Platelet-activating Factor Synthesis Research Articles

Comparison of acceptor and donor substrates in the CoA-independent transacylate reaction in human neutrophils

In human neutrophils (PMN) the ethanolamine-containing phosphoglyceride fraction (PE), principally plasmalogen-linked PE (1- O-alk-1′-enyl-2-acyl- sn-glycero-3-phosphoethanolamine), is the major store of arachidonic acid (AA). Exogenous AA is initially incorporated into 1-acyl-linked phosphoglycerides and is believed to be transferred into the 1-ether-linked phosphoglycerides via the action of a CoA-independent transacylase (CoA-IT). We have investigated the selectivity for both the ‘acceptor’ lysophospholipids and ‘donor’ AA-containing phospholipid substrates in the CoA-IT reaction. Evidence suggests CoA-IT may also participate in the synthesis of platelet activating factor. The transfer of [ 3H]AA from endogenously labeled choline-containing phosphoglycerides (PC) to exogenously added alkenyl-lyso-PE (0–50 μM) was examined in saponin-permeabilized PMN. In these ‘donor’ studies, we observed that [ 3H]AA was transferred from both alkyl- and diacyl-linked PC in a proportional manner. More detailed molecular species analysis showed that [ 3H]AA was deacylated from all the major AA-containing molecular species in both the alkyl and diacyl subclasses with no selectivity for either subclass. To investigate the ‘acceptor’ selectivity, membrane fractions prelabeled with either [ 3H]alkyl-arachidonoyl-PE or -PC were utilized as donor substrates. Various unlabeled lysophospholipids (10 μM) were added and the generation of [ 3H]lyso-PE or -PC was monitored as a measure of CoA-IT activity. Significant subclass preference was observed upon addition of lyso-PE species (1-alkenyl > 1-alkyl > 1-acyl) however, little selectivity was seen with the corresponding lyso-PC species. On the other hand, lysophosphatidylserine, lysophosphatidylinositol, and lysophosphatidic acid all served as poor acceptor substrates in the reaction. These data from PMN are consistent with other evidence that the CoA-IT plays a pivotal role in the enrichment of AA into plasmalogen-linked PE.

Activities of enzymes in platelet activating factor biosynthetic pathways in the gerbil model of cerebral ischemia.

The activities of enzymes in platelet activating factor (PAF) biosynthetic pathways were analyzed in hippocampal and cerebral cortical regions of normal and ischemic gerbil brain to assess changes in enzyme activities and potential modulators that could explain the accentuated production of PAF seen in ischemia. Global forebrain ischemia was produced by bilateral carotid artery ligation, and the effectiveness of the ligation was shown by free fatty acid release and ATP depletion. Specific activities of 1-alkyl-2-acetyl-sn-glycerol (AAG) choline phosphotransferase, 1-alkyl-sn-glycero-3-phosphate (AGP) acetyl transferase, and 1-alkyl-sn-glycero-3-phosphocholine (lyso PAF) acetyl transferase in tissue homogenates were in the ratio 4:1:0.1, respectively. Sham-operated and ischemic or ischemic-reperfused tissues showed similar activities for individual enzymes, indicating that enzyme levels or activation states did not change in ischemic or reperfused tissues. However, small metabolites (relevant to ischemia) added to the in vitro assays did modify enzyme activities. Physiological concentrations of MgATP severely inhibited AGP acetyl transferase activity, and this resulted in the ratio of AGP acyl transferase to AGP acetyl transferase activities changing from 48:1 in the presence of 2.5 mM MgATP to 6:1 in the absence of MgATP. This suggests that falling ATP levels in cerebral ischemia may promote the de novo pathway of PAF biosynthesis by releasing inhibition of AGP acetyl transferase. Lyso PAF acetyl transferase was much less active than AGP acetyl transferase and was also inhibited by MgATP. AAG choline phosphotransferase was not inhibited by MgATP but was inhibited by calcium. However the superior specific activity of the choline phosphotransferase in comparison with the AGP acetyl transferase suggested that the lowered choline phosphotransferase activity in the presence of rising intracellular calcium would not seriously compromise the synthesis of PAF by the de novo route. Both acetyl transferase enzymes were also inhibited by oleoyl CoA.

Ligation of the alpha 2-macroglobulin signaling receptor on macrophages induces synthesis of platelet activating factor.

The binding of receptor-recognized forms of alpha 2-macroglobulin (alpha 2M) to macrophage alpha 2M signaling receptors increases inositol-1,4,5-triphosphate synthesis and induces Ca2+ mobilization. In this report, we demonstrate that ligation of the macrophage alpha 2M signaling receptor is also associated with synthesis of platelet activating factor (PAF) by both the de novo and remodeling pathways. Both alpha 2M-methylamine and a cloned and expressed 20-kDa receptor binding fragment (RBF) from rat alpha 1M+, stimulated macrophage synthesis of PAF from [3H]acetate, [3H]methylcholine, and 1-O-[3H]alkyl lyso-PAF by two- to threefold. PAF levels reached a peak in 20 min after the cells were exposed to alpha 2M-methylamine or RBF; they remained elevated for about 1 h after ligand addition to the cells. When [3H]methylcholine was the substrate, pertussis toxin did not block PAF synthesis, but the protein kinase C inhibitor staurosporin reduced synthesis by 65-70%. Cycloheximide completely abolished the increase in synthesis of PAF by macrophages exposed to alpha 2M-methylamine. By contrast, when [3H]acetate was employed as a precursor, staurosporin or cycloheximide did not abolish the increase in PAF synthesis. These studies suggest that protein kinase C is necessary for the induction of the de novo pathway by alpha 2M-methylamine. Both alpha 2M-methylamine and RBF stimulated the activity of lyso-PAF acetyltransferase by about fourfold. Both ligands also stimulated the activity of PAF acetylhydrolase by about six- to sevenfold, indicating that ligation of the alpha 2M signaling receptor also regulates the degradation of PAF. The ability of receptor-recognized forms of alpha 2M to regulate levels of PAF suggests that alpha 2M-proteinase complexes not only regulate macrophage function by activating intracellular signaling but also may indirectly regulate the function of other cells that cannot bind alpha 2M-proteinase complexes.

Ligation of the α2-macroglobulin signaling receptor on macrophages induces synthesis of platelet activating factor

The binding of receptor-recognized forms of α2-macroglobulin (α2M) to macrophage α2M signaling receptors increases inositol-1,4,5-triphosphate synthesis and induces Ca2+ mobilization. In this report, we demonstrate that ligation of the macrophage α2M signaling receptor is also associated with synthesis of platelet activating factor (PAF) by both the de novo and remodeling pathways. Both α2M-methylamine and a cloned and expressed 20-kDa receptor binding fragment (RBF) from rat α2M+, stimulated macrophage synthesis of PAF from [3H]acetate, [3H]methylcholine, and 1-O-[3H]alkyl lyso-PAF by two- to threefold. PAF levels reached a peak in 20 min after the cells were exposed to α2M-methylamine or RBF; they remained elevated for about 1 h after ligand addition to the cells. When [3H]methylcholine was the substrate, pertussis toxin did not block PAF synthesis, but the protein kinase C inhibitor staurosporin reduced synthesis by 65–70%. Cycloheximide completely abolished the increase in synthesis of PAF by macrophages exposed to α2M-methylamine. By contrast, when [3H]acetate was employed as a precursor, staurosporin or cycloheximide did not abolish the increase in PAF synthesis. These studies suggest that protein kinase C is necessary for the induction of the de novo pathway by α2M-methylamine. Both α2M-methylamine and RBF stimulated the activity of lyso-PAF acetyltransferase by about fourfold. Both ligands also stimulated the activity of PAF acetylhydrolase by about six- to sevenfold, indicating that ligation of the α2M signaling receptor also regulates the degradation of PAF. The ability of receptor-recognized forms of α2M to regulate levels of PAF suggests that α2M-proteinase complexes not only regulate macrophage function by activating intracellular signaling but also may indirectly regulate the function of other cells that cannot bind α2M-proteinase complexes. © 1996 Wiley-Liss, Inc.

Hypoxia attenuates metabolism of platelet activating factor by fetal and newborn lamb lungs

The metabolism of platelet activating factor (PAF) by lungs of near-term fetal and 5- to 9-day-old lambs was studied during normoxia and hypoxia at 37 degrees C in 30 mM Tris buffer. PAF synthesis was studied in lung cytosol and membrane using 250 microM [3H]acetyl CoA, 40 microM lyso-PAF, and 50 micrograms protein. PAF catabolism was studied in lung homogenate (LH) using 50 microM [3H]alkyl-PAF. PAF was extracted and assayed by thin-layer chromatography (TLC) and liquid scintillation spectrometry. Levels of PAF synthesized (nmol/min per mg protein) by fetal lung membrane versus cytosol were 1.35 +/- 0.07 versus 0.61 +/- 0.08, which were greater than those by newborn which were 0.33 +/- 0.07 versus 0.17 +/- 0.03. Hypoxia did not alter PAF synthesis by the lungs. PAF catabolism (nmol lyso-PAF/min per mg protein) by fetal LH was 0.07 +/- 0.01, which increased to 0.24 +/- 0.02 during normoxia. In newborn LH, the rate was 0.24 +/- 0.04 and increased to 0.33 +/- 0.01 during normoxia. PAF catabolism was higher in newborn than in fetal LH. An increase in pO2 augmented PAF catabolism, more in fetal than in newborn LH. Thus rate of PAF synthesis decreases from fetus to newborn, but PAF catabolism increases from fetus to newborn. The higher rate of PAF synthesis coupled with a low rate of PAF catabolism in the hypoxic environment of fetal lungs may predispose the fetus to a high PAF level, which may contribute to the high basal vasomotor tone in fetal lungs. A fall in PAF level with oxygenation, due to increased PAF catabolism, may facilitate the normal fall in pulmonary vascular resistance at birth.

Anti-inflammatory, membrane-stabilizing interactions of salmeterol with human neutrophils in vitro.

1. We have investigated the effects of salmeterol (0.3-50 microM) on several pro-inflammatory activities of human neutrophils in vitro. 2. Oxidant production by FMLP- and calcium ionophore (A23187)-activated neutrophils was particularly sensitive to inhibition by low concentrations (0.3-3 microM) of salmeterol, while the responses of phorbol myristate acetate- and opsonised zymosan-stimulated cells were affected only by higher concentrations (3-50 microM) of the drug. At these concentrations salmeterol is not cytotoxic, nor does it act as a scavenger of superoxide. 3. These anti-oxidative interactions of salmeterol with neutrophils were insensitive to propranolol but could be eliminated by washing the cells, or by pretreatment with low concentrations (1-2 microM) of the pro-oxidative, membrane-destabilizing phospholipids, lysophosphatidylcholine (LPC), platelet activating factor (PAF) and lysoPAF (LPAF). 4. At concentrations of 6.25-50 microM salmeterol interfered with several other activities of stimulated neutrophils, including intracellular calcium fluxes, phospholipase A2 activity and synthesis of PAF. 5. In an assay of membrane-stabilizing activity, salmeterol (25 and 50 microM) neutralized the haemolytic action of LPC, PAF and LPAF. 6. Of the other commonly used beta 2-adrenoceptor agonists, fenoterol, and formoterol, but not salbutamol, caused moderate inhibition of neutrophil oxidant generation by a superoxide-scavenging mechanism. However, unlike salmeterol, these agents possessed only weak membrane stabilizing properties. 7. We conclude that salmeterol antagonizes the pro-inflammatory, pro-oxidative activity of several bioactive lipids implicated in the pathogenesis of bronchial asthma, by a mechanism related to the membrane-stabilizing, rather than to the beta 2-agonist properties of this agent.

Ligation of the α2‐macroglobulin signaling receptor on macrophages induces synthesis of platelet activating factor

Ligation of the α2‐macroglobulin signaling receptor on macrophages induces synthesis of platelet activating factor

Prevention of cyclosporin nephrotoxicity with a platelet-activating factor (PAF) antagonist

Cyclosporin (CsA) is a potent immunosuppressive drug whose main side-effect is nephrotoxicity. In the kidney, CsA induces vasoconstriction with a decrease in renal blood flow (RBF) and glomerular filtration rate (GFR) and a significant increase in renal vascular resistance (RVR). CsA enhances platelet-activating factor (PAF) synthesis in mesangial cells in vitro. PAF, a secondary mediator of anaphylaxis and inflammation, exhibits vasoactive properties in the kidney similar to those of CsA. The in situ autoperfused rat kidney model was used to investigate whether PAF plays a role in the haemodynamic injury induced by CsA. In this model, CsA (40 mg/kg and 20 mg/kg i.v.) induced a significant decrease in RBF and in GFR and an increase in RVR. BN 52021, a potent and specific PAF antagonist (20 mg/kg i.v. bolus dose) induced a significant increase in GFR (137 +/- 32% of initial value, P < 0.05). BN 52021 (20 and 10 mg/kg) also significantly prevented the decline in RBF and GFR induced by CsA. We have demonstrated that the PAF antagonist BN 52021 can minimize the alteration of renal function induced by CsA.

Urinary excretion of platelet-activating factor in human and experimental nephrosis

Platelet-activating factor (PAF) is a phospholipid that has been implicated in the pathogenesis of glomerulonephritis and can be synthesized by glomerular cells in response to different stimuli. PAF increases glomerular permeability to proteins and urinary PAF has been determined to be of renal origin. In order to assess whether urinary PAF can be found augmented in situations of glomerular damage without glomerular leukocyte infiltration, urinary PAF was quantified in human and experimental nephrosis. Urinary PAF was quantified by platelet bioassay and glomerular PAF by incorporation of 3H-acetate into PAF. PAF was characterized by its behaviour on thin-layer chromatography and high performance liquid chromatography and the blockade of its bioactivity by receptor antagonists. Urinary PAF excretion was significantly higher in patients with active idiopathic nephrotic syndrome than in controls (5.8+/-1.5 versus 1.7+/-0.75 mg/24 h; P<0.05) and patients in remission (1.63+/-0.75 ng/24 h; P<0.02). In rats with nephrosis induced by puromycin aminonucleoside there was an early increase in urinary PAF excretion (138+/-19 versus 49+/-22 pg/24 h in controls; P<0.035) that coincided with the augmented glomerular PAF synthesis (67+/-3.4 versus 36+/-1.2 DPM/mg protein in controls; P<0.003). These results suggest that the synthesis of PAF in the kidney may be involved in the pathogenesis of the proteinuria in idiopathic nephrotic syndrome and that urinary PAF excretion may be a good marker of disease activity.

High-density lipoprotein inhibits the synthesis of platelet-activating factor in human vascular endothelial cells

The regulation of platelet-activating factor (PAF) synthesis by serum lipoproteins was investigated in human umbilical vein endothelial cells. High-density lipoprotein (HDL) inhibited PAF synthesis in agonist (thrombin, histamine, and A23187)-stimulated endothelial cells, that was determined by incorporation of [ 3H]acetate into PAF and by bioassay. The inhibition by HDL was increased in a concentration-dependent manner, but was reversed as the concentration of thrombin increased. HDL did not affect the time course of PAF production. HDL lipids suppressed the PAF production to a lesser extent than HDL. The reduction of PAF accumulation by HDL did not result from degradation of PAF but inhibition of PAF synthesis, which was mainly mediated via the blockade of acetyl-CoA:l-alkyl-2-lyso- sn-glycero-3-phosphocholine acetyltransferase activation. HDL did not prevent the release of [ 3H]arachidonic acid in thrombin-stimulated endothelial cells. The binding of 125I-HDL to endothelial cells and its uptake were not enhanced by thrombin stimulation. These results demonstrate that HDL may inhibit the activation of acetyltransferase by thrombin at the cell surface. This observation may explain a part of mechanism of HDL action.

Functional and structural features of plasma platelet-activating factor acetylhydrolase.

Platelet-activating factor (PAF) is a potent pro-inflammatory phospholipid that exerts its effects by binding to a receptor on target cells such as monocytes, polymorphonuclear leukocytes, platelets, and smooth muscle cells1. Other phospholipids that have undergone oxidative fragmentation are structurally similar to PAF, bind to its receptor, and mimic its biological properties. Although PAF synthesis is tightly regulated, the fragmented phospholipids arise via unregulated chemical oxidation.

Platelet-activating factor synthesis and its role in salivary glands.

Platelet-Activating Factor (PAF) was originally described as a substance released from activated basophils. It was then detected in a variety of inflammatory and immune-related cells, e.g. polymorphonuclear leucocytes, monocytes, macrophages and plateletes. PAF is considered to be a mediator in anaphylaxis and inflammatory responses. Moreover, PAF production has been demonstrated in a wider variety of cells and tissues, and it has been suggested that PAF plays various physiological and pathological roles. For example, PAF may play a role in stimulation-secretion coupling including neuron tissues in which PAF increases intracellular free Ca2+ concentration([Ca2+]i)1–6 and modulates neurotransmitter release2,3, 6–8, and is suggested its contribution to long-term potentiation fomation in the hippocampus 9,10 or enhanced neurotransmitter release in pathological conditions such as ischemia or convulsions. Soling et al11 has reported that PAF stimulates amylase release in the exocrine salivary gland and the pancreas. The capacity of cells from exocrine glands to produce PAF in response to physiological stimulations12,13 and the presence of PAF in human saliva14 have also been demonstrated. The present study examines the regulation of PAF biosynthesis and its role in secretory responses in salivary glands.KeywordsSalivary GlandSubmandibular GlandPolymorphonuclear LeucocyteExocrine SecretionPhosphoinositide TurnoverThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Characterization of a secretory phospholipase A2 in human bronchoalveolar lavage fluid.

Phospholipase A2 (PLA2) is a pivotal enzyme involved in the synthesis of the potent lipid inflammatory mediators platelet activating factor (PAF) and the eicosanoids. This study characterizes a PLA2 recovered in the bronchoalveolar lavage fluid (BALF) of healthy adult human subjects. Human BALF PLA2 exhibited characteristics of secretory PLA2s that include an activity that is acid stable, sensitive to reducing agents, and optimally requires millimolar calcium. BALF PLA2 showed marked selectivity for phosphatidylcholine containing arachidonic acid (AA) over linoleic or palmitic acids. Size exclusion chromatography showed the BALF PLA2 protein to be approximately 14 kDa in mass, consistent with it being a secretory form of PLA2. The biological significance of BALF PLA2 was tested by applying BALF concentrates to cultures of the human bronchial epithelial cell line BEAS 2B. Cultures of BEAS 2B cells treated with BALF concentrates released increased amounts of AA and produced higher levels of PAF. These data show that the lining fluid of the human respiratory tract normally contains a secretory PLA2, which may be involved in the formation of lipid inflammatory mediators in normal and pathophysiologic states in the lung.

Quantification of distinct molecular species of platelet activating factor in ulcerative colitis.

The aim of this study was to characterize the synthesis and metabolism of platelet activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) by colonic mucosa from patients with ulcerative colitis and healthy individuals. Tissue was obtained by endoscopic biopsy and by scraping the mucosa from surgical resections. Tissue was assayed for the various molecular species of PAF and its biologically inactive metabolite lyso-PAF using gas chromatography/mass spectrometry. Mucosa from surgical resections for ulcerative colitis contained C16:0 PAF (mean = 156 ng/g of mucosa), but not C18:0 PAF, PAF was not identified in mucosa from normal surgical resections or in endoscopic biopsies from either patients with ulcerative colitis or normal individuals. Both C16:0 lyso-PAF and C18:0 lyso-PAF were found in mucosa from normal and ulcerative colitis surgical resections and in endoscopic biopsies from ulcerative colitis and normal tissue. Levels of lyso-PAF were similar in ulcerative colitis and normal mucosa. Incubation of mucosa from areas of active inflammation in ulcerative colitis with the calcium ionophore A23187 increased the levels of C16:0 PAF by 2-3 fold and also increased the levels of C16:0 lyso-PAF. Addition of 3H-PAF to endoscopic biopsies from either normal individuals or patients with ulcerative colitis resulted in hydrolysis to 3H-lyso-PAF. The data on colonic mucosal levels of PAF are consistent with the results of earlier studies measuring PAF in patients with ulcerative colitis by bioassay. This study examines the synthesis and metabolism of specific molecular species of PAF in ulcerative colitis for the first time.

Platelet-activating factor (PAF): signalling and adhesion in cell-cell interactions.

Signalling by PAF is closely linked to adhesive interactions between cells of the inflammatory and vascular systems. It acts as a juxtacrine signal that alters the activity of beta 2 integrins on myeloid leukocytes (Figure 1), and works in concert with P-selectin at the surfaces of endothelial cells (Figure 2 and text). Observations in models of flow and in vivo support the original experiments using cultured endothelium under static conditions that indicated that PAF acts at this vascular interface. P-selectin modifies and integrates signals delivered through the PAF receptor on monocytes (Figure 4). Adhesion via P-selectin and engagement of beta 2 integrins modify signals leading to PAF synthesis (text and Figure 5). The intimate relationship between adhesive events and signalling by PAF may be a critical determinant in its roles in physiologic and pathologic responses.

ZG-1494 alpha, a novel platelet-activating factor acetyltransferase inhibitor from Penicillium rubrum, isolation, structure elucidation andbiological activity.

A novel inhibitor of platelet-activating factor (PAF) acetyltransferase, an essential enzyme in the remodeling pathway of platelet-activating factor synthesis, was identified by a high throughout screen of natural product extracts of microbial origin. The compound, ZG-1494 alpha, was isolated from an ethyl acetate extract of a culture broth of Penicillium rubrum through bioassay guided fractionation. The structure of ZG-1494 alpha was determined by spectroscopic methods. A key feature of the structure, which is relatively rare among natural products, is the 5-hydroxy-3-pyrrolin-2-one moiety. A 13C-13C INADEQUATE was utilized to unambiguously determine the regiochemistry of this molecule.

Autocrine/paracrine involvement of platelet-activating factor and transforming growth factor-beta in the induction of phosphatidylserine recognition by murine macrophages.

The specific recognition of phosphatidylserine (PS) by macrophages is believed to be one means by which effete and apoptotic cells expressing PS on their outer membrane leaflet are targeted for phagocytosis. The aim of this study was to better understand the autocrine/paracrine factors involved in beta-glucan induction of PS recognition by macrophages. We provide evidence that both platelet-activating factor (PAF) and TGF-beta are involved in beta-glucan induction of PS recognition. This is based on the observations that the PAF receptor antagonist WEB 2086 and Ab against TGF-beta each could partially inhibit beta-glucan-induced PS recognition when used alone and could completely inhibit induction when used in combination. PAF, like TGF-beta, was found to prime macrophages for PS recognition, which could then be triggered by costimulation with a nonspecific phagocytic stimulus, latex particles. We also provide evidence that the priming by PAF and that by TGF-beta can occur independently of each other. This is based on the observations that 1) PAF priming was not blocked by anti-TGF-beta Ab, nor was TGF-beta priming prevented by WEB 2086; and 2) PAF did not increase the steady state level of TGF-beta mRNA, and TGF-beta did not induce PAF synthesis in these cells.

Hypoxia induces PMN adherence to umbilical vein endothelium

In vitro incubation of cultured endothelial cells under hypoxia leads to the activation of these cells and results in an increase of their adhesiveness for neutrophils (PMN). Because of the possible relevance of these observations for pathological situations, we investigated whether adherence of PMN also occurs in an entire vein after its incubation in hypoxic conditions. Human umbilical veins in complete cords were incubated for 2 h in normoxic or hypoxic conditions and the adherence of unstimulated human 51Cr-labelled-PMN was measured under flow conditions. Experiments with human umbilical vein endothelial cells (HUVEC) were performed in parallel for comparison. Morphological studies in scanning electron microscopy were carried out in both in vitro and ex vivo situations. Hypoxia induced an increase in the adherence of PMN either to HUVEC or to the umbilical vein endothelium up to 5- to 6-fold when compared to normoxic conditions (P < 0.001). In both cases, this hypoxia-induced adherence was inhibited by anti-ICAM-1 antibodies or when the PAF (platelet-activating factor) synthesis was blocked during hypoxia by oleic acid. Furthermore, the adherence of PMN was inhibited when PMN were pre-incubated with WEB 2086 (a selective PAF receptor antagonist). These results indicate a crucial role of PAF in this process. Morphological studies confirmed that the number of PMN adherent to hypoxic HUVEC or to the hypoxic umbilical vein endothelium was much greater than the number of PMN on normoxic endothelial cells. Both in vitro and ex vivo, PMN adherent to the hypoxic endothelial cells to the contrary of the ones adherent to normoxic endothelial cells demonstrated membrane foldings typical of an activated state. These results show that in a complete vein, hypoxia induced an increased adhesiveness of endothelial cells for PMN by a similar mechanism to the one observed for cultured endothelial cells. They suggest an active role of endothelial cells in the initiation of the inflammatory response often described in ischemic-reperfused organs.

5-Lipoxygenase Products Modulate the Activity of the 85-kDa Phospholipase A2 in Human Neutrophils

Addition of submicromolar concentrations of arachidonic acid (AA) to human neutrophils induced a 2-fold increase in the activity of a cytosolic phospholipase A2 (PLA2) when measured using sonicated vesicles of 1-stearoyl-2-[14C]arachidonoylphosphatidylcholine as substrate. A similar increase in cytosolic PLA2 activity was induced by stimulation of neutrophils with leukotriene B4 (LTB4), 5-oxoeicosatetraenoic acid, or 5-hydroxyeicosatetraenoic acid (5-HETE). LTB4 was the most potent of the agonists, showing maximal effect at 1 nM. Inhibition of 5-lipoxygenase with either eicosatetraynoic acid or zileuton prevented the AA-induced increase in PLA2 activity but had no effect on the response induced by LTB4. Furthermore, pretreatment of neutrophils with a LTB4-receptor antagonist, LY 255283, blocked the AA- and LTB4-induced activation of PLA2 but did not influence the action of 5-HETE. Treatment of neutrophils with pancreatic PLA2 also induced an increase in the activity of the cytosolic PLA2; this response was inhibited by both eicosatetraynoic acid or LY 255283. The increases in PLA2 activity in response to stimulation correlated with a shift in electrophoretic mobility of the 85-kDa PLA2, as determined by Western blot analysis, suggesting that phosphorylation of the 85-kDa PLA2 likely underlies its increase in catalytic activity. Although stimulation of neutrophils with individual lipoxygenase metabolites did not induce significant mobilization of endogenous AA, they greatly enhanced the N-formylmethionyl-leucyl-phenylalanine-induced mobilization of AA as determined by mass spectrometry analysis. Our findings support a positive-feedback model in which stimulus-induced release of AA or exocytosis of secretory PLA2 modulate the activity of the cytosolic 85-kDa PLA2 by initiating the formation of LTB4. The nascent LTB4 is then released to act on the LTB4 receptor and thereby promote further activation of the 85-kDa PLA2. Since 5-HETE and LTB4 are known to prime the synthesis of platelet-activating factor, the findings suggest that 85-kDa PLA2 plays a role in platelet-activating factor synthesis.

25 Molecular mechanisms of angiogenesis associated to kaposi's sarcoma

Imbalance in the network of soluble mediators may play a pivotal role in the pathogenesis of Kaposi's sarcoma (KS) a multifocal lesion characterized by a prominent angiogenesis. We demonstrated that KS cells grown in vitro produced and in part released platelet activating factor (PAF), a lipid mediator of inflammation and cell-to-cell communication. ILI, TNF and thrombin enhanced the synthesis of PAF. PAF receptor mRNA and specific, high affinity binding site for PAF were present in KS cells. Nanomolar concentration of PAF stimulated the chemotaxis and chemokinesis of KS cells, endothelial cells (EC) and vascular smooth muscle cells. The migration response to PAF was inhibited by WEB 2170, a PAF receptor antagonist. Since PAF activates EC we examined the potential role of PAF as an instrumental mediator of angiogenesis associated with KS. Conditioned medium (CM) from KS cells (KS-CM) or KS cells themselves induced angiogenesis and macrophage recruitment in Matrigel model. These effects were inhibited by treating mice with WEB 2170. The action of PAF was amplified by expression of other angiogenic factors and chemokines: these included basic and acidic FGF, PIGF, VEGF and its specific receptor flk-1 , HGF, KC, and MIP2. Treatment with WEB 2170 abolished the expression of these transcripts within Matrigel containing KS-CM. These results indicate that PAF may cooperate with other angiogenic molecules and chemokines in inducing vascular development, in KS.