Tài liệu Báo cáo Y học: Differential effects of arachidonoyl trifluoromethyl ketone on arachidonic acid release and lipid mediator biosynthesis by human neutrophils pot

Eur J Biochem 269, 3760–3770 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03070.x Differential effects of arachidonoyl trifluoromethyl ketone on arachidonic acid release and lipid mediator biosynthesis by human neutrophils Evidence for different arachidonate pools Alfred N Fonteh Department of Internal Medicine, Division of Pulmonary and Critical Care Medicine, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157, USA The goal of this study was to determine the effects of a putative specific cytosolic phospholipase A2 inhibitor, arachidonyl trifluoromethyl ketone (AACOCF3), on arachidonic acid (AA) release and lipid mediator biosynthesis by ionophore-stimulated human neutrophils Initial studies indicated that AACOCF3 at concentrations 0–10 lM did not affect AA release from neutrophils In contrast, AACOCF3 potently inhibited leukotriene B4 formation by ionophore-stimulated neutrophils (IC50  2.5 lM) Likewise, AACOCF3 significantly inhibited the biosynthesis of platelet activating factor In cell-free assay systems, 10 lM AACOCF3 inhibited 5-lipoxygenase and CoA-independent transacylase activities [3H]AA labeling studies indicated that the specific activities of cell-associated AA mimicked that of leukotriene B4 and PtdCho/PtdIns, while the specific activities of AA released into the supernatant fluid closely mimicked that of PtdEtn Taken together, these data argue for the existence of segregated pools of arachidonate in human neutrophils One pool of AA is linked to lipid mediator biosynthesis while another pool provides free AA that is released from cells Additionally, the data suggest that AACOCF3 is also an inhibitor of CoA-independent transacylase and 5-lipoxygenase Thus, caution should be exercised in using AACOCF3 as an inhibitor of cytosolic phospholipase A2 in whole cell assays because of the complexity of AA metabolism Phospholipases A2 (PLA2) are enzymes that hydrolyse acyl bonds at the sn-2 position of phospholipids generating free fatty acids and lysophospholipid moieties [1] Several mammalian PLA2 isotypes have been cloned and sequenced [2–10] The most characterized of these enzymes are a hormonally regulated, cytosolic high molecular mass enzyme (cPLA2) [11], a calcium-independent PLA2 (iPLA2) [12] and various secretory low molecular mass isotypes (sPLA2) [6] These major PLA2 isotypes have all been implicated in arachidonic acid (AA) mobilization and eicosanoid biosynthesis by inflammatory cells [13–19] Specifically, knockout studies have conclusively linked cPLA2 with lipid mediator formation [20] Because of the importance of PLA2, various approaches have been designed to influence PLA2 levels within cells [21–29] Of various inhibitors that have been used, analogues of AA such as arachidonoyl trifluoromethyl ketone (AACOCF3) have been purported to be specific in inhibiting cPLA2 Thus, AACOCF3 has been used extensively in several cell systems to examine AA release [23,26,30–32] However, no comprehensive study has been undertaken to examine the effects of AACOCF3 on other AA-specific pathways in whole cells, even though this compound has been shown to inhibit other enzyme activities [33,34] In addition to PLA2, other enzymes have been shown to affect arachidonate content and its release from inflammatory cells [35] Incorporation and release of AA is accompanied by remodeling between various phospholipid subclasses [35–39] CoA-dependent and CoA-independent enzymes are responsible for regulating cellular arachidonate levels [40–42] Different forms of an activity (arachidonoyl CoA synthetase) that converts free AA to arachidonoyl-CoA (AA-CoA) at the expense of ATP have been described previously [43–46] Once synthesized, AA-CoA is incorporated into lysophospholipids by CoAdependent acyl transferases [45,47–49] In addition to these CoA-dependent mechanisms, arachidonate is rapidly shuttled from 1-acyl-linked phospholipids to 1-etherlinked phospholipids by CoA-independent transacylase Correspondence to A N Fonteh, Molecular Neurology Program, Huntington Medical Research Institutes, 99, North El Molino Avenue, Pasadena, CA 91101–1830, USA Fax: + 626 795 5774, Tel.: + 626 795 4343, E-mail: afonteh@hmri.org Abbreviations: AA, arachidonic acid; AA-CP, arachidonic acid in cell pellets; AA-SF, arachidonic acid in supernatant fluids; cPLA2, cytosolic phospholipase A2; GPC, sn-glycerol-3-PCho; GPE, sn-glycerol-3-PEtn; GPI, sn-glycero-3-PIns; iPLA2, calcium-independent phospholipase A2; 5-LO, 5-lipoxygenase; NICI-GC/MS, negative ion-chemical ionization gas chromatography/ mass spectrometry; PAF, platelet activating factor; PLA2, phospholipase A2; sPLA2, secretory phospholipase A2; SA, specific activity (Received 14 February 2002, revised 16 May 2002, accepted 24 June 2002) Keywords: arachidonic acid; lipid mediators; neutrophils; phospholipase A2; inhibitor Ó FEBS 2002 Effects of AACOCF3 on lipid mediator biosynthesis (Eur J Biochem 269) 3761 [37–39,50–53] Evidence using cell-free preparations suggests that the selective transfer of arachidonate from 1-alkyl-2-arachidonoyl-GPC (sn-glycero-3-PCho) to lysophospholipid acceptors leads to the formation of 1-alkyl2-lyso-GPC This intermediate can be converted to platelet activating factor (PAF) by acetyl transferase [54–56] Rapid remodeling involving many enzyme activities and phospholipid substrates makes it difficult to study these molecular events [37,57,58] Earlier studies demonstrated that inflammatory cells released relatively large quantities of AA from several phospholipids (PtdEtn > PtdCho > PtdIns) [58,59] However, when cells are pulselabeled, AA release is accompanied by changes in the specific activities (SA) of all major phospholipid subclasses [58,59] These changes in SA are due to rapid acylation, deacylation and remodeling reactions Thus, while PLA2 isotypes may provide most of the AA that is utilised for eicosanoid biosynthesis, reacylation and remodeling between phospholipid subclasses may also be a crucial factor involved in the regulation of free AA levels and the generation of potent lipid mediators The objective of these studies was to determine the effects of a putative cPLA2 inhibitor (AACOCF3) on AA release and lipid mediator biosynthesis Our data suggest that AACOCF3 decreases lipid mediator formation by neutrophils without affecting free AA levels by inhibiting CoA-independent transacylase (CoA-IT) and 5-lipoxygenase (5-LO) activities SA measurements show that lipid mediators and free AA are derived from different phospholipid pools Together, these data suggest that there is segregation of AA pools within neutrophils and caution should always be exercised in the use of AA analogues such as AACOCF3 as specific inhibitors in whole cell assays because of the complexity of AA metabolising pathways and because these compounds influence many enzyme activities Neutrophil isolation and stimulation Neutrophils were obtained from venous blood of healthy human donors as described previously [60] Neutrophils (5 · 106 mL)1) in HBSS were incubated at 37 °C (5 min) prior to stimulation Different concentrations of freshly made AACOCF3 in dimethylsulfoxide were added to the cells before stimulation with 2.5 lM ionophore A23187, for periods of time indicated in the figure legends The incubations were terminated by the addition of methanol/chloroform (2 : 1, v/v) Lipids were extracted from the reaction mixture by the method of Bligh & Dyer [61] In experiments where the quantities of leukotrienes were determined, neutrophils were removed from the supernatant fluid by centrifugation (400 g, min) Prostaglandin B2 (PGB2, 250 ng) was added as an internal standard and eicosanoids were extracted twice from the supernatant fluids using mL ethyl acetate Determination of AA release from stimulated neutrophils After the addition of 100 ng [2H8]AA as an internal standard, solvents were removed from ethanol extracts of supernatant fluids using a stream of nitrogen Fatty acids were then converted to pentafluorobenzyl esters and the molar quantities of free fatty acids were determined by combined negative ion-chemical ionization gas chromatography/mass spectroscopy (NICI-GC/MS), using a Hewlett Packard model 5989 instrument Carboxylate anions (m/z) were monitored at 303 and 311 nm for AA and [2H8]AA, respectively, in the single ion-monitoring mode In experiments where cellular AA was determined, lipids were extracted from cellular pellets A fatty acid enriched fraction was obtained using Bakerbond silica gel disposable columns [62] After solvent removal using a stream of nitrogen, molar quantities of AA were determined as described above MATERIALS AND METHODS Determination of molar quantities of leukotrienes Materials [3H]Acetic acid (3.3 CiỈmmol)1) and [5,6,8,9,11,12,14, 15–3H]AA (76 CiỈmmol)1) were purchased from New England Nuclear (Boston, MA, USA) AACOCF3 was purchased from Biomol (Plymouth Meeting, PA, USA), lipid standards from Avanti Polar Lipids (Birmingham, AL, USA), and Hanks Balanced Salt Solution (HBSS) and NaCl/Pi purchased from Gibco Laboratories (Grand Island, NY, USA) Silica gel G plates were purchased from Analtech (Newark, DE, USA), with silica gel columns purchased from J T Baker Inc (Phillipsburg, NJ, USA) Ionophore A23187 was purchased from Calbiochem (La Jolla, CA, USA), Ficoll-Paque from Pharmacia (Piscataway, NJ, USA) and Dextran 70 from Abbot Laboratories (North Chicago, IL, USA) Arachidonic acid, leukotriene B4 (LTB4) and 20-hydroxy-leukotriene B4 were purchased from Cayman (Ann Arbor, MI, USA) Essentially fattyacid-free human serum albumin was purchased from Sigma (St Louis, MO, USA) Pentafluorobenzyl bromide and diisopropylethylamine were purchased from Alltech/ Applied Science Associate (Deerfield, IL, USA) and HPLC grade solvents purchased from Fisher Scientific (Norcross, GA, USA) PGB2 (250 ng) was added to supernatant fluids as an internal standard prior to sample concentration using a stream of nitrogen Leukotrienes were suspended in 30% methanol in water and injected onto an Ultrasphere ODS column (2.0 · 250 mm, Rainin Instrument Co, Woburn, MA, USA) that had been conditioned in a solvent that consisted of methanol/water/phosphoric acid (550 : 450 : 0.2, v/v/v, pH 5.7) The solvent was delivered at a flow rate of 0.3 mLỈmin)1 and products were monitored (270 and 206 nm) using a Hewlett Packard diode array detection system After min, eicosanoids were eluted from the column by increasing the amount of methanol to 100% over 50 Leukotrienes and free AA were collected and the radioactivity in these fractions determined by liquid scintillation counting Molar quantities of leukotrienes were determined by UV spectroscopy PAF biosynthesis The incorporation of [3H]acetate into PAF was used to quantitate PAF biosynthesis in human neutrophils Briefly, neutrophils in HBSS (5 · 106 mL)1) were incubated with lCiỈmL)1 [3H]acetate for 15 at 37 °C The cells were Ó FEBS 2002 3762 A N Fonteh (Eur J Biochem 269) stimulated in the presence and absence of 10 lM AACOCF3 and reactions terminated by the addition of methanol/ chloroform (2 : 1, v/v) After phospholipid extraction, PAF was isolated by TLC on silica gel G developed in chloroform/methanol/acetic acid/water (50 : 25 : : 4, v/v/v/v) Radioactivity on TLC plates was detected using a Bioscan radioactivity imaging system (Washington, DC, USA) and the amount of radioactivity migrating with PAF was determined by zonal scraping, followed by liquid scintillation counting Determination of 5-LO activity Neutrophils (5 · 106 mL)1) were stimulated as described above Cells were removed from supernatant fluids by centrifugation (400 g, min) and cell pellets were suspended in mL of 50 mM phosphate buffer containing mM dithiothreitol, 1.6 mM EDTA, lgỈmL)1 leupeptin, lgỈmL)1 pepstatin and 0.5 mM phenylmethanesulfonyl fluoride Cells were then broken by sonication (10 s, three times) using a model W-220 sonicator (Heat System Ultrasonic Inc., Farmingdale, NY, USA) set at a power scale of two and 10% output Unbroken cells were removed from sonicates by centrifugation (10 000 g, 10 min) Cytosolic and pellet fractions were obtained after ultracentrifugation of sonicates (100 000 g, 60 min) 5-LO activity was determined in cytosolic fractions in a final volume of mL of 200 mM Tris/HCl (pH 7.5) containing 1.8 mM ATP, 1.6 mM EDTA and 10 mM CaCl2 5-LO activity was initiated by the addition of [3H]AA (100 nCiỈnmol)1) and 2.6 nmol hydroxy-9-cis-11-trans-octadecadienioc acid as a hydroxyperoxide activator 5-LO products [5-hydroxyeicosatetraenoic acid (HETE), LTB4 and 20-OH-LTB4] and AA were isolated by reverse phase HPLC as described previously [16] and radioactivity determined by liquid scintillation counting Determination of CoA-IT activity Neutrophils were suspended in CoA-IT sonication buffer [50 mM Hepes buffer, pH 7.4, containing mM EDTA and 20% sucrose (w/v)] Cells were broken using a probe sonicator as described above, and cytosolic and membrane fractions obtained after ultracentrifugation (100 000 g, 60 min, °C) The membrane fraction was diluted in NaCl/Pi containing mM EGTA with 10 lg total protein utilised for determining CoA-IT activity The reaction was initiated by the addition of [3H]1-alkyl-2-lyso-GPC (0.1 lCi) and nmol 1-O-hexadecyl-2-lyso-GPC in a final volume of 100 lL After 10 at 37 °C, the reaction was stopped and lipids were extracted [61] Phospholipids were separated by TLC on silica gel G developed in chloroform/ methanol/acetic acid/water (50 : 25 : : 4, v/v/v/v) The product ([3H]1-alkyl-2-acyl-GPC) was visualized by radioscaning (Bioscan), scrapped and quantified by liquid scintillation spectroscopy [3H]AA was removed by washing cells three times with HBSS containing 0.25 mgỈmL)1 human serum albumin Cells were incubated in a water bath (37 °C, 15 min) to allow complete reacylation of cellular free AA After stimulation with ionophore, cells were removed from supernatant fluids by centrifugation (400 g, min) Supernatant fluids were added to vol of ethanol and molar quantities of leukotriene and AA determined by HPLC and NICI-GC/MS, respectively Lipids in the cell pellets were extracted and glycerolipid classes were isolated using normal phase HPLC as described previously [63] Radioactivity was determined in a portion of the isolated fractions by liquid scintillation counting using a Beckman liquid scintillation counting system (Fullerton, CA, USA) Portions of PtdCho and PtdEtn fractions were hydrolysed using 10 U of Grade Bacillus cereus phospholipase C (Boehringer Mannheim) for 2.5 h Diradylglycerols obtained from phospholipase C hydrolysis were converted to acetate derivatives [63] 1-Acyl-, 1-alkyl-, and 1-alk-1¢-enylsubclasses were separated by TLC on silica gel G developed in benzene/hexane/ether (50 : 25 : 4, v/v/v) Molar quantities of AA in phospholipid classes and subclasses were determined after base hydrolysis by NICI-GC/MS as described above SA in phospholipid classes and subclasses were calculated and expressed as radioactivity (nCi)Ỉnmol)1 arachidonate Determination of SA of AA and leukotrienes The neutral lipid fraction obtained from normal phase HPLC was separated into classes by TLC using silica gel G developed in hexane/ether/formic acid (90 : 60 : 6, v/v/v) Radioactivity in products was determined using a radiochromatogram imaging system (Bioscan) The region corresponding to free fatty acids was scraped into vials while an equal amount was used to determine molar quantities of arachidonate by NICI-GC/MS SA of cellular AA was calculated and expressed as radioactivity (nCi)Ỉnmol)1 arachidonate Leukotrienes and free AA were isolated by reverse phase HPLC, as described above Fractions corresponding to leukotrienes and free AA were collected and the amount of radioactivity in each determined by scintillation counting Free AA was also converted to pentafluorobenzylesters and the molar quantity of AA determine by NICI-GC/MS SA of leukotrienes and AA from supernatant fluids (AA-SF) were calculated and expressed as radioactivity (nCi)Ỉnmol)1 Statistical analysis All data are expressed as the means ± SEM of separate experiments Statistics (P-values) were obtained from Student’s t-test for paired samples Notations used on figures and legends are for P < 0.05 [3H]AA labeling of glycerolipids and determination of specific activities (SA) RESULTS Human neutrophils were labeled by adding [3H]AA (1 lCi per · 107 cells) complexed to fatty-acid-free human serum albumin (0.25 mgỈmL)1) in HBSS for 0.5 h Unincorporated Effect of AACOCF3 on AA release from neutrophils During activation of neutrophils, AA is released from phospholipid pools by cPLA2 Our studies and those of Ó FEBS 2002 Effects of AACOCF3 on lipid mediator biosynthesis (Eur J Biochem 269) 3763 Fig Dose-dependent effects of AACOCF3 on AA release Human neutrophils incubated with different concentrations of AACOCF3 for were stimulated for with 2.5 lM ionophore A23187 Stimulation was stopped by centrifugation and molar quantities of AA in the supernatant fluid (AA-SF) and AA associated with cell pellet (AA-CP) were determined by NICI-GC/MS as described in Materials and methods These data are the mean ± SEM of six separate experiments (*P < 0.05 compared to lM AACOCF3) Wykle and colleagues have shown that AA levels and lipid mediator formation are also modulated by changes in CoAIT activity [53,64–69] As cPLA2 and CoA-IT are selective for AA, we hypothesized that AACOCF3 may influence AA levels in activated neutrophils by modulating these activities Therefore, we examined AA levels within cells or in supernatant fluids of neutrophils that had been stimulated in the presence of different concentrations of AACOCF3 As shown in Fig 1, AACOCF3 (0–10 lM) did not inhibit AA release from stimulated neutrophils Paradoxically, there was an increase in AA levels within neutrophils and in supernatant fluids as the concentration of AACOCF3 was increased These data suggested that AACOCF3 at these concentrations did not affect PLA2 activity However, at higher concentrations ( 20 lM), AACOCF3 reduced AA release from A23187-stimulated neutrophils (78.7 ± 54.9 pmol per 10 million neutrophils, n ¼ 4, for AA in supernatant fluids and 178 ± 23.8 pmol per 10 million neutrophils, n ¼ 4, for cellular AA) To make sure that AACOCF3 was presented for sufficient time to penetrate the cells and inhibit cellular enzymes, neutrophils were incubated with 10 lM AACOCF3 for different periods of time As shown in Fig 2A, AA released into supernatant fluids was not inhibited even after neutrophils were incubated with AACOCF3 for 60 Similarly, cellular free AA levels were not inhibited under similar conditions (Fig 2B) Together, these data suggest that there are two types of AA pools in neutrophils, one that is not inhibited by low concentrations of AACOCF3 and another that is inhibited by high concentrations of AACOCF3 The increase in AA at low concentrations of AACOCF3 could also suggest that an AACOCF3-sensitive pool was destined for product formation, and the inhibition of this product formation may have accounted for the build up of free AA Fig Time-dependent effects of AACOCF3 on AA release Human neutrophils incubated with 10 lM AACOCF3 for were stimulated for different periods of time with 2.5 lM ionophore A23187 Stimulation was stopped by centrifugation and molar quantities of AA-SF (A) for cells stimulated with or without AACOCF3 were determined by NICI-GC/MS as described in Materials and methods Similarly, AA-CP (B) with or without AACOCF3 was determined These data are the mean ± SEM of six separate experiments Influence of AACOCF3 on lipid mediator biosynthesis To determine whether the increase in AA levels was due to a decrease in AA-derived mediators, we examined leukotriene biosynthesis AACOCF3 inhibited the biosynthesis of LTB4 and 20-OH-LTB4 at a concentration (10 lM) that did not affect AA release (Fig 3) These data suggest that free AA and AA destined for leukotriene formation are derived from different phospholipid pools or are regulated by different signaling pathways As LTB4 and PAF share a common precursor [13,70], we next determined whether AACOCF3 also influenced PAF synthesis Ionophore A23187-induced [3H]acetate incorporation into PAF (Fig 4) Pre-incubation with 10 lM 3764 A N Fonteh (Eur J Biochem 269) Ó FEBS 2002 Fig Influence of AACOCF3 on leukotrienes biosynthesis Human neutrophils incubated without or with 10 lM AACOCF3 for were stimulated for with 2.5 lM ionophore A23187 Cell stimulation was stopped by centrifugation and molar quantities of LTB4 and 20-OH-LTB4 in supernatant fluids were determined by reverse phase HPLC as described in Materials and methods These data are the mean ± SEM of five separate experiments (*P < 0.05 compared to lM AACOCF3) Fig Effects of AACOCF3 on PAF formation Human neutrophils incubated without or with 10 lM AACOCF3 for were stimulated for with 2.5 lM ionophore A23187 PAF biosynthesis assessed by [3H]acetate incorporation was determined as described in Materials and methods These data are the mean ± SEM of five separate experiments (*P < 0.05 compared to lM AACOCF3) AACOCF3 resulted in significant inhibition of radiolabeled PAF formation Similar to LTB4, these data suggest that the formation of the PAF precursor, lyso PAF, was independent of free AA release Influence of AACOCF3 on enzyme activities An explanation for the inhibition of LTB4 and PAF biosynthesis independent of AA inhibition is that AACOCF3 may inhibit other enzymes that are directly Fig Influence of AACOCF3 on 5-lipoxygenase activity Human neutrophils incubated without or with 10 lM AACOCF3 for were stimulated for with 2.5 lM ionophore A23187 Cytosol and membrane fractions were prepared by ultracentrifugation 5-LO activity in membranes from unstimulated neutrophils (Control) or ionophore A23187-stimulated (A23187) neutrophils (A) and A23187stimulated neutrophils in the presence of 10 AACOCF3 (B) was determined as described in Materials and methods Radioactivity (DPM) coeluting with 5-LO products (LTB4, 5-HETE) or with free AA is indicated by arrows These data are representative of three separate experiments linked to the formation of these lipid mediators while higher concentrations of AACOCF3 are required to inhibit the PLA2 activity responsible for AA release in whole cells We next examined the effects of AACOCF3 on 5-LO and CoAIT activities 5-LO activity In unstimulated cells, the bulk of radioactivity resided in the AA peak After ionophore stimulation, there was an increase in radiolabel coeluting with 5-LO products, LTB4 and 5-HETE (Fig 5A) In the presence of 10 lM AACOCF3, there was a significant decrease (89.8 ± 5.6%, n ¼ 3) in 5-LO product formation (Fig 5B) These studies suggest that AACOCF3 at a concentration that does not affect AA release in whole neutrophils effectively inhibits 5-LO activity Ó FEBS 2002 Effects of AACOCF3 on lipid mediator biosynthesis (Eur J Biochem 269) 3765 Fig Influence of AACOCF3 on CoA-IT activity Membrane and cytosolic fractions were prepared from human neutrophils by ultracentrifugation Total membrane protein (10 lg) was incubated with different concentrations of AACOCF3 for Subsequently, CoAIT activity was initiated by adding [3H]1-alkyl-2-lyso-GPC Phospholipids were isolated by TLC and radiolabel in PtdCho determined by liquid scintillation counting These data are the mean ± SEM of four separate experiments (*P < 0.05 compared to lM AACOCF3) CoA-IT activity Inhibition of CoA-IT by AACOCF3 could potentially account for the decrease in PAF formation We tested this hypothesis by measuring microsomal CoA-IT activity As shown in Fig 6, microsomes prepared from neutrophils contained CoA-IT activity (4.06 0.15 nmolặ mg)1ặmin)1, n ẳ 4) AACOCF3 dose-dependently inhibited CoA-IT activity (IC50  7.5 lM) These data suggest that inhibition of CoA-IT activity by AACOCF3 might account for the decrease in PAF formation Relationship between SA of phospholipids, arachidonic acid and leukotrienes To obtain further evidence for different AA pools, neutrophils were labeled with [3H]AA for 30 to achieve nonisotopic equilibrium This strategy differentially labels the major phospholipid pools within neutrophils and thus allows comparisons to be made between the SA of phospholipids and products Five minutes after stimulation, molar quantities of arachidonate in phospholipid subclasses are reduced except in 1-alk1-enyl-2-AA-GPC (Table 1) 1-Alk-1-enyl-2-AA-GPE and 1-alkyl-2-AA-GPC account for most of the AA released from PtdEtn and PtdCho, respectively In PtdCho and PtdEtn subclasses, the rank order of SA is 1-acyl-2-AA-GPC (51.7 ± 12.5 nCiỈnmol)1, n ¼ 3) > 1-alkyl-2-AA-GPC (7.2 ± 0.8 nCiỈnmol)1) > 1-acyl-2-AAGPE (3.9 ± 0.8 nCiỈnmol)1) > 1-alk-1-enyl-2-AA-GPE (0.6 ± 0.2 nCiỈnmol)1) During cell activation, there is a decrease in the SA in 1-acyl-linked phospholipids (38.1 ± 9.33% decrease in 1-acyl-2-AA-GPC; 22.7 ± 2.2% decrease in 1-acyl-2-AA-GPI and 26.9 ± 10.8% decrease in 1-acyl-2-AA-GPE) Concomitantly, there is an increase in the SA in 1-ether-linked phospholipid subclasses (19.0 ± 6.0% increase in 1-alkyl-2-AAGPC; 48.3 ± 10.3% increase in 1-alk-1-enyl-2-AA-GPE) There is a twofold increase in SA of PtdEtn (Fig 7) These data suggest that a remodeling process involving the release of low SA AA from ether-linked phospholipid subclasses accompanied the incorporation of this AA into 1-acyl-linked phospholipid subclasses (mainly in PtdCho/PtdIns) Conversely, the increase in the SA of ether-linked PtdEtn and PtdCho subclasses suggested that high SA AA from 1-acyl-linked phospholipids (mainly PtdCho and PtdIns) was being remodeled into the etherlinked subclasses (1-alkyl-2-AA-GPC and 1-alk-1-enyl-2AA-GPE) Examination of SA of products indicated that PtdCho/ PtdIns were the likely sources of AA that was utilized for leukotriene biosynthesis (Fig 7) In contrast, PtdEtn that accounts for the bulk of free AA released from neutrophils had a SA that was significantly lower than the SA of leukotrienes and thus did not contribute AA for leukotriene biosynthesis The SA of AA associated with cell pellet (AA-SP) closely resembled that of PtdCho/PtdIns and leukotrienes and was different from AA that was released into the supernatant fluid (AA-SF) Likewise, the SA of AA-SF mimicked that of PtdEtn and was different from that of PtdCho/PtdIns Together, these data suggest that there is segregation of AA pools within neutrophils Table Changes in arachidonate content of phospholipids after A23187 stimulation Human neutrophils were incubated without (control) or with ionophore A23187 for Glycerolipids were extracted and phospholipids (PtdEtn, PtdIns, PtdCho) isolated by normal phase HPLC PtdEtn and PtdCho subclasses (1-acyl-, 1-alkyl-, 1-alk-1-enyl-) were separated as described under Materials and methods Molar quantities of arachidonate were determined after base hydrolysis by NICI-GC/MS Net release of AA was then determined These data are the mean ± SEM of three separate experiments (*P < 0.05 compared to control) Arachidonate content (nmolỈ10)7 cells) Phospholipids Control 1-Acyl-2-AA-GPC 1-Alkyl-2-AA-GPC 1-Alk-1-enyl-2-AA-GPC 1-Acyl-2-AA-GPI 1-Acyl-2-AA-GPE 1-Alkyl-2-AA-GPE 1-Alk-1-enyl-2-AA-GPE 0.495 1.655 0.102 1.829 0.858 1.279 5.491 ± ± ± ± ± ± ± A23187 0.066 0.224 0.013 0.041 0.235 0.470 0.857 0.237 0.550 0.191 0.991 0.720 1.142 2.661 ± ± ± ± ± ± ± Net Release 0.070* 0.058* 0.141 0.131* 0.285 0.472 0.458* )0.258 )1.105 +0.089 )0.838 )0.138 )0.137 )2.830 3766 A N Fonteh (Eur J Biochem 269) Fig Relationship between specific activity of phospholipids and products Human neutrophil phospholipids were differentially labeled with [3H]-AA After stimulation with 2.5 lM ionophore A23187 for different periods of time, cell pellets and supernatant fluids were obtained by centrifugation Phospholipids (PtdCho, PtdIns and PtdEtn) were isolated by normal phase HPLC, while products in the supernatant fluid (AA-SF, LTB4 and 20-OH-LTB4) were isolated by reverse phase HPLC The radioactivity recovered in phospholipids, AA-SF, AA-CP and leukotrienes was determined by liquid scintillation counting and the SA (radioactivity) determined as described in Materials and methods These data are the mean ± SEM of three separate experiments (*P < 0.05 compared to AA-CP, LTB4 and 20-OH LTB4; **P < 0.05 compared to AA-SF) DISCUSSION An important finding of the present study is that there are at least two distinct arachidonate pools in human neutrophils One AA pool (from PtdCho/PtdIns) is linked to lipid mediator formation while another AA pool that is not linked with product formation is closely associated with PtdEtn The following key pieces of data support these observations: (a) concentrations of AACOCF3 < 10 lM not inhibit AA release from ionophore-stimulated neutrophils Paradoxically, there is a slight increase in AA levels at these concentrations It requires > 10 lM AACOCF3 for AA release to be effectively inhibited (b) At concentrations of AACOCF3 that not inhibit AA release, there is > 85% inhibition of LTB4 and PAF biosynthesis suggesting that AA release may not be linked to lipid mediator biosynthesis (c) AACOCF3 inhibits 5-LO activity at concentrations that are not effective in decreasing AA release (d) CoA-IT activity that generates lyso PAF, an intermediate of PAF biosynthesis, is inhibited by AACOCF3 at concentrations that not affect AA release A decrease in LTB4 biosynthesis concomitant with an increase in free AA suggest that AA destined for LTB4 biosynthesis is regulated differently from AA that remains free (e) Further evidence for the segregation of AA pools within neutrophils is provided by studies showing that AA Ó FEBS 2002 Fig Proposed mechanism for incorporation, remodeling, release and lipid mediator biosynthesis in human neutrophils Free AA is converted to AA-CoA and incorporated into 1-acyl-linked phospholipid subclasses by AA-CoA synthetase (a) and AA-CoA-dependent acyl transferases (b), respectively Under resting conditions, AA is remodeled from 1-acyl-linked phospholipids to 1-ether-linked phospholipids by CoA-IT activity (c) During cell activation, the remodeling process is accelerated due to the formation of 1-alk1-enyl-2-lyso-GPE by PLA2 (d) Enhanced remodeling is accompanied by an increase in the formation of 1-alkyl-2-lyso-PAF (e), which is converted, to PAF by acetyl transferase (f) AA from PtdCho/ PtdIns is simultaneously utilised by 5-LO (g) to form LTB4, which is further metabolised to 20-OH-LTB4 Low concentrations of AACOCF3 prevent PAF and LTB4 formation by inhibiting CoA-IT and/or 5-LO Higher concentrations of AACOCF3 prevent AA release from PtdEtn resulting in a decrease in AA and lipid mediators (§, enzyme activities inhibited by low concentrations of AACOCF3; /, enzyme activity inhibited by higher concentrations of AACOCF3; Y, low SA AA released into the supernatant fluid and re-incorporated into PtdCho/PtdIns) destined for leukotriene biosynthesis has a higher SA than AA that is released from the biggest arachidonate pool in PtdEtn A schematic representing AA metabolism in human neutrophils is shown in Fig (major enzymes are labeled alphabetically, a–g) AA is constantly released and remodeled in cells This process is slow and well controlled in unstimulated cells and is accelerated when cells are stimulated Once presented to cells, AA is incorporated into cellular glycerophospholipids by a distinct pathway Free AA is initially converted to AA-CoA by AA-CoA synthetase (a) [46] Inhibitors of AA-CoA synthetase prevent AA incorporation into cellular lipids leading to a build-up of free cellular AA within cells [71] Without inhibitors, AACoA is acylated to 1-acyl-linked phospholipid subclasses by CoA-dependent acyl transferase (b) Once in 1-acyl-linked subclasses, AA is transferred to the larger pools found in ether-linked subclasses by CoA-IT (c) [35] Similar to Ó FEBS 2002 Effects of AACOCF3 on lipid mediator biosynthesis (Eur J Biochem 269) 3767 AA-CoA synthetase, inhibition of CoA-IT results in an increase in free AA and a corresponding build-up of AA in triacylglycerols that can be prevented by co-incubation of cells with CoA-synthetase inhibitors [72,73] Whereas inhibition of CoA-IT results in a decrease in lipid mediator formation, changes in AA-CoA synthetase may or may not accompany a decrease in lipid mediator formation [53,71,74] These data suggest that the initial incorporation of AA into phospholipids may not always be critical in mediator generation by stimulated cells, while the capacity of cells to remodel AA via CoA-IT is closely linked to mediator formation Specific activity measurements have shown that the major lipid mediators produced by human neutrophils, PAF and LTB4, share the same common precursor, 1-alkyl-2-AA-GPC [70] The present data using AACOCF3 are in agreement with these earlier studies by demonstrating that inhibition of LTB4 and PAF biosynthesis by AACOCF3 occurs without concomitant inhibition of AA release Generation of the common precursor pool by CoA-IT occurs by the transfer of AA from 1-acyl-linked phospholipid to 1-ether linked phospholipid classes During cell activation, PLA2 (Fig 8, d) releases AA from mainly 1-alk-1-enyl-2-AA-GPE resulting in the formation of 1-alk-1-enyl-2-lyso-GPE Wykle and colleagues have shown that 1-alk-1-enyl-2-lyso-GPE enhances PAF formation by a CoA-IT-dependent generation of lyso PAF [55] Thus, while PLA2 isotypes may not be directly involved in generating lyso PAF, the activity of PLA2 on PtdEtn may indirectly drive the remodeling process that generates the PAF precursor by providing AA acceptor molecules As AA release is not affected, these data suggest that some activities responsible for AA metabolism in whole cells are more sensitive to AACOCF3 than PLA2 While CoA-IT may explain how PAF is formed, the release of AA destined for LTB4 cannot easily be explained by a decrease in AA remodeling alone There are several mechanisms that may account for a decrease in LTB4 formation First, CoA-IT may have an intrinsic lipase activity that is inhibited by AACOCF3 and this process prevents AA release from 1-alkyl-2-AA-GPC with the corresponding decrease in LTB4 formation Secondly, CoA-IT may be linked to AACOCF3-sensitive PLA2 isotypes (Fig 8, e) whose activities are also increased during cell activation Putative candidates include iPLA2 (group VI PLA2), which is very sensitive to AACOCF3, or isoforms of cPLA2 that may be more sensitive to AACOCF3 Presently, four isoforms of cPLA2 have been sequenced and cloned [3,7,8] Determining which cPLA2 isoform(s) is(are) expressed in neutrophils and its sensitivity to AACOCF3 will be critical in elucidating the interplay between remodeling and AA release Thirdly, the selective transfer of AA to 1-alkyl-2-AA-GPC under resting conditions by CoA-IT may be disrupted when cells are activated Evidence for this possibility comes from studies showing that AA constitutes the bulk of the fatty acid at the sn-2 position of 1-alkyl-2-acyl-GPC under resting conditions [70] Upon cell activation, most of this AA is replaced by other fatty acids Finally, because cell activation is accompanied by an increase in PAF biosynthesis via acetylation of 1-alkyl-2-lyso-GPC by acetyl transferase (Fig 8, f) activity, competition for 1-alkyl-2-lyso-GPC intermediate by various transferases may prevent reacylation of AA As CoA-IT has not been cloned and characterized, our knowledge of its interaction with PLA2 isotypes and other transferases and its role in AA release, LTB4 and PAF biosynthesis will remain rudimentary While the major focus of our study has been on CoAIT because of its role in AA remodeling and regulating lipid mediator biosynthesis, it is important to note that AACOCF3 may also inhibit other AA metabolizing enzymes that control lipid mediator formation For example, our studies show that AACOCF3 effectively inhibits 5-LO (Fig 8, g) activity and this inhibition could account for the decrease in leukotriene biosynthesis AACOCF3 also inhibited the incorporation of exogenous AA into neutrophils, possibly via AA-CoA synthetase or CoA-dependent-acyl transferase (data not shown) Inhibition of these enzyme activities that are linked to the control of AA levels within cells could lead to the depletion of cellular AA that would have been utilized for leukotriene biosynthesis Further studies that favor AACoA synthetase and CoA-acyl transferase activities are required to fully identify the role of these enzymes in lipid mediator formation Overall, these data highlight the role of two main activities (5-LO, CoA-IT) in lipid mediator biosynthesis and the complex nature of AA metabolism Whereas 5-LO and CoA-IT are directly linked to LTB4 and PAF formation respectively, PLA2 isotypes hydrolyse AA from a phospholipid pool that is not linked to mediator 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(Fig 8, f) activity, competition for 1-alkyl-2-lyso-GPC intermediate by various... constitutes the bulk of the fatty acid at the sn-2 position of 1-alkyl-2-acyl-GPC under resting conditions [70] Upon cell activation, most of this AA is replaced by other fatty acids Finally,