with spin labeling. Taken together, these findings indicate that pulmonary surfactant, particularly the DPPC component, can inhibit LPS-stimulated PAF production via perturbation of the cell membrane, which inhibits the activity of specific membrane-associated enzymes involved in PAF biosynthesis.

Roles of Cytosolic Phospholipase A2 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 A2 knock-out (cPLA2-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, cPLA2-KO mice were deficient in the PAF production. These results provide the direct evidence while cPLA2 is essential in the production of PAF, PAF receptor deficiency has little effect on the PAF production.

S7-2 - Regulation of PAF biosynthesis and its role in secretion in salivary glands

S7-2 - Regulation of PAF biosynthesis and its role in secretion in salivary glands

Pharmacological evidence of a role for platelet activating factor as a modulator of vasomotor tone and blood pressure

The purpose of the present study was to investigate the role of platelet-activating factor (PAF, 1- O-hexadecyl-2-acetyl- sn-glyceryl-3-phosphorylcholine), a phospholipid mediator synthesized by endothelial and smooth muscle cells, in the modulation of vascular tone and blood pressure. In pentobarbitone-anaesthetised rabbits, unloading of the carotid sinus baroreceptors by a bilateral carotid artery occlusion elicited a reflex rise in arterial pressure which was markedly potentiated by pretreating the animals with the PAF receptor antagonists WEB 2086 [3-4-(2-chlorphenyl)-9-methyl-6 H-thieno-3,2 f-1,2,4-triazolo-4,3 a-1,4-diazepin-2-yl-(4-morpholinyl)- l-propanone; 2, 5 or 10 mg kg −1, i.v.] or BN 52021 (ginkgolide B; 0.1, 0.3 or 1.0 mg kg −1, i.v.). The increases in systemic vascular resistance induced by noradrenaline (30 μg kg −1, i.v.) or by the central activation of the sympathetic nervous system with glutamate (1 mg kg −1, intracerebroventricular) were also significantly potentiated in animals pretreated with WEB 2086 (5 mg kg −1, i.v.). In contrast, pretreatment with the cyclooxygenase inhibitor indomethacin (3 mg kg −1, i.v.) did not affect the haemodynamic actions of noradrenaline, thus excluding the possibility that prostacyclin may modulate the potentiating effect. To further confirm that PAF is released during systemic vasoconstriction, the cardiovascular PAF receptors were desensitized by the daily administration of PAF (3 μg kg −1, i.v.) for seven days. This procedure significantly reduced the intensity and duration of the hypotensive response to a subsequent PAF injection (3 μg kg −1, i.v.). In desensitized animals, the hypertensive response to bilateral carotid artery occlusion was potentiated to the same extent as in the animals treated with PAF receptor antagonists. Inhibition of PAF biosynthesis by pretreatment of the animals with the phospholipase A 2 inhibitor mepacrine (5 mg kg −1, i.v.) also enhanced the increase in blood pressure elicited by carotid artery occlusion. We conclude that PAF is involved in the acute but not basal modulation of vasomotor tone and, hence, arterial pressure, probably by a negative feedback mechanism triggered by important increases in the vascular tone.

Differential metabolism of exogenous platelet-activating factor by glandular epithelial and stromal cells of rabbit endometrium.

Significant changes in platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine) concentration have been observed in rabbit endometrium during the preimplantation period, but, under in vitro conditions, constitutive PAF biosynthesis by isolated endometrial tissues was not easily demonstrable. Relative changes in enzymes involved in the synthesis and metabolism of PAF in the tissues may account for this disparity. In addition, during this period of preimplantation, marked changes in PAF receptor concentration have been noted. The present study examines the factors that may modulate the metabolism of exogenous [3H]PAF in the endometrium of rabbits on day 6 of pregnancy. Since preferential [3H]PAF binding in situ by the glandular epithelial, but not by the stromal, cells was demonstrated, their cell-specific metabolism of exogenous [3H]PAF was also examined. After entry into the endometrial cell, [3H]PAF was rapidly metabolized by the sequential action of cytosolic Ca(2+)-independent acetylhydrolase to [3H]lyso-PAF and this was in turn acylated by membrane-associated transacylase to [3H]alkylacyl-glycerylphosphorylcholine. PAF resynthesis was not observed and, in stromal cells, there was a significant build-up of [3H]lyso-PAF, suggesting that lyso-PAF:acetyl-CoA acetyl-transferase may be a limiting factor. In the glandular epithelial cells, however, there was a significant accumulation of a neutral lipid without a significant build-up of [3H]lyso-PAF or [3H]PAF. The neutral lipid co-migrated with the product of phospholipase C-catalysed metabolism of PAF and authentic 1-O-hexadecyl-2-acetyl-glycerol. In addition, the elution times of phospholipase C digestion of C18 PAF and the neutral lipid produced by cellular metabolism of [3H]PAF, determined by gas chromatography/flame ionization detection, were similar. It seems that it is the synthesis of the neutral lipid from reacetylated [3H]lyso-PAF that prevented [3H]PAF accumulation under in vitro conditions. This is the first documentation of the synthesis of this lipid in the mammalian uterus. The lipid may serve as the precursor for de novo PAF synthesis in the glandular epithelial cells during endometrial proliferation.

Lyso-PAF:acetyl-CoA acetyltransferase and CDP-choline cholinephosphotransferase activities in the rabbit endometrium

Endometrial platelet-activating factor (PAF, 1- O-alkyl-2-acetyl- sn-glycero-3-phos-phorylcholine) levels change significantly during the pre-implantation period in the rabbit uterus, but under in vitro culture conditions, constitutive PAF biosynthesis by isolated endometrial tissues is not easily demonstrable. Rapid metabolism of PAF relative to its synthesis may account for this disparity because we have recently shown that in stromal cells there is a significant build-up of lyso-PAF suggesting that lyso-PAF:acetyl-CoA acetyltransferase might be a limiting factor. In the glandular epithelial cells however, the lyso-PAF build-up was replaced by a significant accumulation of a neutral lipid which was tentatively identified as 1- O-hexadecyl-2-acetylglycerol. It was hypothesized that, during endometrial growth and development, this lipid might serve as the substrate for the alkylacetylglycerol CDP-choline cholinephosphotransferase enzyme for PAF synthesis via the de novo pathway. We have therefore examined the activities of lyso-PAF:acetyl-CoA acetyltransferase and the CDP-cholinephosphotransferase enzymes. Microsomal preparations containing lyso-PAF:acetyl-CoA acetyltransferase activity catalyzed the incorporation of [3H]acetyl-CoA lyso-PAF into two distinct lipid products. One co-migrated with authentic PAF and the other with 1- O-hexadecyl-2-acetylglycerol, the latter being formed subsequent to PAF formation. The alkylacetylglycerol CDP-choline cholinephosphotransferase enzyme, which would potentially utilize the alkylacetylglycerol synthesized via the remodeling pathway, was also demonstrable. Unlike the species present in other tissues however, it was found to be sensitive to the presence of 10 mM DTT. The diacylglycerol CDP-choline cholinephosphotransferase species was also demonstrable and supported the synthesis of both PAF and phosphatidylcholine, in the absence of DTT, when only the synthesis of phosphatidylcholine was expected. It is hypothesized that the rabbit endometrium possesses active enzymes which may catalyze PAF synthesis via both the de novo and ‘remodeling’ pathways.

The CoA-independent transacylase in PAF biosynthesis: tissue distribution and molecular species selectivity

Microsomal membranes from six different rat tissues (spleen, lung, kidney, brain, testis, and liver) were found to possess CoA-independent transacylase activity that could both acylate lyso-[ 3 H] PAF (1-[ 3 H] hexadecyl-2- lyso-sn- glycero-3- phosphocholine) and then deacylate the 1-[ 3 H] hexadecyl-2- acyl-sn- glycero-3- phosphocholine product via the transacylation of added exogenous 1-alk-1′-enyl-2-lyso- sn-glycero-3-phosphoethanolamine. Platelet-activating factor ( 1-[ 3 H] hexadecyl-2- acetyl-sn- glycero-3- phosphocholine) was produced when acetyl-CoA was added to the spleen microsomes during generation of lyso-[ 3H]PAF by the transacylases. More extensive studies with subcellular fractions from spleen revealed that, in addition to microsomes, the transacylase activities were also present in the 15 000 × g membrane fraction but not in the cytosol. Analysis of molecular species of 1-[ 3 H] hexadecyl-2- acyl-sn- glycero-3- phosphocholine before and after addition of 1-alk-1′-enyl-2-lyso- sn-glycero-3-phosphoethanolamine as the acyl acceptor demonstrated a high selectivity for polyunsaturated fatty acids (> 3 double bonds/acyl group) in both the acylation and deacylation processes that occurred in testicular microsomal membranes. The transfer of acyl groups by the transacylase appeared to be equally effective for either arachidonic or docosapentaenoic( n − 6) fatty acids, whereas linoleic and oleic fatty acids were not transferred from 1-[ 3 H] hexadecyl-2- acyl-sn- glycero-3- phosphocholine following the addition of 1-alk-1′-enyl-2-yso- sn-glycero-3-phosphoethanolamine. Similar experiments with the membrane fraction of undifferentiated HL-60 cells showed that arachidonic acid supplementation of intact cells enhanced both the CoA-independent transacylation of lyso-[ 3H]PAF and the subsequent deacylation of 1-[ 3 H] hexadecyl-2- acyl-sn- glycero-3- phosphocholine caused by addition of 1-alk-1′-enyl-2-acyl- sn-glycero-3-phosphoethanolamine. Differentiation of the HL-60 cells into a neutrophil-like form had no effect on the transacylase activity. Our results indicate the PAF-related transacylase is widely distributed among tissues and, although highly selective for polyunsaturated acyl groups, does not discriminate selectively among the polyunsaturates.

Cellular and Topical in vivo Inflammatory Murine Models in the Evaluation of Inhibitors of Phospholipase A&lt;sub&gt;2&lt;/sub&gt;

Several novel inhibitors of human synovial fluid phospholipase A2 (HSF-PLA2) were evaluated in cellular models of inflammatory mediator release (murine macrophage and human neutrophil) and topical in vivo inflammatory skin models in mice to ascertain the scope of effects which might be observed for PLA2 inhibitors. Potent inhibition of HSF-PLA2 in vitro can be observed with compounds such as scalaradial and ellagic acid, which both have IC50 values of 0.02 microM (using autoclaved [3H]-arachidonic-acid (AA)-labelled Escherichia coli membranes as substrate). Luffariellolide, a manoalide analog, and aristolochic acid are less potent (IC50 = 5 and 46 microM, respectively) in this assay. An interesting observation is that ellagic acid in cellular assays does not inhibit macrophage eicosanoid production and only 30% inhibition of PAF biosynthesis can be obtained at 50 microM in the human neutrophil. Possibly due to its irreversible mechanism of action, scalaradial retained its potent activity in both the macrophage (IC50 for PGE2 production = 0.05 microM) and neutrophil assays (IC50 for PAF biosynthesis = 1 microM). Aristolochic acid is active in these cellular assays (macrophage IC50 = 2.5 microM and neutrophil IC50 = 100 microM), but is consistently less active than either scalaradial or luffariellolide. The relative potencies of these compounds were determined in several murine in vivo inflammatory models such as oxazolone contact hypersensitivity, AA-induced ear edema and phorbol ester (PMA)-induced ear edema. In the mouse model of oxazolone contact hypersensitivity, these PLA2 inhibitors have little effect (< or = 30% inhibition at 400 micrograms/ear) with scalaradial and luffariellolide being less effective than either aristolochic or ellagic acid. PMA-induced ear edema was effectively inhibited by scalaradial, luffariellolide and aristolochic acid (ED50 = 70, 50 and 50 micrograms/ear, respectively) whereas ellagic acid was less effective (ED50 = 230 micrograms/ear). In AA-induced ear edema, these PLA2 inhibitors had minimal effects, as would be expected for compounds which inhibit PLA2. These results, especially those of ellagic acid, suggest that caution should be taken in the extrapolation of potency against a purified human extracellular type PLA2 to the scope of activities these compounds might have in the cellular and in vivo models. The consistency of scalaradial and luffariellolide may be inherent to their irreversible mechanism of action, which is a factor to be accounted for in the extrapolation of enzyme data to cellular and in vivo models.

Effects of organometals on cellular signaling. II. Inhibition of reincorporation of free arachidonic acid and influence on paf-acether synthesis by triethyllead.

Organometal compounds affect many enzymes, especially those containing SH-groups as acyl- and acetyltransferases involved in lysophospholipid reacylation. In HL-60 cells, organotin and -lead compounds stimulate phospholipase A2 activity, contributing thus to increase the level of lysophospholipids. In the present study, we have tested whether paf-acether (paf) biosynthesis was affected by treatment with triethyllead (Et3PbCl) in HL-60 cells. Et3PbCl inhibits the incorporation of exogenous arachidonic acid in the presence of high (> or = 50 microM) but not low concentrations (< or = 1 microM). High concentrations of the lead compound are unable to induce paf formation by itself, however, lower concentrations (< or = 10 microM) acted synergistically with TPA or fMLP to stimulate paf formation. Whereas unstimulated cells produced 0.4 pmole paf/2 x 10(6) cells, the stimulation with low fMLP (0.1 microM) resulted in the synthesis of 1.7 pmole and with low TPA (2 ng/ml) in 0.5 pmole paf. Preincubation of the cells with 10 microM Et3PbCl for 20 to 30 min increased the amount of paf formed by these cells to 3.3 pmole after treatment with 0.1 microM fMLP and 1.5 pmole after TPA. Furthermore, the results showed an inhibition of acetyltransferase (the key enzyme of paf synthesis) by the high and not by low concentrations of the lead compound. We conclude that low concentrations of Et3PbCl (< or = 10 microM) may act as a synergistic inducer of paf synthesis initiated via a receptor-coupled stimulation.