Lyso-phospholipids exert a major injurious effect on lung cell membranes during Acute Respiratory Distress Syndrome (ARDS), but the mechanisms leading to their in vivo generation are still unknown. Intratracheal administration of LPS to guinea pigs induced the secretion of type II secretory phospholipase A2 (sPLA2-II) accompanied by a marked increase in fatty acid and lyso-phosphatidylcholine (lyso-PC) levels in the bronchoalveolar lavage fluid (BALF). Administration of LY311727, a specific sPLA2-II inhibitor, reduced by 60% the mass of free fatty acid and lyso-PC content in BALF. Gas chromatography/mass spectrometry analysis revealed that palmitic acid and palmitoyl-2-lyso-PC were the predominant lipid derivatives released in BALF. A similar pattern was observed after the intratracheal administration of recombinant guinea pig (r-GP) sPLA2-II and was accompanied by a 50-60% loss of surfactant phospholipid content, suggesting that surfactant is a major lung target of sPLA2-II. In confirmation, r-GP sPLA2-II was able to hydrolyze surfactant phospholipids in vitro. This hydrolysis was inhibited by surfactant protein A (SP-A) through a direct and selective protein-protein interaction between SP-A and sPLA2-II. Hence, our study reports an in vivo direct causal relationship between sPLA2-II and early surfactant degradation and a new process of regulation for sPLA2-II activity. Anti-sPLA2-II strategy may represent a novel therapeutic approach in lung injury, such as ARDS. (

The olive oil-based emulsion was well tolerated, maintained a normal EFA status, and may be more suitable for prevention of lipid peroxidation than the soybean-oil-based emulsion.

Type II-secreted phospholipase A 2 (type II-sPLA 2 ) is expressed in smooth muscle cells during atherosclerosis or in response to interleukin-1␤. The present study shows that the induction of type II-sPLA 2 gene by interleukin-1␤ requires activation of the NFB pathway and cytosolic PLA 2 /PPAR␥ pathway, which are both necessary to achieve the transcriptional process. Interleukin-1␤ induced type II-sPLA 2 gene dose-and time-dependently and increased the binding of NFB to a specific site of type II-sPLA 2 promoter. This effect was abolished by proteinase inhibitors that block the proteasome machinery and NFB nuclear translocation. Type II-sPLA 2 induction was also obtained by free arachidonic acid and was blocked by either AACOCF 3 , a specific cytosolic-PLA 2 inhibitor, PD98059, a mitogen-activated protein kinase kinase inhibitor which prevents cytosolic PLA 2 activation, or nordihydroguaiaretic acid, a lipoxygenase inhibitor, but not by the cyclooxygenase inhibitor indomethacin, suggesting a role for a lipoxygenase product. Type II-sPLA 2 induction was obtained after treatment of the cells by 15-deoxy-⌬ 12,14 -dehydroprostaglandin J 2 , carbaprostacyclin, and 9-hydroxyoctadecadienoic acid, which are ligands of peroxisome proliferator-activated receptor (PPAR) ␥, whereas PPAR␣ ligands were ineffective. Interleukin-1␤ as well as PPAR␥-ligands stimulated the activity of a reporter gene containing PPAR␥-binding sites in its promoter. Binding of both NFB and PPAR␥ to their promoter is required to stimulate the transcriptional process since inhibitors of each class block interleukin-1␤-induced type II-sPLA 2 gene activation. We therefore suggest that NFB and PPAR␥ cooperate at the enhanceosome-coactivator level to turn on transcription of the proinflammatory type II-sPLA 2 gene.

In order to study the role played by the fetoplacental unit in providing the human fetus with arachidonic acid, Δ⁵- and Δ⁶-desaturase activities were studied in microsomes from human fetal liver and placenta after 18 and 22 weeks of gestation. We evidenced for the first time Δ⁵- and Δ⁶-desaturase activities in fetal liver microsomes. As in adult liver, Δ⁶-desaturation is the rate-limiting step of arachidonic acid synthesis. No activity was found in the placenta. Arachidonic acid concentrations were higher in fetal serum than in maternal serum while the opposite was observed for linoleic acid. The fetal liver microsomal content in arachidonic acid was low. Taken together the data suggest that arachidonic acid is supplied to the fetus through a preferential transfer across the placenta.

Extracellular ATP is a pro-inflammatory mediator involved in the release of prostaglandin from articular chondrocytes, but little is known about its effects on intracellular signaling. ATP triggered the rapid release of prostaglandin E 2 (PGE 2 ) by acting on P2Y 2 receptors in rabbit articular chondrocytes. We have explored the signaling events involved in this synthesis. ATP significantly increased arachidonic acid production, which involved the activation of the 85-kDa cytosolic phospholipase A 2 (cPLA 2 ) but not a secreted form of PLA 2 , as demonstrated by various PLA 2 inhibitors and translocation experiments. We also showed that ATP induced the phosphorylation of p38 and ERK1/2 mitogen-activatedprotein kinases (MAPKs). Both PD98059, an inhibitor of the ERK pathway, and SB203580, an inhibitor of p38 MAPK, completely inhibited the ATP-induced release of PGE 2 . Finally, dominant-negative plasmids encoding p38 and ERK transfected alone into the cells impaired the ATP-induced release of PGE 2 to about the same extent as both plasmids transfected together. These results suggest that PGE 2 production induced by ATP requires the activation of both ERK1/2 and p38 MAPKs. Thus, ATP acts via P2Y 2 -purine receptors to recruit cPLA 2 by activating both ERK1/2 and p38 MAPKs and stimulates the release of PGE 2 from articular chondrocytes.

The activity of the [2831; 1103] promoter of the human cyclooxygenase-2 gene in cultured rabbit chondrocytes is stimulated 2.9^0 sequence binds with a low affinity both the p50/p50 homodimeric and p50/p65 heterodimeric forms of NF-kB but has no role in the regulation of the human COX-2 promoter in chondrocytes.

Conjectural results have been reported on the capacity of inflammatory secreted phospholipase A2 (sPLA2) to hydrolyse mammalian membrane phospholipids. Development of an assay based on the release of non-esterified fatty acids by the enzyme acting on the organized phospholipid mixture constituting the membrane matrix has led to the identification of two prominent effectors, sphingomyelin (SPH) and annexin. Recombinant human type II sPLA2 hydrolyses red-cell membrane phospholipids with a marked preference for the inner leaflet. This preference is apparently related to the high content of SPH in the outer leaflet, which inhibits sPLA2. This inhibition by SPH is specific for sPLA2. Cholesterol counteracts the inhibition of sPLA2 by SPH, suggesting that the SPH-to-cholesterol ratio accounts in vivo for the variable susceptibility of cell membranes to sPLA2. Different effects were observed of the presence of the non-hydrolysable D-alpha-dipalmitoyl phosphatidylcholine (D-DPPC), which renders the membranes rigid but does not inhibit sPLA2. Annexin VI was shown, along with other annexins, to inhibit sPLA2 activity by sequestering the phospholipid substrate. The present study has provided the first evidence that annexin VI, in concentrations that inhibit hydrolysis of purified phospholipid substrates, stimulated the hydrolysis of membrane phospholipids by sPLA2. The activation requires the presence of membrane proteins. The effect is specific for type II sPLA2 and is not reproducible with type I PLA2. The activation by annexin VI of sPLA2 acting on red cell membranes results in the preferential release of polyunsaturated fatty acids. It suggests that type II sPLA2, in conjunction with annexin VI, might be involved in the final step of endocytosis and/or exocytosis providing the free polyunsaturated fatty acids acting synergistically to cause membrane fusion.

The abundant secretion of type IIA secreted phospholipase A(2) (sPLA(2)) is a major feature of the inflammatory process of atherosclerosis. sPLA(2) is crucial for the development of inflammation, as it catalyses the production of lipid mediators and induces the proliferation of smooth muscle cells. We have analysed the activation of sPLA(2) transcription by cAMP and interleukin-1beta (IL-1beta), and shown that the 500 bp region upstream of the transcription start site of the rat sPLA(2) gene is implicated in activation by synergistically acting cAMP and IL-1beta. We transiently transfected and stimulated rat smooth muscle cells in primary culture and measured the promoter activities of serial and site-directed deletion mutants of sPLA(2)-luciferase constructs. A distal region, between -488 and -157 bp, bearing a CAAT/enhancer binding protein (C/EBP)-responsive element (-242 to -223) was sufficient for cAMP/protein kinase A-mediated sPLA(2) promoter activation. We find evidence for the first time that activation of the sPLA(2) promoter by IL-1beta requires activation of an Ets-responsive element in the -184 to -180 region of the distal promoter via the Ras pathway and a nuclear factor-kappaB site at positions -141 to -131 of the proximal promoter. We also used electrophoretic mobility shift assays to identify five binding sites for the Sp1 factor; a specific inhibitor of Sp1, mithramycin A, showed that this factor is crucial for the basal activity of the sPLA(2) promoter.