PAF Synthesis Research Articles

Rational design of new in situ reduction of Ni(II) catalytic system for low‐cost and large‐scale preparation of porous aromatic frameworks

AbstractPorous aromatic framework 1 (PAF‐1) is an extremely representative nanoporous organic framework owing to its high stability and exceptionally high surface area. Currently, the synthesis of PAF‐1 is catalyzed by the Ni(COD)2/COD/bpy system, suffering from great instability and high cost. Herein, we developed an in situ reduction of the Ni(II) catalytic system to synthesize PAF‐1 in low cost and high yield. The active Ni(0) species produced from the NiCl2/bpy/NaI/Mg catalyst system can effectively catalyze homocoupling of tetrakis(4‐bromophenyl)methane at the room temperature to form PAF‐1 with high Brunauer–Emmett–Teller (BET)‐specific surface area up to 4948 m2 g−1 (Langmuir surface area, 6785 m2 g−1). The possible halogen exchange and dehalogenation coupling mechanisms for this new catalytic process in PAF's synthesis are discussed in detail. The efficiency and universality of this innovative catalyst system have also been demonstrated in other PAFs' synthesis. This work provides a cheap, facile, and efficient method for scalable synthesis of PAFs and explores their application for high‐pressure storage of Xe and Kr.

Physiological and Biochemical Consequences of Exposure of Neonatal Rats to Chronic Hypoxia

We studied effect of chronic hypoxia (CH) on expression of platelet activating factor receptor (PAFR) by neonatal rats (pups). We hypothesized that PAFR antagonist will prevent pulmonary hypertension (PH) in pups exposed to CH. Pups were placed in an air-tight chamber ventilated with 13% oxygen, hypoxia (Hpx) or room air normoxia (Nmx) from 1d to 22d of age. Three groups of pups were studied (each group, n=10-14 pups): Group1, pups in Nmx; Group2, pups in Hpx given 5mg/kg PAFR receptor antagonist, WEB 2170, IP, every other day for 22d, (Hpx+WEB); Group3, pups in Hpx control. Hemotocrit, RV/LV+S, PAF binding, PAF synthesis, and PAFR expression were determined. Hyx control group had 2-fold higher RV/LV+S than Nmx group and PAFR antagonist decreased RV/LV+S to the Nmx control value. Lungs of pups in Hpx expressed more PAFR protein than Hpx+WEB and Nmx groups. Additionally, Hpx increased PAF synthesis and PAFR binding whereas WEB treatment decreased PAFR binding, but produced no difference in PAF synthesis compared to Nmx group. Hpx increased NF-kB p65 and TLR4 expression. WEB treatment abrogated expression or NF-kB p65 and TLR4 proteins. Our findings show that chronic hypoxia induces expression of PAFR, NF-kB p65 and TLR4 by pups’ lungs and suggest that increased PAFR expression may be responsible for the right ventricular hypertrophy and PH. Thus a PAFR antagonist may offer a therapeutic intervention for CH-induced PH in human neonates.

Role of spinal LPCAT2, an inducible PAF synthesis enzyme on development and maintenance of painful peripheral neuropathy models in mice

Role of spinal LPCAT2, an inducible PAF synthesis enzyme on development and maintenance of painful peripheral neuropathy models in mice

Fibronectin (FN) decreases glomerular lesions and synthesis of tumour necrosis factor-alpha (TNF-alpha), platelet-activating factor (PAF) and FN in proliferative glomerulonephritis.

We have studied the effect of therapy with plasma FN on glomerular synthesis of PAF, TNF-alpha and FN, in experimental proliferative glomerulonephritis. Glomerular PAF, TNF-alpha and FN production were increased in rats with nephritis. Peak glomerular PAF production preceded, while peak glomerular TNF-alpha bioactivity coincided with maximal proteinuria. Rats treated with FN (5 mg/kg per 48 h) for 15 days had less proteinuria, glomerular and interstitial cell infiltration and glomerular PAF, TNF-alpha and FN synthesis than non-treated rats. In order to characterize further the mechanisms of action of FN, healthy rats were injected with either FN or saline. Peripheral blood mononuclear cells and neutrophils from healthy rats injected with FN secreted less TNF-alpha and PAF, respectively, than those obtained from saline-treated rats. Our data suggest that the beneficial effect of FN may be related to decreased number of glomerular leucocytes and decreased synthesis of inflammatory mediators and extracellular matrix.

Angiopoietins-1 and -2 are both capable of mediating endothelial PAF synthesis: intracellular signaling pathways

Angiopoietins-1 and -2 are both capable of mediating endothelial PAF synthesis: intracellular signaling pathways

Stress-induced platelet-activating factor synthesis in human neutrophils

Platelet-activating factor (1- O-alkyl-2-acetyl- sn-glycero-3-phosphocholine; PAF) is a potent inflammatory mediator produced by cells in response to physical or chemical stress. The mechanisms linking cell injury to PAF synthesis are unknown. We used liquid chromatography-tandem mass spectrometry to investigate stress-induced PAF synthesis in human neutrophils. PAF synthesis induced by extracellular pH 5.4 correlated with the activation of a stress-activated kinase, p38 mitogen-activated protein kinase (MAPK), and was blocked by the p38 MAPK inhibitor SB 203580. A key enzyme of PAF synthesis, acetyl-CoA:lysoPAF acetyltransferase, which we have previously shown is a target of p38 MAPK, was also activated in an SB 203580-sensitive fashion. Another MAPK pathway, extracellular signal-regulated kinase-1/2 (ERK-1/2), was also activated. Surprisingly, the pharmacological blockade of the ERK-1/2 pathway with PD 98059 did not block, but rather enhanced, PAF accumulation. Two unexpected actions of PD 98059 may underlie this phenomenon: an augmentation of stress-induced p38 MAPK phosphorylation and an inhibition of PAF catabolism. The latter effect did not appear to be due to a direct inhibition of PAF acetylhydrolase. Finally, similar results were obtained using another form of cellular stress, hypertonic sodium chloride. These data are consistent with a model in which stress-induced PAF accumulation is regulated positively by p38 MAPK and negatively by ERK-1/2. Such a model contrasts with the PAF accumulation induced by other forms of stimulation, which we and others have found is up-regulated by both p38 MAPK and ERK-1/2.

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.

Protective effect of ketoprofen lysine salt on interleukin-1beta and bradykinin induced inflammatory changes in hamster cheek pouch microcirculation.

The purpose of this study was to assess the efficacy of topically applied ketoprofen lysine salt (KLS), a cyclooxygenase inhibitor, against the inflammatory changes induced by interleukin-1beta (IL-1beta) and bradykinin (BK) in hamster cheek pouch microcirculation. In addition, we characterised the pharmacological regulation of IL-1beta activity in this model. Male Syrian hamsters were used. Microcirculation was visualised by fluorescent microscopy. Leukocyte adhesion, permeability, perfused capillary length (PCL) and capillary red blood cell (RBC) velocity were evaluated. KLS (25 microg/ml/min to 1.6 mg/ml/min) was topically applied for 3 min before topically administered IL-1beta (1microg/ml) and BK (10(-4) M). Monoclonal anti-mouse IL-1beta receptor antagonist (200 ng/ml), BK-B2 receptor antagonist (10(-6) M), PAF inhibitor (10(-5) M) and cycloheximide (10 microg/ml) were added topically 15, 10, 15 and 60 min, respectively, before IL-1beta (1 microg/ml). IL-1beta caused a significant increase in microvascular permeability, a decrease in capillary RBC velocity followed by increased leukocyte adhesion in postcapillary venules. BK caused a marked increase in leukocyte adhesion and no decrease in PCL and RBC velocity. Treatment with KLS significantly inhibited both the leukocyte adhesion and microvascular leakage induced by the two mediators. The inflammatory effects induced by IL-1beta were reduced by blockade of IL-1beta receptors and by a BK-B2 receptor antagonist but were not affected by a PAF antagonist and protein synthesis inhibition. These results demonstrate that KLS is effective in preventing early inflammatory changes induced by both IL-1beta and BK in the capillary network. Prostaglandin release and BK are essential components for IL-beta mediated responses, whereas neither PAF nor new protein synthesis appear to be linked to the early inflammatory changes induced by IL-1beta.

Maturational Changes in Ovine Pulmonary Metabolism of Platelet-Activating Factor: Implications for Postnatal Adaptation

We recently reported that PAF acetylhydrolase (PAF-Ah) mRNA level and PAF-Ah activity in lamb lungs are up-regulated in the immediate newborn period, thereby facilitating the fall in postnatal PAF levels as well as a fall in pulmonary vascular resistance (B. O. Ibe, F. C. Sardar, and J. U. Raj, Mol Genet Metab 69:46–55, 2000). We have studied hypoxia effects on PAF synthesis and PAF-Ah activity in fetal lamb pulmonary arterial smooth muscle cells (FPASMC) and endothelial cells (FPAEC). We also studied PAF synthesis by platelets, and PAF-Ah activity in plasma of perinatal lambs at different ages. PAF synthesis (means ± SEM, pmol/106 cells) by SMC in baseline was 168 ± 27 and increased 3-fold on stimulation with A23187. Hypoxia augmented A23187-stimulated PAF synthesis by 30%. In FPAEC, baseline synthesis was 0.54 ± 0.062 and increased 3-fold to 1.72 ± .34. Hypoxia had no effect on PAF synthesis by EC. FPASMC produced over 300-fold more PAF than FPAEC. PAF synthesis by platelets was 47.02 ± 7.1, 63.4 ± 6.6, 71.5 ± 9.9, and 62.2 ± 5.2 for fetal, and newborn lambs <2 h, <1 day, and 6–12 days, old, respectively. PAF synthesis by platelets of <1 day-old lambs was different from that of fetal lambs. PAF-Ah activity (nmol lyso-PAF/min/mg protein) by FPASMC in normoxia was 3.41 ± 0.38 which was 50% higher than the rate in hypoxia. Activity in FPAEC was 1.75 ± 0.37 which was not different from hypoxia. PAF-Ah activity in fetal lamb plasma was 47.83 ± 6.87 which was different from 155.32 ± 12.10, the activity in plasma of newborn <1 day old. Activity in the other perinatal lambs did not differ from fetal or newborn <1 d. Our data suggest that lower pulmonary vascular PAF synthesis in normoxia together with higher PAF-Ah activity during immediate postnatal period is necessary to ensure rapid catabolism of PAF in vivo so as to facilitate postnatal adaptation of the pulmonary and systemic circulations.

Regulation of VEGF-induced endothelial cell PAF synthesis: role of p42/44 MAPK, p38 MAPK and PI3K pathways.

1. Vascular endothelial growth factor (VEGF) is a potent angiogenic and inflammatory mediator. We have recently shown that this latter effect requires the activation of Flk-1 receptor and subsequent endothelial cell (EC) PAF synthesis. However, the intracellular events that regulate EC PAF synthesis upon Flk-1 stimulation by VEGF remain to be elucidated. 2. Using specific inhibitors and Western blot analysis, we herein report that in bovine aortic endothelial cells (BAEC), VEGF induces the synthesis of PAF through the cascade activation of Flk-1 receptor, phospholipase Cgamma (PLCgamma), protein kinase C (PKC) and p42/44 mitogen-activated protein kinases (MAPK). 3. Moreover, we demonstrate that VEGF-mediated PAF synthesis requires the activation of p38 MAPK, likely by directing the conversion of lyso-PAF to PAF. 4. Interestingly, we observed that VEGF also promoted the activation of the phosphatidyl inositol-3-phosphate kinase (PI3K) pathway, and that its blockade potentiated PAF synthesis following a VEGF treatment. Consequently, it appears that the PI3K pathway acts as a negative regulator of EC PAF synthesis. 5. Taken together, these results allow a better understanding of the intracellular events activated upon EC stimulation by VEGF, and shed a new light on the mechanisms by which VEGF induces PAF synthesis.

VEGF stimulation of endothelial cell PAF synthesis is mediated by group V 14 kDa secretory phospholipase A2.

1. Vascular endothelial growth factor (VEGF) is a potent inducer of inflammation, and we have shown that this latter effect is mediated through endothelial cell (EC) PAF synthesis. Since the phospholipid remodelling pathway enzymes (CoA-independent transacylase, CoA-IT; phospholipase A2, PLA2; and lyso-PAF acetyltransferase, lyso-PAF-AT) may participate in PAF synthesis, we assessed their contribution to VEGF-induced PAF synthesis in bovine aortic EC (BAEC) and human umbilical vein EC (HUVEC). 2. VEGF enhanced BAEC and HUVEC PAF synthesis by up to 28 and 4 fold above basal levels respectively. 3. A pretreatment with a CoA-IT and lyso-PAF-AT inhibitor (Sanguinarin; 500 nM) blocked VEGF-induced PAF synthesis by 95%, a specific CoA-IT inhibitor (SKF45905; 10 - 50 microM) was without effect, confirming the crucial role of the PLA2 and lyso-PAF-AT. 4. Treatment with secreted PLA2 (sPLA2) inhibitors which have been shown to inhibit both groups IIA and V sPLA2 (SB203347; 10 microM and LY311727; 100 microM) blocked EC PAF synthesis by up to 90%, whereas selective inhibition of group IIA sPLA2 (LY311727; 1 microM) had no significant effect. 5. RT - PCR and Western blot analyses demonstrated the presence of group V sPLA2 whereas group IIA sPLA2 was undetected in EC. 6. Treatment with cytosolic and calcium-independent PLA2 inhibitors (Arachidonyl trifluoromethyl ketone, Bromoenol lactone, Methyl arachydonyl fluorophosphate, up to 50 microM) did not prevent but rather potentiated the VEGF effect on EC PAF synthesis. 7. These results provide evidence that with VEGF activation of EC cells, the group V sPLA2 provides substrate for EC PAF formation.

The pathogenesis of clostridial myonecrosis

These pieces of evidence can be assimilated into a molecular and cellular model of pathogenesis which is initiated by direct toxin effects upon venous capillary endothelial cell function, leading to expression of pro-inflammatory mediators and adhesion molecules, and initiation of platelet aggregation. Toxin-induced hyperadhesion of leukocytes (see above section) with enhanced respiratory burst activity (due to toxins directly or to toxin-induced IL-8 or PAF synthesis by host cells) and toxin-induced chemotaxis deficits could result in neutrophil-mediated vascular injury. Direct toxin-induced cytopathic effects on EC may also contribute to vascular abnormalities associated with gas gangrene. Over prolonged incubation periods, PLC at sublytic concentrations causes EC to undergo profound shape changes similar to those described following prolonged TNF or interferon gamma exposure. In vivo, conversion of EC to this fibroblastoid morphology could contribute to the localized vascular leakage and massive swelling observed clinically with this infection. Similarly, the direct cytotoxicity of PFO could disrupt endothelial integrity and contribute to progressive edema both locally and systemically. Thus, via the mechanisms outlined above, both PLC and PFO may cause local, regional and systemic vascular dysfunction. For instance, local absorption of exotoxins within the capillary beds could affect the physiological function of the endothelium lining the postcapillary venules, resulting in impairment of phagocyte delivery at the site of infection. Toxin-induced endothelial dysfunction and microvascular injury could also cause loss of albumin, electrolytes, and water into the interstitial space resulting in marked localized edema. These events, combined with intravascular platelet aggregation and leukostasis, would increase venous pressures and favor further loss of fluid and protein in the distal capillary bed. Ultimately, a reduced arteriolar flow would impair oxygen delivery thereby attenuating phagocyte oxidative killing and facilitating anaerobic glycolysis of muscle tissue. The resultant drop in tissue pH, together with reduced oxygen tension, might further decrease the redox potential of viable tissues to a point suitable for growth of this anaerobic bacillus. As infection progresses and additional toxin is absorbed, larger venous channels would become affected, causing regional vascular compromise, increased compartment pressures and rapid anoxic necrosis of large muscle groups. When toxins reach arterial circulation, systemic shock and multiorgan failure rapidly ensue, and death is common.

Effects of W-3 fatty acids on PAF synthesis in trinitro benzene sulfonic acid (TNB)-induced colitis in rats

Effects of W-3 fatty acids on PAF synthesis in trinitro benzene sulfonic acid (TNB)-induced colitis in rats

The sensitivity of bovine corneal epithelial lyso-PAF acetyltransferase to cyclooxygenase and lipoxygenase inhibitors is independent of arachidonate metabolites.

Lyso-platelet-activating factor (PAF) acetyltransferase is a critical regulatory step in PAF synthesis. PAF accumulates in the cornea in response to injury and is a potent inflammatory mediator that stimulates corneal cyclooxygenase but not lipoxygenase reactions. 12(S)-hydroxyeicosatetraenoic (HETE) acid, a major lipoxygenase product of mammalian corneas, is also generated during injury. In the bovine corneal epithelium, both PAF acetyltransferase and 12-lipoxygenase are microsomal enzymes. A potential interaction between these two lipid mediators was, therefore, examined. PAF acetyltransferase activity was assayed by determining radioactivity in the trichloroacetic acid-precipitated complex of [3H]PAF bound to albumin, formed after incubation of corneal epithelial microsomes with lyso-PAF and [3H]acetyl CoA. Lipoxygenase metabolism by bovine corneal epithelial microsomes was also studied. While 12(S)-HETE did not activate lyso-PAF acetyltransferase, PAF synthesis was decreased when microsomes were treated with lipoxygenase inhibitors. The IC50 values for nordihydroguaiaretic acid (NDGA), and baicalein were 75 and 105 microM, respectively. The IC50 value for CDC, a more potent inhibitor of platelet 12-lipoxygenase, was greater than 200 microM. The results indicate that concentrations suppressing lyso-PAF acetyltransferase activity exceed those required to inhibit lipoxygenases from bovine corneal epithelia. Similarly, concentrations of aspirin and indomethacin, cyclooxygenase inhibitors that decrease PAF formation, were greater than those reported to block prostaglandin generation. A number of other compounds, some structurally similar to the lipoxygenase inhibitors that suppress lyso-PAF acetyltransferase, and others unrelated chemically but known as anti-oxidant and cationic-chelators, also inhibited lyso-PAF acetyltransferase. This suggests that lyso-PAF acetyltransferase activity is inhibited by mechanisms independent of lipoxygenase and cyclooxygenase metabolites.

Identification of sn-2 acetyl glycerophosphocholines in human keratinocytes

Evidence is accumulating suggesting that platelet-activating factor plays a role in inflammatory dermatoses. Mass spectrometric methods were used to examine the molecular species of sn-2 acetyl glycerophosphocholines (GPC) synthesized by primary cultures of human neonatal foreskin-derived keratinocytes. Ionophore-stimulated keratinocytes synthesize both 1-alkyl and 1-acyl sn-2 acetyl-GPC, and the relative amounts were as follows: hexadecyl>palmitoyl>octadecyl>stearoyl at the sn-1 position. PAF synthesis in the keratinocyte-derived cell line HaCaT was inhibited by dexamethasone, suggesting that the anti-inflammatory effects of glucocorticosteroids in inflammatory dermatoses might be in part related to the inhibition of the synthesis of mediators such as PAF.

Escherichia coli hemolysin and Staphylococcus aureas alpha-toxin potently induce neutrophil adhesion to cultured human endothelial cells.

Abstract Adhesion of polymorphonuclear leukocytes (PMN) to endothelial cells is an essential step in inflammatory reactions. We characterized the effects of two important bacterial exotoxins, Escherichia coli hemolysin (HlyA) and Staphylococcus aureus alpha-toxin (S. alpha-toxin) on PMN adhesion to cultured HUVEC. Both toxins increased adherence of human PMN to HUVEC in a dose- and time-dependent manner, peaking after 30 min at 0.01 hemolytic units/ml HlyA or 0.5 microg/ml S. alpha-toxin. Pretreatment of HUVEC with anti-P-selectin mAbs or of PMN with anti-CD11b/CD18 mAb reduced HlyA- and S. alpha-toxin-related cell adhesion significantly. Increased P-selectin expression on toxin-treated endothelial cells was demonstrated by cell surface ELISA. Compared with endotoxin, HlyA and S. alpha-toxin did not induce the expression of E-selectin, ICAM-1, or VCAM-1. FACS analysis showed increased CD11b/CD18 expression on HlyA-, but not on S. alpha-toxin-stimulated PMN. Platelet-activating factor, an important costimulatory factor for PMN adhesion and activation, was also active in the exotoxin-stimulated adhesion system, as evidenced by studies using the platelet-activating factor receptor antagonist BN50727. HPLC analysis of endothelial cell extracts confirmed enhanced toxin-mediated PAF synthesis. The capacity of exotoxins to stimulate PMN adhesion to endothelial cells may be relevant in patients with severe local or systemic bacterial infections.

PAF synthesis

PAF synthesis

Activation of 85 kDa PLA2 by eicosanoids in human neutrophils and eosinophils.

The initial step in the remodeling pathway of PAF synthesis is catalyzed by phospholipase A2 (PLA2).1 It has generally been accepted that PLA2 acts directly on 1-0-alkyl-2-arachidonoyl-sn-glycero-3-phosphocholine (alkyl-2-AA-GPC) to form lyso-PAF (1-alkyl-2-lyso-GPC) which is then acetylated to yield PAF. More recent studies suggest that PAF synthesis may also be initiated indirectly through the action of CoA-independent transacylase following the hydrolysis of l-0-alk-1′-enyl-2-AA-sn-glycero-3-phosphoethanolamine (alkenyl-2-AA-GPE) by PLA2.2,3 In the putative indirect pathway, an accumulation of alkenyl-2-lyso-GPE triggers the transfer of AA from alkyl-2-AA-GPC to the alkenyl-2-lyso-GPE thus forming lyso-PAF. It seems logical that both the direct and indirect pathways may contribute to PAF synthesis. However, the relative physiological importance of the two pathways has not been established.

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.

Enzymes of platelet activating factor synthesis in brain

In this review, evidence is summarized for the production of PAF in brain, in response to stimulation associated with pathology. As well, there is a growing literature on the duality of actions of this lipid autocoid upon nervous tissue, indicated by extracellular and intracellular actions and binding sites for PAF in brain. The metabolic routes to PAF can be divided into the de novo and remodelling pathways of synthesis. The de novo route consists of 1-alkyl glycerophosphate acetyltransferase, and the subsequent actions of distinct phosphohydrolase and cholinephosphotransferase activities. This acetyltransferase can be activated by phosphorylation, and inhibited by MgATP and fatty acyl CoA thioesters, inhibitions which have particular relevance to brain ischemia. There is also evidence that the cholinephosphotransferase is controlled by phosphorylation, and regulated by levels of CDP-choline. The remodelling pathway to PAF relies upon the actions of phospholipase A2 or CoA-independent transacylases to generate the 1-alkyl glycerophosphorylcholine, as substrate for a distinct acetyltransferase. Following stimulation, rising intracellular calcium may trigger arachidonate selective cytosolic phospholipase activity which leads to increased PAF synthesis. The 1-alkyl glycerophosphocholine acetyltransferase activity is quite small in brain in comparison with the de novo acetyltransferase activity, and is also controlled by phosphorylation. Evidence has been presented for the actions of both pathways in brain, in response to biologically relevant stimulation pertinent to the disease state.