5-Lipoxygenase Products Modulate the Activity of the 85-kDa Phospholipase A(2) in Human Neutrophils (original) (raw)
Related papers
2004
Phospholipase A2 regulation of arachidonic acid levels Availability of free arachidonic acid (AA) is widely recognized as a rate-limiting step in the formation of prostaglandins. This fatty acid is an intermediate of a reacylationldeacylation cycle of membrane phospholipids, the so-called Lands pathway, in which the fatty acid is cleaved from phospholipid by phospholipase Azs (PLA z) and reincorporated by acyltransferases. Whereas in resting cells reacylation dominates, in stimulated cells the dominant reaction is the PLArmediated deacylation. Nevertheless, increased AA reacylation during cellular activation is still very significant, as manifested by the fact that only a minor portion of the free AA released by PLA z is converted into eicosanoids, the remainder being effectively incorporated back into phospholipids. Phagocytic cells generally contain multiple PLA 2 s [1, 2]. Thus the challenge in recent years has been both to identify these PLAzs and to clarify their roles in AA metabolism. A general mechanism for PLAz-regulated AA metabolism in resting and activated cells has emerged from the studies by several laboratories [3, 4], and involves participation of all three major classes of PLA z , namely cPLA z (cytosolic PLA z), iPLA z (Ca 2 + independent PLA z) and sPLA z (secreted PLA z) (Fig. 1). In resting conditions, iPLA 2 accounts for most of the PLA z activity of cells. iPLA z is therefore the dominant PLA z involved in the liberation of fatty acids, including AA, during the continuous recycling of membrane phospholipids that takes place under these conditions. Since, as indicated above, the rate of AA release by iPLA z is lesser than the rate of its reacylation back into phospholipids, no net accumulation of free fatty acid occurs. Stimulation of the cells by receptor agonists results in the activation of cPLA z , which then becomes the dominant PLA 2 involved in AA release. Under these conditions, the rate of AA release clearly exceeds that of reincorporation into phospholipids; hence net accumulation of AA occurs that is followed by its conversion into different oxygenated compounds, collectively called the eicosanoids.
British Journal of Pharmacology, 2006
Background and Purpose: The biosynthesis of leukotrienes (LT) and platelet-activating factor (PAF) involves the release of their respective precursors, arachidonic acid (AA) and lyso-PAF by the group IVA PLA 2 (cPLA 2 a). This paper aims at characterizing the inhibitory properties of the cPLA 2 a inhibitor pyrrophenone on eicosanoids and PAF in human neutrophils (PMN). Experimental Approach: Freshly isolated human PMN were activated with physiological and pharmacological agents (fMLP, PAF, exogenous AA, A23187 and thapsigargin) in presence and absence of the cPLA 2 a inhibitor pyrrophenone and biosynthesis of LT, PAF, and PGE 2 was measured. Key Results: Pyrrophenone potently inhibited LT, PGE 2 and PAF biosynthesis in PMN with IC 50 s in the range of 1-20 nM. These inhibitory effects of pyrrophenone were specific (the consequence of substrate deprivation), as shown by the reversal of inhibition by exogenous AA and lyso-PAF. Comparative assessment of pyrrophenone, methyl-arachidonoyl-fluorophosphonate (MAFP) and arachidonoyl-trifluoromethylketone (AACOCF 3) demonstrated that pyrrophenone was more specific and 100-fold more potent than MAFP and AACOCF 3 for the inhibition of LT biosynthesis in A23187-activated PMN. The inhibitory effect of pyrrophenone on LT biosynthesis was reversible as LT biosynthesis was recovered when pyrrophenonetreated PMN were washed with autologous plasma. No alteration of phospholipase D (PLD) activity in fMLP-activated PMN was observed with up to 10 mM pyrrophenone, suggesting that the cPLA 2 a inhibitor does not directly inhibit PLD. Conclusions and Implications: Pyrrophenone is a more potent and specific cPLA 2 a inhibitor than MAFP and AACOCF 3 and represents an excellent pharmacological tool to investigate the biosynthesis and the biological roles of eicosanoids and PAF.
British Journal of Pharmacology, 2006
Background and Purpose: The biosynthesis of leukotrienes (LT) and platelet-activating factor (PAF) involves the release of their respective precursors, arachidonic acid (AA) and lyso-PAF by the group IVA PLA 2 (cPLA 2 a). This paper aims at characterizing the inhibitory properties of the cPLA 2 a inhibitor pyrrophenone on eicosanoids and PAF in human neutrophils (PMN). Experimental Approach: Freshly isolated human PMN were activated with physiological and pharmacological agents (fMLP, PAF, exogenous AA, A23187 and thapsigargin) in presence and absence of the cPLA 2 a inhibitor pyrrophenone and biosynthesis of LT, PAF, and PGE 2 was measured. Key Results: Pyrrophenone potently inhibited LT, PGE 2 and PAF biosynthesis in PMN with IC 50 s in the range of 1-20 nM. These inhibitory effects of pyrrophenone were specific (the consequence of substrate deprivation), as shown by the reversal of inhibition by exogenous AA and lyso-PAF. Comparative assessment of pyrrophenone, methyl-arachidonoyl-fluorophosphonate (MAFP) and arachidonoyl-trifluoromethylketone (AACOCF 3 ) demonstrated that pyrrophenone was more specific and 100-fold more potent than MAFP and AACOCF 3 for the inhibition of LT biosynthesis in A23187-activated PMN. The inhibitory effect of pyrrophenone on LT biosynthesis was reversible as LT biosynthesis was recovered when pyrrophenonetreated PMN were washed with autologous plasma. No alteration of phospholipase D (PLD) activity in fMLP-activated PMN was observed with up to 10 mM pyrrophenone, suggesting that the cPLA 2 a inhibitor does not directly inhibit PLD. Conclusions and Implications: Pyrrophenone is a more potent and specific cPLA 2 a inhibitor than MAFP and AACOCF 3 and represents an excellent pharmacological tool to investigate the biosynthesis and the biological roles of eicosanoids and PAF.
Biochimica et biophysica acta, 1995
Phospholipase A2 (PLA2) activity in the soluble fraction of rat kidney yielded three peaks on DEAE cellulose column chromatography. From these three, we purified two PLA2 isoforms to near-homogeneity. Both had a molecular weight of approx. 14,000 on SDS-PAGE, and immunochemical and enzymological studies indicated that one is a 14 kDa type I PLA2 and the other a 14 kDa type II PLA2. RNA blot analysis confirmed that rat kidney contains both types of PLA2 and that administration of lipopolysaccharides and mercury chloride into rats increased type II PLA2 mRNA levels in kidney. When cultured rat mesangial cells were incubated with purified type I or type II PLA2 in combination with the calcium ionophore A23187 at suboptimal condition, augmentation of prostaglandin E2 production was observed. Type I and type II forms of PLA2 may play a role in arachidonate metabolism in rat kidney.
Journal of Biological Chemistry, 2002
The abbreviations used are: PLA 2 , phospholipase A 2 ; GIIA PLA 2 , group IIA phospholipase A 2 ; sPLA 2 , secretory phospholipase A 2 ; cPLA 2 , Ca 2ϩ-dependent cytosolic phospholipase A 2 (ϭ group IV PLA 2); cPLA 2 ␣, cPLA 2  and cPLA 2 ␥, ␣,  and ␥ isoforms of cPLA 2 ; PMN, polymorphonuclear leukocyte (neutrophil); MAFP, methylarachidonyl fluorophosphonate; PGE 2 , prostaglandin E 2 ; FACS, fluorescence-activated cell sorter; RT, reverse transcriptase; R-PE, rhodamine-phycoerythrin; LTB 4 , leukotriene B 4 ; FITC, fluorescein isothiocyanate; BPI, bactericidal/permeability-increasing protein; MPO, myeloperoxidase; NGAL, neutrophil gelatinase-associated lipocalin; OZ, serum opsonized zymosan; BSA, bovine serum albumin; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; PVDF, polyvinylidene difluoride; h, human.
Journal of Biological Chemistry, 2003
Here we report cellular arachidonate (AA) release and prostaglandin (PG) production by novel classes of secretory phospholipase A 2 s (sPLA 2 s), groups III and XII. Human group III sPLA 2 promoted spontaneous AA release, which was augmented by interleukin-1, in HEK293 transfectants. The central sPLA 2 domain alone was sufficient for its in vitro enzymatic activity and for cellular AA release at the plasma membrane, whereas either the unique Nor C-terminal domain was required for heparanoid-dependent action on cells to augment AA release, cyclooxygenase-2 induction, and PG production. Group III sPLA 2 was constitutively expressed in two human cell lines, in which other sPLA 2 s exhibited different stimulus inducibility. Human group XII sPLA 2 had a weak enzymatic activity in vitro and minimally affects cellular AA release and PG production. Cells transfected with group XII sPLA 2 exhibited abnormal morphology, suggesting a unique functional aspect of this enzyme. Based on the present results as well as our current analyses on the group I/II/V/X sPLA 2 s, general properties of cellular actions of a full set of mammalian sPLA 2 s in regulating AA metabolism are discussed.
Journal of Biological Chemistry, 1998
We examined the relative contributions of five distinct mammalian phospholipase A 2 (PLA 2) enzymes (cytosolic PLA 2 (cPLA 2 ; type IV), secretory PLA 2 s (sPLA 2 s; types IIA, V, and IIC), and Ca 2؉-independent PLA 2 (iPLA 2 ; type VI)) to arachidonic acid (AA) metabolism by overexpressing them in human embryonic kidney 293 fibroblasts and Chinese hamster ovary cells. Analyses using these transfectants revealed that cPLA 2 was a prerequisite for both the calcium ionophore-stimulated immediate and the interleukin (IL)-1-and serum-induced delayed phases of AA release. Type IIA sPLA 2 (sPLA 2-IIA) mediated delayed AA release and, when expressed in larger amounts, also participated in immediate AA release. sPLA 2-V, but not sPLA 2-IIC, behaved in a manner similar to sPLA 2-IIA. Both sPLA 2 s-IIA and-V, but not sPLA 2-IIC, were heparin-binding PLA 2 s that exhibited significant affinity for cell-surface proteoglycans, and site-directed mutations in residues responsible for their membrane association or catalytic activity markedly reduced their ability to release AA from activated cells. Pharmacological studies using selective inhibitors as well as co-expression experiments supported the proposal that cPLA 2 is crucial for these sPLA 2 s to act properly. The AA-releasing effects of these sPLA 2 s were independent of the expression of the M-type sPLA 2 receptor. Both cPLA 2 , sPLA 2 s-IIA, and-V were able to supply AA to downstream cyclooxygenase-2 for IL-1-induced prostaglandin E 2 biosynthesis. iPLA 2 increased the spontaneous release of fatty acids, and this was further augmented by serum but not by IL-1. Finally, iPLA 2derived AA was not metabolized to prostaglandin E 2. These observations provide evidence for the functional cross-talk or segregation of distinct PLA 2 s in mammalian cells in regulating AA metabolism and phospholipid turnover.
Biochemical and Biophysical Research Communications, 1988
The 15-lipoxygenase pathway is the predominant pathway for arachidonic acid metabolism in homogenates of human lung (l), in isolated human airway epithelial cells (2) and in human eosinophils (3,4), and keratinocytes(5). Although some functions of arachidonate18lipoxygenase metabolites have been described(6), little is known about the isolation or modulation of human 15-lipoxygenase. The 5lipoxygenase of human leukocytes has been purified to homogeneity(7), and has been separated from 15lipoxygenase using anion exchange chromatography(7,8). Our study provides new evidence that human leukocyte 15-lipoxygenase activity may be entirely accounted for by eosinophils. Using this observation, we have developed a method for purifying human 15-lipoxygenase with cation-exchange and hydrophobicinteraction chromatography using eosinophils obtained from patients treated with interleukin-2. The kinetic properties, pH dependence and divalent cation dependence of the 15-lipoxygenase are described. Our results indicate that human leukocyte 15-lipoxygenase differs from 5-lipoxygenase in both electrostatic and hydrophobic properties. In addition, we have found that phosphatidylcholine stimulates, and ATP inhibits, 15lipoxygenase activity. These observations suggest a regulatory role for both phosphatidylcholine and