On the expression of cytosolic calcium-independent phospholipase A 2 (88 kDa) in immature and mature myeloid cells and its role in leukotriene synthesis in human granulocytes (original) (raw)
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The Journal of biological chemistry, 1990
The existence of an intracellular phospholipase A2 (PLA2) involved in the production of 1-O-alkyl-sn-glycero-3-phosphocholine and free arachidonic acid has been repeatedly postulated. Using 1-O-hexadecyl-2-[3H]arachidonoyl-sn-glycero-3-phosphocholine as a substrate and a series of conventional and high-pressure liquid chromatographic techniques, we have purified a PLA2 from the soluble fraction of differentiated human monocytic U937 cells. The enzyme has been purified nearly 2000-fold to homogeneity. The purified enzyme has a molecular mass of 56 kDa, under reducing conditions, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The enzyme activity has a pH optimum of 8.0 and is calcium concentration-dependent. The EC50 for the activation of the enzyme activity by calcium is 300 nM. When the cells were homogenized in the presence of the calcium chelator EGTA (0.2 mM), the enzyme was found to be soluble (more than 90% of the activity in the 100,000 x g supernatant)...
Journal of Biological Chemistry, 2002
energy transfer; HBSS, Hanks' balanced salt solution; hIIaPLA 2 , human group IIa PLA 2 ; hVPLA 2 , human group V PLA 2 ; HPLC, high performance liquid chromatography; iPLA 2 , group VI Ca 2ϩ-independent PLA 2 ; 5-LO, 5-lipoxygenase; LTB 4 , leukotriene B 4 ; LTB 4 DMA, LTB 4 dimethyl amide; lyso-PC, 1-palmitoyl-2-hydroxy-snglycero-3-phosphocholine; MAP kinase, mitogen-activated protein kinase; MEK; mitogen-activated protein kinase/extracellular signalregulated kinase kinase; OA, oleic acid; PED6, N-((6-(2,4-dinitrophenyl)amino)hexanoyl)-2-(4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3phosphoethanolamine triethylammonium salt; PGB 2 , prostaglandin B 2 ; SAPC, 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine; sPLA 2 , secretory PLA 2 .
Journal of leukocyte biology, 1992
An interleukin 3 (IL-3)-dependent macrophage-like cell line, 11-1-B3, was newly established from CBA/J mouse bone marrow cell cultures. Assay of eicosanoids in the culture supernatants of the intact and [3H]arachidonic acid (AA)-prelabeled cells showed that, after stimulation with the Ca2+ ionophore A23187, the 11-1-B3 cells synthesized and released relatively large amounts of prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) but not LTC4. In addition, 11-1-B3 cells showed Ca(2+)-dependent and alkaline pH-optimal phospholipase A2 (PLA2) activity that preferentially hydrolyzed cleavage of sn-2-arachidonyl- but not sn-2-oleoylphosphatidylcholine. The cellular enzyme was distributed with 90% of the activity in the cytosol and 10% in the membrane fraction. Treatment of cells with A23187 for 5-10 min resulted in five- to sevenfold increases in the membrane-associated PLA2 but activity in the cytosol was unchanged. This increase in membrane-associated enzyme activity was transient, return...
Journal of Biological Chemistry, 1986
We have previously reported that platelet-activating factor (PAF) is present in human amniotic fluid obtained from women in labor. We have also demonstrated that PAF, lyso-PAF, and alkyl acyl-an-glycero-3-phosphocholine (AA-GPC) are present in human amnion tissue. In the reported study, we have investigated the enzymes involved in PAF metabolism in amnion tissue and their regulation. A phospholipase AZ activity has been demonstrated in amnion tissue which cleaves alkyl acyl (long-chain) an-glycero-3-phosphocholine. The enzyme activity is not altered by Ca2+ and is distinctly different from the phospholipase A2 that we have previously characterized in this tissue. Amnion tissue contains acetyltransferase activity which requires CaZf and is associated with the microsomal fraction. Acetylhydrolase is also present in the cytosolic fraction of amnion tissue. Acetylhydrolase activity has also been demonstrated in amniotic fluid. The affinities of acetyltransferase (for lyso-PAF) and acetylhydrolase (for PAF) were unaffected by Ca'+. In the presence of Ca '+ however, the specific activity of acetyltransferase was increased four-to fivefold while that of acetylhydrolase was unaffected. Acetyltransferase and acetylhydrolase activities in fetal membranes and decidua were similar and were unchanged with gestational age. The possible role of PAF in the initiation of human parturition is discussed. o 19% Academic press, I,,~.
Cellular Signalling, 1991
Human neutrophils pre-incubated with granulocyte-macrophage-colony-stimulating factor (GM-CSF) exhibit an enhanced mobilization of calcium in response to secondary stimuli such as chemotactic factors. The mechanisms underlying this priming effect of GM-CSF were examined. It was first demonstrated that the additional calcium mobilized by chemotactic factors in GM-CSF-treated cells was derived from intracellular stores and was associated neither with an increased permeability to calcium nor with production of inositol 1,4,5-trisphosphate. These results indicated that GM-CSF called upon a novel mechanism in order to enhance the mobilization of calcium in human neutrophils. The growth factor has recently been shown to prime phospholipase D leading to an enhanced activation by chemotactic factors and an augmented production of phosphatidic acid. Furthermore the ability of exogenous phosphatidie acid to mobilize calcium in cell types other than neutrophils has been previously demonstrated. Therefore, we examined the potential involvement of phospholipase D in the priming of the calcium response by GM-CSF in human neutrophils. Inhibition of the production of the fMet-Leu-Phe-stimulated production of phosphatidic acid by ethanol or wortmannin had only marginal effects on the concurrent mobilization of calcium. However, the priming of the mobilization of calcium by GM-CSF was greatly decreased in cells treated with either ethanol or wortmannin. These results provide strong support for the hypothesis that the production of phosphatidic acid, which is enhanced in GM-CSF-treated cells, is linked to an increased mobilization of intracellular calcium. These results may have relevance to the mechanism of action of GM-CSF in mature haematopoeitic cells as well to the mitogenic activity of other growth factors.
Calcium‐independent phospholipase A2 mediates proliferation of human promonocytic U937 cells
2008
One of the products of a calcium-independent phospholipase A 2 (iPLA 2 ) attack of plasmenylcholine, lysoplasmenylcholine, has previously been shown to activate cAMP-dependent protein kinase (PKA). Because endothelial cells respond to some agonists in part by the activation of iPLA 2 , the present study was designed to determine whether double-stranded RNA (dsRNA), the primary activator of the antiviral response in endothelial cells, elicits cAMP response element binding protein (CREB) phosphorylation through a mechanism mediated by iPLA 2 . dsRNA stimulated CREB phosphorylation in bovine pulmonary artery endothelial cells that was inhibited by the iPLA 2 inhibitor, bromoenol lactone, and the PKA inhibitor, H-89. Additionally, the product of iPLA 2 hydrolysis of plasmenylcholine and lysoplasmenylcholine elicited CREB phosphorylation in bovine pulmonary endothelial cells. Taken together, the present studies suggest that dsRNA as well as other agonists of endothelial cells elicit signaling mechanisms that include in part CREB phosphorylation mediated stranded RNA-stimulated endothelial cells. J. Lipid Res. 2003. 44: 1686-1691.
Prostaglandins & other lipid mediators, 1999
Ca 2ϩ-independent phospholipase A 2 (iPLA 2) is involved in the incorporation of arachidonic acid (AA) into resting macrophages by the generation of the lysophospholipid acceptor. The role of iPLA 2 in AA remodeling in different cells was evaluated by studying the Ca 2ϩ dependency of AA uptake from the medium, the incorporation into cellular phospholipids, and the effect of the iPLA 2 inhibitor bromoenol lactone on these events. Uptake and esterification of AA into phospholipids were not affected by Ca 2ϩ depletion in human polymorphonuclear neutrophils and rat fibroblasts. The uptake was Ca 2ϩ independent in chick embryo glial cells, but the incorporation into phospholipids was partially dependent on extracellular Ca 2ϩ. Both events were fully dependent on extra and intracellular Ca 2ϩ in human platelets. In human polymorphonuclear neutrophils, the kinetics of incorporation in several isospecies of phospholipids was not affected by the absence of Ca 2ϩ at short times (Ͻ30 min). The involvement of iPLA 2 in the incorporation of AA from the medium was confirmed by the selective inhibition of this enzyme with bromoenol lactone, which reduced Յ50% of the incorporation of AA into phospholipids of human neutrophils. These data provide evidence that suggests iPLA 2 plays a major role in regulating AA turnover in different cell types.
European Journal of Biochemistry, 1999
Recently, we reported the human 88-kDa calcium-independent phospholipase A 2 (iPLA 2 ) cDNA sequence, as well as extensive alternative splicing of the iPLA 2 mRNA. In this report we identified the gene coding for iPLA 2 , which was localized on chromosome 22q13.1. The gene consists of at least 17 exons spanning . 69 kb. Based on the iPLA 2 gene organization the splice variants can be explained. The putative promotor for the iPLA 2 gene lacks a TATA-box and contains a CpG island as well as several potential Sp-1-binding sites. Furthermore, the 5 H -flanking region also contains one medium reiteration frequency repeat (MER53) and an Alu repetitive sequence. Northern blot analysis of iPLA 2 mRNA in various human tissues demonstrated tissue-specific expression of four distinct iPLA 2 transcripts. The native human 3.2-kb iPLA 2 transcript was predominantly expressed in heart, brain, skeletal muscle, prostate, testis, thyroid and spinal cord, and to a lesser extent in peripheral blood leucocytes, stomach, trachea and bone marrow. Studies on the subcellular localization of the native iPLA 2 protein were performed in COS-7 cells overexpressing this enzyme. The cytosolic fraction of untransfected and cells overexpressing iPLA 2 contained equal amounts of calcium-independent PLA 2 activity. However, the membrane fraction displayed a 5.5-fold increased activity in iPLA 2 overexpressing cells. This increased calcium-independent PLA 2 activity correlated with the presence of iPLA 2 immunoreactive protein in the membrane fraction, indicating that this form of iPLA 2 protein was membrane associated. Studies of iPLA 2 in rat vascular smooth muscle cells verified the membrane association of this form of iPLA 2 . The major difference between this form of iPLA 2 enzyme and the soluble forms of iPLA 2 studied previously is the presence of 54 additional amino acid residues derived from exon 9. We suggest that the addition of these 54 amino acids leads to a membrane-associated protein. In summary, these results demonstrate that alternative splicing of the human iPLA 2 transcript generates multiple iPLA 2 isoforms with distinct tissue distribution and cellular localization.
2000
It has been demonstrated that equine neutro- phils, but not eosinophils, require exogenous arachidonic acid for calcium ionophore A23187-induced leukotriene synthesis. Because cytosolic phospholipase A 2 (cPLA 2 ) plays an essential role in leukotriene formation in leukocytes, we investigated the presence of a functional cPLA 2 in equine neutrophils. To determine whether cPLA 2 from neutrophils was catalytically active, we purified the enzyme . 6,500 fold with 3% recovery from equine neutrophils. The full-length cDNA sequence encoded a 749-amino acid protein. The de- duced amino acid sequence demonstrated 95% identity with human and mouse cPLA 2 , as well as 83 and 73% identity with chicken and zebra fish cPLA 2 protein, respectively. The equine cPLA 2 possessed some properties that distinguished the equine enzyme from the human enzyme. First, the en- zyme activity of the equine cPLA 2 was differently influenced by cations as compared with the human cPLA 2 . Second, the equine neutr...