A Decrease in Remodeling Accounts for the Accumulation of Arachidonic Acid in Murine Mast Cells Undergoing Apoptosis*

Alfred N. FontehDagger, Tiffany LaPorteDagger, Dennis SwanDagger, and M. Allen McAlexanderDagger§

From the Department of Internal Medicine, Pulmonary and Critical Care Medicine, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27154

Received for publication, July 21, 2000, and in revised form, September 26, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The goal of this study was to examine arachidonic acid (AA) metabolism by murine bone marrow-derived mast cells (BMMC) during apoptosis induced by cytokine depletion. BMMC deprived of cytokines for 12-48 h displayed apoptotic characteristics. During apoptosis, levels of AA, but not other unsaturated fatty acids, correlated with the percentage of apoptotic cells. A decrease in both cytosolic phospholipase A2 expression and activity indicated that cytosolic phospholipase A2 did not account for AA mobilization during apoptosis. Free AA accumulation is also unlikely to be due to decreases in 5-lipoxygenase and/or cyclooxygenase activities, since BMMC undergoing apoptosis produced similar amounts of leukotriene B4 and significantly greater amounts of PGD2 than control cells. Arachidonoyl-CoA synthetase and CoA-dependent transferase activities responsible for incorporating AA into phospholipids were not altered during apoptosis. However, there was an increase in arachidonate in phosphatidylcholine (PC) and neutral lipids concomitant with a 40.7 ± 8.1% decrease in arachidonate content in phosphatidylethanolamine (PE), suggesting a diminished capacity of mast cells to remodel arachidonate from PC to PE pools. Further evidence of a decrease in AA remodeling was shown by a significant decrease in microsomal CoA-independent transacylase activity. Levels of lyso-PC and lyso-PE were not altered in cells undergoing apoptosis, suggesting that the accumulation of lysophospholipids did not account for the decrease in CoA-independent transacylase activity or the induction of apoptosis. Together, these data suggest that the mole quantities of free AA closely correlated with apoptosis and that the accumulation of AA in BMMC during apoptosis was mediated by a decreased capacity of these cells to remodel AA from PC to PE.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Mast cells play a vital role in acute and chronic allergic inflammation by virtue of their ability to release inflammatory mediators and cytokines upon antigen activation (1-5). Arachidonic acid (AA)1 located at the sn-2-position of membrane phospholipids is the precursor of one important class of inflammatory mediators, collectively known as eicosanoids (e.g. PGD2, TXB2, LTC4, and LTB4) (6). During mast cell activation, AA is rapidly released and utilized for the formation of eicosanoids (7). This can be accomplished by one or more of several PLA2 enzymes with different molecular characteristics that have been described in mast cells (8-10). These enzymes show different selectivity toward the hydrolysis of fatty acids at the sn-2-position or phosphobase moieties at the sn-3-position of phospholipids and include a relatively high molecular mass cytosolic PLA2 (cPLA2; 70-110 kDa) and several low molecular mass or secretory PLA2s (~14 kDa) (11-15).

In mast cells and other inflammatory cells, low levels of free AA are maintained during resting conditions by both CoA-dependent and CoA-independent mechanisms (16-19). Free AA is initially converted to arachidonoyl CoA by arachidonoyl-CoA synthetase and is then rapidly incorporated into 1-acyl-2-lysophospholipids by CoA-dependent acyltransferase (20, 21). AA in 1-acyl-linked phospholipids is then gradually remodeled into 1-ether-linked phospholipids by CoA-independent transacylase (CoA-IT), leading to large amounts of AA in ether lipid pools (22-25).

Recent studies suggest that mast cell numbers are primarily controlled by IL-3 and SCF as mast cells have been shown to undergo apoptosis in the absence of these cytokines (26-29). Until now, neither the inflammatory capacity nor the ability to regulate AA levels of mast cells that are undergoing apoptosis has been determined. To begin to understand how mast cells metabolize AA during apoptosis, we have utilized an in vitro model of mast cell apoptosis induced by cytokine depletion. Our data suggest that there is an increase in free AA levels outside mast cells undergoing apoptosis. Importantly, the mole quantities of AA, but not other unsaturated fatty acids, correlated with the number of apoptotic cells and were inversely proportional to the number of live cells. This accumulation of AA can be explained by a decrease in CoA-IT activity, which results in a diminished capacity of mast cells to remodel AA from phosphatidylcholine (PC) to phosphatidylethanolamine (PE).


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials

Octadeuterated [5,6,8,9,11,12,14,15-2H]AA ([2H8]AA) and trideuterated stearic acid ([2H3]SA) were purchased from Biomol Research Laboratory (Plymouth Meeting, PA). LTB4, [2H4]LTB4, PGD2, [2H4]PGD2, TXB2, and [2H4]TXB2 were purchased from Cayman (Ann Arbor, MI). Essentially fatty acid-free human serum albumin (HSA), essential and nonessential amino acids, mouse IgE anti-dinitrophenol (IgE anti-DNP), heat-inactivated fetal bovine serum, ionophore A23187, malondialdehyde (MDA), and N-methyl-2-phenylindole were purchased from Sigma. Methoxylamine HCl (MOX in pyridine), acetonitrile, pentafluorobenzyl bromide (20% in acetonitrile), diisopropylethylamine (20% in acetonitrile), and N,O-bis(trimethylsilyl)-trifluoroacetamide were purchased from Pierce. RPMI 1640 cell culture medium and Hanks' balanced salt solution (HBSS) were purchased from Life Technologies, Inc. PC, phosphatidylinositol (PI), phosphatidylserine (PS), and PE were purchased from Serdary Research Laboratories (Englewood Cliffs, NJ). HPLC grade organic solvents were purchased from Fisher. A rabbit antipeptide antibody for cPLA2 was kindly provided by Hoffman-LaRoche. Dr. A. Sane (Wake Forest University School of Medicine) kindly provided cPLA2 cDNA. Murine recombinant stem cell factor (SCF) expressed in Escherichia coli was a generous gift from Amgen (Seven Oaks, CA). Recombinant IL-3 was purchased from Genezyme (Cambridge, MA). [3H]AA (200 Ci/mmol), [Me3H]choline hydrochloride (85 Ci/mmol), [1-3H]ethanolamine hydrochloride (30 Ci/mmol), and palmitoyl-2-[1-14C-]arachidonoyl-sn-glycero-3-phosphocholine (55 mCi/mmol) were purchased from American Radiolabel Chemical, Inc. (St. Louis, MO). 1-[3H]Alkyl-2-lyso-GPC (56 Ci/mmol) was provided by Dr. R. Wykle (Wake Forest University School of Medicine).

Mast Cell Culture and Activation

BMMC were obtained from CBA/J mice (Jackson Laboratories, Bar Harbor, ME) and grown in RPMI 1640 culture medium (Life Technologies, Inc.) supplemented with 10% (v/v) fetal calf serum, 50 µM 2-mercaptoethanol, 1% essential amino acids, 1% nonessential amino acids, 2 mM L-glutamine, 5 µg/ml gentamycin, and 1% (v/v) penicillin/streptomycin. The culture medium was enriched twice a week with 50% WEHI supernatant fluid as a source of IL-3 for 3 weeks. Cell viability (>90%) was determined by trypan blue exclusion.

BMMC were maintained in culture media with or without cytokines (0-100 ng/ml SCF or 100 ng/ml IL-3) for different periods of time as indicated in the figure legends. Before stimulation with antigen, BMMC were sensitized for 24 h with 0.5 µg/ml IgE anti-2,4-dinitrophenol (Sigma) in culture media. BMMC in HBSS containing calcium, 0.1 mg/ml gelatin, and 0.01 mg/ml fatty acid-free HSA were stimulated with antigen (2 µg/ml) or ionophore A23187 (1 µM) for 5 min at 37 °C. Cells were rapidly removed from supernatant fluids by centrifugation (400 × g for 5 min), and the mole quantities of fatty acids and eicosanoids were determined by negative ion chemical ionization gas chromatography/mass spectroscopy (NICI-GC/MS) as described below.

Assessment of Apoptosis in BMMC

Caspase Activity-- BMMC were cultured without or with cytokines for 24 h. Protease activity in BMMC homogenate was determined using ApoAlertTM CPP32 Colorimetric Assay Kit (CLONTECH) as directed by the manufacturers. Protease activity, defined as the amount of CPP32 required to produce 1 pmol of chromophore (p-nitroanilide, pNA) per min at 25 °C, was determined using a standard curve established using different concentrations of pNA at 405 nm. In order to establish that the signal detected was attributable to protease activity, a control assay was performed in which homogenates from cells that had been placed in cytokine-depleted media were pretreated with a CPP32 inhibitor, Asp-Glu-Val-Asp-aldehyde, prior to the addition of CPP32 substrate (Asp-Glu-Val-Asp-p-nitroanilide).

Annexin V Binding to Mast Cells-- The reorientation of plasma membrane phospholipids was monitored using the TACSTM annexin V-FITC binding kit from Trevigen (Gaithersburg, MD). Briefly, 1 × 106 BMMC were cultured without cytokines or with IL-3 or SCF for 24 h. BMMC were collected by centrifugation (400 × g, 10 min) and then washed once in ice-cold (4 °C) phosphate-buffered saline without Ca2+ and Mg2+. The cells were resuspended in 100 µl of binding buffer (10 mM HEPES, pH 7.4, containing 0.15 M NaCl, 5 mM KCl, 1 mM MgCl2, and 1.8 mM CaCl2) containing annexin V-FITC and 0.5 µg of propidium iodide for 15 min at room temperature in the dark. Binding buffer (400 µl) was then added to the mixture, and flow cytometry was performed using a Coulter Epics XL-MCL flow cytometer. The percentages of total cells that did not bind propidium iodide or annexin-FITC (live cells), cells that bound annexin-FITC alone (early apoptosis), cells that bound both propidium iodide and annexin-FITC (late apoptosis), and cells that bound mainly propidium iodide (necrotic cells) were determined, and the results are presented in the form of dot plots.

DNA Ladder Formation-- BMMC were lysed in 10 mM Tris/HCl, pH 7.6, containing 0.2% Triton X-100 and 15 mM EDTA. The lysate was treated with 50 µg of proteinase K (Promega) overnight at 50 °C. Subsequently, DNA was precipitated by centrifugation (15,000 rpm, 10 min on a microcentrifuge) after the addition of 1 ml of cold 2-propanol and 0.1 ml of NaCl (5 M). The DNA was resolved on 2.0% agarose gels by electrophoresis, and DNA fragments were detected after ethidium bromide staining by UV visualization.

[3H]Thymidine Incorporation into BMMC-- BMMC were placed in growth medium with or without cytokines. Thymidine incorporation was determined by incubating 1 × 106 BMMC with 1 µCi of [3H]thymidine (Amersham Pharmacia Biotech) for 1 h at 37 °C. Unincorporated label was removed by washing (twice) the cells with HBSS containing 0.25 mg/ml HSA. The cell pellet was lysed using 0.2 N NaOH (0.25 ml for 1 h). DNA was precipitated using 15% trichloroacetic acid (1 ml) overnight at 4 °C. Cellular DNA was then trapped on glass microfiber filters (Whatman GF/C). Free cellular [3H]thymidine was removed from the filters by washes (4 ml, three times), and the amount of radioactivity in DNA was determined by liquid scintillation counting.

Quantitation of Free Fatty Acids in Supernatant Fluids

After the addition of trideuterated stearic acid (100 ng of [2H3]SA) as an internal standard for the analysis of linoleic acid (LA) and oleic acid (OA) and the addition of octadeuterioarachidonic acid ([2H8]AA, 100 ng) as an internal standard for the analysis of AA, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) to an ethanolic extract of the supernatant fluids, solvents were removed under a stream of nitrogen. Fatty acids were then converted to pentafluorobenzyl esters, and the mole quantities were determined by NICI-GC/MS using a Hewlett Packard model 5989 instrument. Carboxylate anions (m/z at 279, 281, 286, 303, 301, 327, and 311 for LA, OA, [2H3]SA, AA, EPA, DHA, and [2H8]AA, respectively) were monitored by gas chromatography/mass spectroscopy. Mole quantities of fatty acids were determined from standard curves obtained for each fatty acid using [2H3]SA and [2H8]AA as internal standards.

Determination of Lipid Peroxidation

BMMC were maintained in culture media without cytokines or with cytokines for 24 h. BMMC were washed (three times) using ice-cold HBSS containing 0.25 mg/ml HSA. Subsequently, cells were lysed in 200 µl of HPLC grade H2O by repeated (four times) freezing using liquid N2 and thawing using a 37 °C water bath. Lipid peroxidation was determined by measuring the mole quantities of MDA, an end product of the peroxidation of unsaturated fatty acids (30). Briefly, lysates (200 µl, 5 × 106 BMMC) were added to a 10 mM solution of N-methyl-2-phenylindole (650 µl) freshly made in acetonitrile/methanol (3:1, v/v). After the addition of 150 µl of 12 N HCl, the mixture was incubated at 45 °C for 45 min. The A586 was determined for samples and 0-10 µM MDA standards. The molar amounts of MDA in samples were calculated using an extinction coefficient (gradient of concentration versus A586) obtained using 0-10 µM MDA standards.

Quantitation of Eicosanoids in Supernatant Fluids

After stimulation of BMMC, [2H4]PGD2, [2H4]TXB2, and [2H4]LTB4 (10 ng of each) were added to supernatant fluids as internal standards. Eicosanoids were converted to methoxime-pentafluorobenzyl ester trimethylsilyl ether derivatives. Derivatized eicosanoids were then extracted with hexane and analyzed using combined NICI-GC/MS. Carboxylate anions for LTB4 (m/z 479), [2H4]LTB4 (m/z 483), PGD2 (m/z 524), [2H4]PGD2 (m/z 528), TXB2 (m/z 614), and [2H4]TXB2 (m/z 618) were analyzed in the single ion-monitoring mode.

Determination of cPLA2 Activity

BMMC were washed (twice) with HBSS containing 0.25 HSA and 5 mM dithiothreitol. Cells were then suspended at 107/ml in sonication buffer (10 mM HEPES, pH 7.4, containing 80 mM KCl, 1 mM EDTA, 1 mM EGTA, 40 µg/ml leupeptin, 25 µg/ml pepstatin, 1 mM phenylethylsulfonyl fluoride, 10 mM NaF, 0.2 mM Na2VO3, and 5 mM dithiothreitol). Sonication (10 s, three times) was performed using a probe sonicator (Heat System Inc.) at a power setting of 2 and 10% output. Protein content of fractions was determined using the Coomassie Plus protein assay reagent (Pierce). cPLA2 activity was determined using 400 pmol sonicated-vesicles of 1-palmitoyl-2-[1-14C]arachidonoyl-sn-glycero-3-phosphocholine as substrate and 50 µg of protein from BMMC sonicates. cPLA2 activity was initiated by the addition of substrate to fractions that had been preincubated for 15 min at 37 °C in an assay mixture that contained 5 mM dithiothreitol. This preincubation was necessary for eliminating residual secretory PLA2 activity. The PLA2 reaction was stopped after 15 min at 37 °C by extracting lipids by the method of Bligh and Dyer (31). Free fatty acids were isolated from phospholipids by TLC on silica gel G developed in hexane/diethyl ether/formic acid (90:60:6, v/v/v). The radioactivity in lipids was located using a radiochromatogram imaging system (Bioscan Inc., Washington, D. C.). Free AA and phospholipids were isolated using TLC zonal scraping, and the radioactivity was determined utilizing liquid scintillation counting. cPLA2 activity was calculated and expressed as pmol of AA released/mg of protein/min.

SDS-Polyacrylamide Gel Electrophoresis and Western Blot Analysis of cPLA2

Amounts of cPLA2 protein were determined in total lysates of 1-2 million BMMC (32). Briefly, BMMC were incubated in lysis buffer (100 mM Tris/HCl, pH 7.5, containing 0.1 M NaCl, 2 mM EDTA, 1% Nonidet P-40, 1 mM Na3VO4, 50 mM NaF, 0.1 mM N-tosyl-L-phenylalanine chloromethyl ketone, 0.1 mM quercetin, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, and 1 µg/ml leupeptin) for 10 min on ice. After removal of nuclei by centrifugation, extracts were mixed with an equal volume of 2× loading buffer (0.125 M Tris, pH 6.8, 4% SDS, 20% glycerol, 10% mercaptoethanol, 0.05% bromphenol blue) and boiled for 5 min. Proteins were separated by SDS-polyacrylamide gel electrophoresis on a 4-20% polyacrylamide gel. Separated proteins were transferred onto nitrocellulose membranes, and the blots were incubated overnight with an anti-cPLA2 antibody (1 µg/ml). Detection of cPLA2 was accomplished using horseradish peroxidase conjugate anti-rabbit IgG and enhanced chemiluminescence reagents (Pierce).

RNA Isolation Northern Analysis of cPLA2 from BMMC

Total RNA was extracted from BMMC cultured with different concentrations of SCF (0-100 ng/ml) using RNazol (Tel Test Inc., Friendswood, TX). RNA (10 µg) resolved by electrophoresis on formaldehyde-containing agarose was transferred onto GeneScreen Plus (PerkinElmer Life Sciences) membranes by capillary blotting. The cPLA2 cDNA probe was labeled using a nick translation labeling kit (PerkinElmer Life Sciences) following the recommendations of the manufacturer. The membranes were then prehybridized in Quikhyb (Strategene, La Jolla, CA) for 15 min at 68 °C before the direct addition of the labeled cDNA probe (1.25-1.5 × 106 cpm/ml) to the hybridization solution. After hybridization for 1 h at 68 °C, excess probe was washed (twice) with 1× sodium citrate (0.15 M NaCl and 15 mM Na3C6H5O7·2H2O) containing 0.1% SDS (SSC) at 25 °C for 1 min followed by a 15-min wash using 0.2× SSC, 0.1% SDS at 65 °C. Signals were detected by exposing the blot to x-ray film with intensifying screens at -80 °C.

Determination of AA Incorporation into BMMC

BMMC were maintained in culture without or with cytokines. After 24 or 48 h, the cells were removed from culture media and placed in HBSS containing 1 µCi of [3H]AA/107 BMMC for 30 min at 37 °C. Unincorporated label was removed by washing (three times) the cells in HBSS containing 0.25 mg/ml HSA. Glycerolipids were then extracted from the cells, and individual glycerolipid classes were isolated by normal phase HPLC (33, 34). Briefly, lipid extracts were reconstituted in loading buffer (hexane/2-propanol/water, 4:5.4:0.3, v/v/v) and then loaded onto an Ultrasphere Silica column (Rainin Instrument Co., Woburn, MA) that had been conditioned with hexane/2-propanol/ethanol/50 mM phosphate buffer (pH 7.4)/acetic acid (490:367:100:30:0.6, v/v/v/v/v). After 5 min, the amount of phosphate was increased from 3 to 5% over a 5-min period. This solvent composition was then maintained until all major phospholipid classes had been eluted from the column. In radiolabeled assays, 1-min fractions were collected, and the amount of radioactivity in each lipid class was determined by liquid scintillation counting.

The neutral lipid fraction was separated into monoglycerides, diglycerides, free fatty acid, and triglycerides by TLC on silica gel G using hexane/diethyl ether/formic acid (90:60:6, v/v/v).

Determination of Mole Quantities of AA in Phospholipid Classes and Subclasses

BMMC glycerolipids extracted by the method of Bligh and Dyer were separated into classes by normal phase HPLC as described above. [2H8]AA (100 ng) was added to a fraction of each glycerolipid that was subsequently subjected to base hydrolysis (34). Fatty acids were then converted to pentafluorobenzyl esters, and the mole quantities of free fatty acids were determined by NICI-GC/MS. Carboxylate anions (m/z) were monitored at 303 and 311 for AA and [2H8]AA, respectively, in the single ion monitoring mode, and mole quantities of AA were determined from standard curves obtained using [2H8]AA as an internal standard.

To determine the distribution of arachidonate into phospholipid subclasses, PC and PE fractions from normal phase HPLC were hydrolyzed using 10 or 25 units of grade 1 Bacillus cereus phospholipase C (Roche Molecular Biochemicals) for 2.5 and 6 h, respectively. Diradylglycerides obtained from the phospholipase C hydrolysis were converted to diradylglyceride acetates using acetic anhydride (500 µl) and pyridine (35). 1-Acyl, 1-alkyl, and 1-alk-1'-enyl subclasses were separated by TLC on silica gel G developed in benzene/hexane/diethyl ether (50:25:4, v/v/v). Each subclass was extracted from the silica gel, and the mole quantities of arachidonate were determined by NICI-GC/MS as described above.

Determination of CoA-IT Activity

For cell free assays, BMMC were cultured without or with cytokines for 24 h and then washed (three times) using HBSS containing 0.25 mg/ml HSA. The cells were then suspended in CoA-IT sonication buffer (50 mM HEPES buffer, pH 7.4, containing 1 mM EDTA and 20% sucrose (w/v)). Subsequently, the cells were broken by sonication using a probe sonicator (Heat System, Inc.) as described above for cPLA2 assays. Cytosolic and membrane fractions were obtained after ultracentrifugation (100,000 × g, 1 h, 4 °C). The membrane fraction was diluted in phosphate-buffered saline containing 1 mM EGTA, and 10 µg of total protein was utilized for CoA-IT activity determination. The reaction was initiated by the addition of 1-[3H]alkyl-2-lyso-GPC (0.1 µCi) containing 1 nmol of 1-O-hexadecyl-2-lyso-GPC in a final volume of 100 µl. After 10 min at 37 °C, the reaction was stopped, and lipids were extracted. Phospholipids were separated by TLC on silica gel G developed in chloroform/methanol/acetic acid/water (50:25:8:4, v/v/v/v). The product (1-[3H]alkyl-2-acyl-GPC) was visualized by radioscaning (Bioscan), scrapped, and then quantified by liquid scintillation spectroscopy.

For whole cell assays, BMMC were pulse-labeled with [3H]AA for 30 min (1 µCi/107 cells). Unincorporated label was removed by washing BMMC (three times) using HBSS containing 0.25 mg/ml fatty acid-free HSA. The cells were then placed in fatty acid-enriched cell culture media (10% fetal calf serum) in the absence or presence of IL-3 or SCF (100 ng/ml). After different periods of time, BMMC were removed from cell culture media, and glycerolipid classes were obtained by normal phase HPLC as described above. The amount of radiolabel in each lipid class was then determined by liquid scintillation counting.

Determination of Lyso-PC and Lyso-PE Levels

BMMC (106/ml) were incubated with 0.5 µCi of [Me3H]choline or 0.5 µCi of [1-3H]ethanolamine for the determination of lyso-PC and lyso-PE, respectively. After 24 h in culture media, unincorporated label was removed by washing the cells (twice) using sterile HBSS containing 0.25 mg/ml HSA. The cells were then cultured in the absence or presence of IL-3 or SCF (100 ng/ml) for a further 24 h. Phospholipids were extracted from cells as described above. The phospholipid extract was resuspended in 50 µl of chloroform containing 10 µg each of PC and lyso-PC or PE and lyso-PE. Lyso-PC and lyso-PE were isolated by TLC on silica gel G using chloroform/methanol/acetic acid/water (50:25:8:4, v/v/v/v). The radioactivity in phospholipids (PC or PE) and lysophospholipids (lyso-PC or lyso-PE) were determined by liquid scintillation counting.

Effects of Exogenous AA on Cellular AA Metabolism

BMMC (106/ml) were labeled with [3H]AA for 24 h to obtain isotopic labeling of the major lipid classes (24). After removal of unincorporated label, cells were maintained in culture in the presence of increasing concentrations of exogenous AA (0-50 µM). After 24 h, the distribution of radiolabel into lipid classes was determined by TLC as described above.

Statistical Analysis

Data are expressed as the means ± S.E. of separate experiments. Statistics (p values) were obtained using Student's t test for paired samples. Notations used on figures and legends are an asterisk for p < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Apoptosis of BMMC in Cytokine-depleted Media-- Our previous studies showed that BMMC cultured in IL-3/SCF maintained low intracellular levels of AA (36). However, the regulation of AA has not been described when these cells are undergoing apoptosis. Initial experiments were designed to obtain a reproducible model of mast cell apoptosis. In these experiments, caspase activity was examined in BMMC cultured without or with IL-3/SCF for 24 h. BMMC placed in cytokine-depleted media had higher protease activity (14.5 ± 0.9 CPP32 units, n = 3) compared with cells maintained in cytokines (4.8 ± 1.1 CPP32 units). This protease activity was reduced to control levels (4.4 ± 0.1 CPP32 units) by a caspase inhibitor. These initial data suggested that a marker of apoptosis (caspase) was increased when BMMC were placed in cytokine-depleted media for 24 h.

To determine the percentage of apoptotic cells, the orientation of PS on the surface of mast cells was examined by annexin V binding. When BMMC were placed in cytokine-depleted media for 24 h, there was an increase in the percentage of annexin-FITC binding cells (Fig. 1A). Consistent with this observation, maintaining BMMC in cytokine-depleted media caused a significant increase in the percentage of apoptotic cells concomitant with a decrease in live cells (Table I). Further evidence of apoptosis was obtained by examining DNA fragmentation. As shown in Fig. 1B, BMMC cultured for 24 h with IL-3 or SCF maintained intact high molecular weight DNA. By contrast, DNA extracted from cells placed in cytokine-depleted media displayed distinct DNA fragmentation patterns. Finally, we determined the capacity of BMMC to synthesize new DNA. As shown in Fig. 1C, the incorporation of radiolabeled thymidine into cellular DNA decreased (>80%) when BMMC were placed in cytokine-depleted media for as little as 12 h. Taken together, these data suggest that BMMC placed in cytokine-depleted media rapidly stop proliferating and then undergo apoptosis.



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Fig. 1.   Cytokine depletion results in apoptosis of BMMC. A, BMMC were maintained with IL-3 (left panel) or SCF (middle panel) or without cytokines (NONE, right panel) for 24 h. Annexin V binding was determined by flow cytometry as described under "Experimental Procedures." Quadrants 1, 2, 3, and 4 represent dead cells, cells in late apoptosis, live cells, and cells in early apoptosis, respectively. These data are representative of eight separate experiments. B, DNA was extracted from BMMC that had been placed in cell culture media for 24 h with IL-3, SCF, or without cytokines (NONE). DNA was extracted from 2 × 106 BMMC and resolved by agarose gel electrophoresis followed by ethidium bromide staining as described under "Experimental Procedures." These data are representative of five separate experiments. C, BMMC were placed in growth media without () or with 100 ng/ml IL-3 (open circle ). After different periods of time in culture, the incorporation of [3H]AA thymidine into 1 × 106 BMMC was determined. These data are the mean ± S.E. of triplicate determinations and are representative of five separate experiments.


                              
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Table I
Cytokine depletion induces apoptosis of BMMC
BMMC were cultured without cytokines (None) or with IL-3 or SCF (100 ng/ml) for 24 h. Annexin FITC binding to mast cells was determined by flow cytometry. The percentage of necrotic cells (quadrants 1 of Fig. 1A), late and early apoptotic cells (quadrants 2 and 4, respectively, of Fig. 1A), and live cells (quadrants 2 of Fig. 1A) was determined. These data are the mean ± S.E. of four separate experiments.

Mole Quantities of Arachidonic Acid Correlate with the Percentage of Apoptotic Cells-- As described above, mast cells placed in cytokine-depleted media undergo apoptosis. It is known that the location of PS on the surface of cells is a marker of apoptosis (37). However, it has not been established whether changes in the phospholipid microenvironment are accompanied by changes in fatty acids within or outside mast cells. Therefore, subsequent studies examined the mole quantities of various unsaturated fatty acids within mast cells or released into the culture media. Removal of cytokines resulted in a significant increase in AA levels within mast cells, while levels of other unsaturated fatty acids (LA, OA) did not change (Table II). Subsequent studies examined free fatty acids outside mast cells and compared these levels with the percentage of live or dead (annexin V binding) cells. As shown in Fig. 2A, resting levels of AA outside mast cells were directly proportional to the percentage of apoptotic cells (Fig. 2A) and inversely proportional to the percentage of live cells (Fig. 2B). In both cases, there was correlation between the percentages of dead or live cells and the mole quantities of AA in cell culture media (R2 = 0.76 and 0.92, respectively). In contrast, mole quantities of other unsaturated fatty acids (LA, OA, EPA, and DHA) were not related to the percentage of dead (Fig. 2, C, D, E, and F, respectively) or living cells (data not shown). These data suggest that the selective accumulation of AA within or outside mast cells is closely associated with apoptosis.


                              
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Table II
Free fatty acid levels within BMMC
BMMC were cultured without (None) or with 100 ng/ml SCF or 100 ng/ml IL-3 for 24 h. Cellular lipids were extracted, and levels of three unsaturated fatty acids (LA, OA, and AA) were determined by NICI-GC/MS. These data are the mean ± S.E. of four separate experiments.



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Fig. 2.   Apoptosis of mast cells correlate with AA accumulation in culture media. Mole quantities of unsaturated free fatty acids in the culture media of BMMC cultured without or with cytokines for different periods of time (12-48 h) were determined by NICI-GC/MS. The percentages of dead cells or living cells were determined for each condition by flow cytometry. A and B show the relationship between the mole quantities of AA and the percentage of dead and live BMMC, respectively. C, D, E, and F show the relationship between the percentage of dead cells and the mole quantities of free LA, OA, EPA, and DHA, respectively. These data were collected from four separate experiments.

To further examine the effects of unsaturated fatty acids on apoptosis, BMMC were incubated with different concentrations (0-50 µM) of LA, OA, or AA for 24 h, and apoptosis was examined by annexin-FITC binding. The percentage of apoptotic cells increased dose-dependently when AA was added to BMMC for 24 h (6.1 ± 5.6 and 12.6 ± 0.4% at 25 and 50 µM AA, respectively, n = 3, p < 0.05 at 50 µM AA). Likewise, 25 and 50 µM AA increased the percentage of necrotic cells by 26.7 ± 5.7 and 46.6 ± 2.6% (n = 3, p < 0.05), respectively. In contrast, two other unsaturated fatty acids, LA and OA, did not affect the percentage of dead cells at similar concentrations. These data suggest that induction of apoptosis is selective for AA, and not other unsaturated fatty acids when BMMC are maintained in cytokine-depleted media.

Mast Cells Undergoing Apoptosis Release More AA into Supernatant Fluid-- Potential explanations for the accumulation of AA within or outside BMMC include an increase in AA-releasing activities (cPLA2), a decrease in AA incorporation and remodeling activities or a decrease in the conversion of free AA to metabolites. Thus, we examined the stimulus-coupled AA release from BMMC that were undergoing apoptosis. BMMC placed in cytokine-depleted media released 757.2 ± 112.3 (n = 5) pmol of AA/5 × 106 BMMC upon stimulation with antigen for 5 min. This level of AA release was significantly higher than AA released from cells placed in IL-3 (259.3 ± 31.7 pmol/5 × 106 BMMC) or SCF (156.8 ± 50.6 pmol/5 × 106 BMMC). Ionophore A23187 stimulation also resulted in more AA release from cytokine-depleted cells compared with cytokine-treated cells (data not shown). Taken together, these data suggest that cells cultured without cytokines have the capacity to release significantly more AA than cells cultured with cytokines.

Increase in Lipid Peroxidation when Mast Cells Are Undergoing Apoptosis-- Several studies suggest that peroxidation of unsaturated fatty acids is linked to apoptosis (38-40). To determine whether AA accumulation is accompanied by lipid peroxidation in BMMC, we examined the accumulation of MDA, the major product of lipid peroxidation reactions, within BMMC induced to undergo apoptosis by cytokine depletion. When BMMC were cultured in IL-3 or SCF-supplemented culture media, levels of MDA (6.70 ± 0.71 nmol/5 × 106 BMMC, n = 6 and 6.78 ± 1 nmol/5 × 106 BMMC, n = 6, respectively) were not altered within a 24-h incubation period. In contrast, there was a significant increase in MDA levels (15.43 ± 1.89 nmol/5 × 106 BMMC, n = 6, p < 0.05) within BMMC that were induced to undergo apoptosis by cytokine withdrawal. Taken together, these data suggest that AA accumulation within BMMC undergoing apoptosis induced by cytokine withdrawal is accompanied by an increase in lipid peroxidation.

cPLA2 Activity and Expression Does Not Correlate with Mole Quantities of AA-- As described above, BMMC placed in cytokine-depleted media for 24 h released more AA upon activation. Since stimulus-coupled release of AA is associated with cPLA2 activation, we next examined cPLA2 expression and activity as a possible explanation for the accumulation of AA outside mast cells. As shown in Fig. 3A (upper panel), cPLA2 mRNA decreased when BMMC were placed in cytokine-depleted media for 24 h. Western analysis also revealed that there was a decrease in cPLA2 protein when mast cells were placed in cytokine-depleted media (Fig. 3B). Likewise, cPLA2 activity was lower in cells cultured without cytokines (Fig. 3C). Taken together, these data suggest that changes in cPLA2 levels are not related to the accumulation of AA in cell culture media or within BMMC when these cells are undergoing apoptosis.



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Fig. 3.   Reduction in cPLA2 expression, protein level, and activity during BMMC apoptosis. A, total RNA was extracted from BMMC cultured with increasing concentrations of SCF. RNA (10 µg) was resolved by electrophoresis on formaldehyde-containing agarose and then transferred onto GeneScreen Plus membranes by capillary blotting. GeneScreen Plus membranes were probed using a labeled cPLA2 cDNA probe (upper panel) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (lower panel) as described under "Experimental Procedures." Signals were detected by exposing the blot to x-ray film with intensifying screens at -80 °C. These blots are representative of five separate experiments. B, BMMC were placed in culture media with increasing concentrations of SCF for 24 h. SDS-PAGE was performed using a 4-20% gel, and proteins were blotted onto polyvinylidene difluoride membranes. Immunodetection of cPLA2 was accomplished using anti-cPLA2 monoclonal antibody and peroxidase-conjugated anti-rabbit IgG, followed by enhanced chemiluminescence. These data are representative of five separate experiments. C, BMMC were cultured with increasing concentrations of SCF for 24 h. cPLA2 activity was determined using 50 µg of homogenate as described under "Experimental Procedures." GAPDH, glyceraldehyde-3-phosphate dehydrogenase. These data are the mean ± S.E. of four separate experiments performed in duplicate. *, p < 0.05.

Assessment of Eicosanoid Formation by BMMC Undergoing Apoptosis-- To determine whether the accumulation of AA outside mast cells was due to a decrease in their capacity to form products, levels of eicosanoids (PGD2 and LTB4) were examined by NICI-GC/MS. Stimulation by either antigen increased PGD2, while PGD2 levels remained unchanged after ionophore A23187 stimulation of BMMC undergoing apoptosis. There was no significant difference in LTB4 biosynthesis by BMMC grown with or without cytokines (Table III). These data suggest that impairment of eicosanoid biosynthesis is not responsible for the accumulation of free AA outside BMMC that are undergoing apoptosis.


                              
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Table III
Formation of eicosanoids by BMMC undergoing apoptosis
BMMC cultured without (None) or with IL-3 for different for 24 h were not stimulated (CT) or stimulated with antigen (Ag) or with ionophore A23187 for 5 min at 37 °C. Mole quantities of PGD2 and LTB4 released into supernatant fluids were determined by NICI-GC/MS as described under "Experimental Procedures." These data are the mean ± S.E. of four separate experiments.

Determination of Arachidonic Acid Incorporation into Lipids-- Our previous data indicated that BMMC rapidly incorporated trace amounts of AA predominantly into PC and PI pools (24). Under resting conditions, AA is slowly remodeled from these early pools to PE (24). To determine whether this incorporation process was blocked in cells undergoing apoptosis, cells were pulse-labeled with [3H]AA, and the incorporation of AA into lipids was monitored. BMMC cultured without cytokines, with IL-3 or SCF for 24 h, incorporated the same amount of radiolabel (0.414 ± 0.097, 0.519 ± 0.148, and 0.512 ± 0.159 µCi/107 BMMC, respectively, n = 5). When the incorporation of [3H]AA into lipid classes was examined, cells cultured without cytokines accumulated more AA in neutral lipids (Fig. 4A) than cells cultured with IL-3 or SCF (Fig. 4, B and C, respectively). In contrast, [3H]AA incorporation into PE was lower in cytokine-depleted BMMC, while there was no difference in the incorporation of [3H]AA into PI/PS or PC fractions when cells were maintained without or with cytokines (Fig. 4). Thus, impairment of AA incorporation into PC and PI does not account for the accumulation of AA when BMMC are undergoing apoptosis.



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Fig. 4.   Influence of cytokine depletion on [3H]AA incorporation into BMMC lipids. BMMC were maintained in culture without or with cytokines (IL-3 or SCF) for 24 h. BMMC were subsequently pulse-labeled with [3H]AA (0.1 µCi/1 × 106, 37 °C for 30 min). The cells were then washed (three times) with HBSS containing 0.25 mg/ml fatty acid-free HSA. Lipids were extracted, and individual classes (NL, unknown fraction (UK), PE, PI/PS, and PC) were isolated by normal phase HPLC as described under "Experimental Procedures." Radioactivity in lipid classes was determined by liquid scintillation counting. These data are representative of three separate experiments.

Distribution of Arachidonate into Phospholipid Classes and Subclasses of BMMC-- BMMC grown in cytokine-enriched media contained arachidonate in PE (41.21 ± 5.40%, n = 5) > PC (30.95 ± 1.96) > PI (18.32 ± 2.94%) > neutral lipids (NL; 7.98 ± 2.56%). The mole quantity of arachidonate was reduced in PE concomitant with an increase in PC when BMMC were maintained in cytokine-depleted media (Fig. 5A). Changes in the distribution of AA within PE subclasses (1-acyl-2AA-GPE and 1-alk-1-enyl-2-AA-GPE) and PC subclasses (1-acyl-2-AA-GPC and 1-alkyl-2-AA-GPC) mirrored the changes in their respective subclasses (Fig. 5B). Compared with BMMC grown in cytokine-supplemented culture media (IL-3 or SCF), there was a significant decrease in arachidonate content of PE (40.7 ± 8.1%; n = 3) in BMMC maintained in cytokine-depleted media (Fig. 5C). Concomitantly, there was an increase in the arachidonate content in NL (84.2 ± 12.8%, n = 3) and PC (50.2 ± 18.1%, n = 3). These data reveal that the cellular distribution of AA is altered when BMMC are undergoing apoptosis, such that PC, and not PE, becomes the predominant arachidonate-containing lipid pool and there is ~2-fold increase in the arachidonate mass in NL.



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Fig. 5.   Mass distribution of arachidonate into lipid classes and subclasses after cytokine depletion. A, BMMC were placed in culture media without cytokines for different periods of time. Lipids were extracted, and individual classes were isolated by normal phase HPLC. The mole quantity of arachidonate in NL (), PE (triangle ), PI/PS (black-triangle), and PC (black-square) was determined after base hydrolysis by NICI-GC/MS (A). Data are shown as the percentage of total cellular arachidonate that is found in each lipid class and are the mean ± S.E. of five separate experiments. B, PE and PC subclasses were isolated by TLC as described under "Experimental Procedures." Mole quantities of arachidonate in PE subclasses (1-acyl-2-AA-GPE () and 1-alk-1-enyl-2-AA-GPE (open circle )) and PC subclasses (1-acyl-2-AA-GPC (black-square) and 1-alkyl-2-AA-GPC ()) were determined as described under "Experimental Procedures." These data are shown as the percentage of arachidonate in each subclass and are the mean ± S.E. of three separate experiments (p < 0.05). C, lipids were extracted from BMMC before (0 h) or after the cells had been cultured for 48 h without or with cytokines (IL-3 or SCF). Lipid classes were isolated by normal phase HPLC. The mole quantity of arachidonate in NL, PE, PI/PS, or PC of BMMC was determined at time 0 (black-square), after 48 h of cytokine depletion (), and after 48 h in IL-3 () or in SCF () by NICI-GC/MS. Data are shown as the percentage of total cellular arachidonate that is found in each lipid class and represent the mean ± S.E. of five separate experiments.

Determination of CoA-independent Transacylase Activity-- As described above, the major difference between cells cultured with cytokines and cells undergoing apoptosis is the decrease in arachidonate content of PE concomitant with an increase in arachidonate content of PC and NL. Additionally, free AA levels are higher in BMMC undergoing apoptosis. AA that is initially incorporated into PC is remodeled to PE by CoA-IT activity, and the inhibition of CoA-IT has been shown to result in AA accumulation in several cell types (41). Thus, a decrease in CoA-IT activity could account for the decrease in AA content of PE and the accumulation of AA within mast cells undergoing apoptosis. We tested this hypothesis by determining AA remodeling in whole cells and also measuring microsomal CoA-IT activity. BMMC pulse-labeled with [3H]AA incorporated arachidonate in rank order PC > PI/PS > PE > NL. In the absence of cytokines, there was an increase of radiolabel in NL when cells are placed in culture media for 24 and 48 h. By contrast, [3H]AA was maintained at the same level in NL cultured with IL-3 or SCF (Fig. 6A). Compared with pulse-labeled cells, there was a slight increase in [3H]AA in PE in cytokine-depleted cells at 24 and 48 h. However, the percentage of [3H]AA in PE in cytokine-depleted BMMC was significantly lower than the levels found in BMMC cultured with IL-3 or SCF (Fig. 6B). [3H]AA levels in PI/PS and PC decreased slightly with time in all three groups at 24 and 48 h. However, there was no significant difference between [3H]AA in PC and PI/PS from cytokine-depleted and IL-3- or SCF- supplemented BMMC (Fig. 6, C and D). Interestingly, the increase in the mole quantities of arachidonate in PC (Fig. 5C) is not observed in the pulse experiments (Fig. 4) or in the pulse/chase experiments in Fig. 6D, probably because the mass amounts of [3H]AA are very small compared with the bulk of unlabeled arachidonate in PC. Moreover, most of [3H]AA that would have resided in PC is shunted to the neutral lipid fraction in apoptotic BMMC when these labeling strategies are utilized. However, the decrease of [3H]AA in PE suggests that the activity (CoA-IT) responsible for transferring/remodeling AA to PE is reduced in BMMC undergoing apoptosis. To further show that the decrease in arachidonate content in PE was due to a decrease in CoA-IT activity, microsomal fractions were prepared from cells cultured without or with cytokines. CoA-IT activity was lower in cells placed in cytokine-depleted media (0.13 ± 0.05 pmol/mg/min) compared with BMMC maintained in IL-3 (1.10 ± 0.09 pmol/mg/min, n = 8, p < 0.05) or SCF (0.71 ± 0.16 pmol/mg/min) for 24 h. A similar CoA-IT activity profile was determined after 48 h (data not shown). These data suggest that a decrease in CoA-IT accounts for the decrease in AA remodeling and the accumulation of free AA when BMMC undergo apoptosis following cytokine depletion.



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Fig. 6.   In situ CoA-independent transacylase activity. BMMC were pulse-labeled with [3H]AA, and unincorporated label was removed by washing cells (three times) using HBSS containing 0.25 mg/ml fatty acid-free HSA. The cells were then placed in fatty acid-enriched cell culture media (10% fetal bovine serum) without cytokines (), with IL-3 (), or with SCF(black-square). After 24 or 48 h in culture, the percentage of radioactivity in NL (A), PE (B), PI/PS (C), and PC (D) was determined as described under "Experimental Procedures." These data are the mean ± S.E. of four separate experiments. *, p < 0.05.

To determine whether the decrease in CoA-IT activity, or the induction of apoptosis during cytokine depletion could be accounted for by an increase in lysophospholipid levels within BMMC, phospholipid head groups were radiolabeled, and the accumulation of radioactivity in lyso-PC or lyso-PE was determined. When cells were placed in cytokine-depleted media, less [3H]choline was incorporated into lyso-PC compared with cytokine-supplemented cells (5757 ± 1781, 6763 ± 1909, and 9245 ± 2085 dpm/5 × 106 BMMC (n = 3) for no cytokine, IL-3, and SCF, respectively). Similarly, BMMC labeled with [1-3H]ethanolamine incorporated less radioactivity into lyso-PE (1160 ± 181, 1233 ± 164, and 1524 ± 215 dpm/5 × 106 BMMC (n = 3) for no cytokine, IL-3, and SCF, respectively). However, expressed as a percentage of total radioactivity, there was no difference in the amount of lyso-PC (16.7 ± 0.4, 13.8 ± 2.1, and 17.0 ± 2.2% of total radioactivity (n = 3) for no cytokine, IL-3, and SCF, respectively) or lyso-PE (7.6 ± 0.7, 5.8 ± 0.2, and 5.8 ± 0.2% of total radioactivity (n = 3) for no cytokine, IL-3, and SCF, respectively) in cells cultured in cytokine-depleted media when compared with cells grown with IL-3 or SCF. These data suggest that the decrease in CoA-IT observed during apoptosis is not due to an increase in the levels of endogenous substrate (lyso-PC or lyso-PE). In addition, these data suggest that lysophospholipids do not contribute to apoptosis of BMMC induced by cytokine depletion.

AA Accumulation in Neutral Lipid Classes-- Our previous studies suggested that inflammatory cells exposed to high concentrations of AA shuttled excess AA into neutral lipids, with triglycerides being the predominant class (42, 43). To determine whether a similar pathway existed in BMMC, the neutral lipid fractions (Fig. 6A) were further resolved into individual classes. As shown in Table IV, there was a significant increase in [3H]AA in all neutral lipid fractions obtained from cells cultured without cytokines. However, the bulk of [3H]AA resided in the TG pool. These data suggest that BMMC undergoing apoptosis accumulate more AA into neutral lipid classes than do live cells.


                              
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Table IV
Distribution of AA in neutral lipid classes
BMMC were pulse-labeled with [3H]-AA for 30 min and then placed in culture media without (None) or with 100 ng/ml SCF or IL-3 for 24 h. Lipids were extracted and separated into classes by normal phase HPLC. The NL fraction was further separated into individual into monoglycerides (MG), diglycerides (DG), free fatty acids (FFA), and TG by TLC. These data are the percentage of arachidonate that is found in each neutral lipid class and are the mean ± S.E. of five separate experiments.

Roles of Exogenous AA on Cellular AA Metabolism-- To determine whether changes in AA metabolism described above were due to the accumulation of AA in the cell culture media, BMMC were radiolabeled to isotopic equilibrium using [3H]AA. The cells were maintained in culture with different concentrations of exogenous AA. As shown in Fig. 7, the bulk of the radiolabel in BMMC resided in phospholipid classes (94.6 ± 0.4%, n = 3). The addition of exogenous AA resulted in a decrease in [3H]AA in phospholipid classes. Concomitantly, there was a dose-dependent accumulation of radiolabel in TGs and in free fatty acids. The bulk of the TG was cell-associated while most of the free AA was released into the cell culture media. These data suggest that adding exogenous AA to BMMC can induce changes in cellular AA metabolism similar to those observed during cytokine depletion.



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Fig. 7.   Effects of exogenous AA on endogenous AA metabolism. BMMC were labeled with [3H]AA to isotopic equilibrium. After removal of unincorporated label, the cells were then placed in whole media containing IL-3 and supplemented with different concentrations of exogenous AA for 24 h. Levels of cellular [3H]arachidonate were determined in phospholipids (black-triangle), free fatty acids (), and triglycerides (black-square). These data are the mean ± S.E. of three separate experiments. *, p < 0.05.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The present study demonstrates an accumulation of AA within cells and in the cell culture media of mast cells undergoing apoptosis induced by cytokine depletion. Of a series of polyunsaturated fatty acids measured, only the levels of AA directly correlated with the percentage of dead cells. Conversely, the percentage of live cells was inversely proportional to the mole quantities of AA found within apoptotic mast cells or in cell culture media. These initial data suggested that AA metabolism played a role in apoptosis. There are several mechanisms that could potentially account for the accumulation of AA by mast cells undergoing apoptosis (Fig. 8). These include a decrease in AA uptake and incorporation into phospholipids (Fig. 8a), a decrease in AA remodeling (Fig. 8b), an increase in AA release from phospholipid pools (Fig. 8c), or a decrease in the capacity of mast cells to convert AA to bioactive lipid products (Fig. 8d).



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Fig. 8.   Proposed mechanism accounting for AA accumulation in mast cells undergoing apoptosis. Processes that increase cellular AA levels within mast cells include a decrease in the uptake and incorporation of cellular AA into phospholipids (a), a decrease in CoA-IT activity resulting in a decrease in AA remodeling (b), an increase in PLA2 activity that results in the rapid hydrolysis of AA from cellular phospholipids (c), or the inhibition of cyclooxygenase (COX) and/or 5-lipoxygenase (5-LO) (d). Hydrolysis of PC during BMMC activation may also lead to AA accumulation (c, dashed line). When AA levels are increased, the cells respond by incorporating free AA into neutral lipids via de novo TG synthesis (e). Cellular AA may also be autoxidized to form products, which may induce apoptosis of mast cells (f).

Previous studies from our laboratory and others have indicated that different isotypes of PLA2 are involved in the release of AA from mast cells (8, 44-46). While secretory PLA2s are known to be nonspecific in hydrolyzing fatty acids in cell-free systems, it appears that secretory PLA2 also recruits cPLA2 that accounts for the selective mobilization of AA during cell activation (32). The importance of cPLA2 in AA mobilization from mast cells has recently been confirmed using targeted disruption of the cPLA2 gene in mice (46). In these studies, neither immediate nor delayed prostanoid formation was observed in mast cells obtained from cPLA2 knockout mice. In another set of studies, cells that overexpressed a mutated cPLA2 (Ser505 right-arrow Ala505) exhibited greatly diminished capacity to release AA compared with cells overexpressing wild type cPLA2 (47). Since the activation of cPLA2 is important in AA release, its activation represents a possible mechanism by which high levels of AA would be found within or in the supernatant fluid around mast cells. However, our data suggest that, while there is an increase in AA levels within mast cells that are undergoing apoptosis, there is a decrease in cPLA2 mRNA, protein and activity. Therefore, cPLA2 is not responsible for the accumulation of AA when mast cells are undergoing apoptosis. Other studies have shown that cPLA2 is decreased in cells undergoing apoptosis induced by Fas activation or by contact inhibition (48-51). The decrease in cPLA2 was shown to be due to a caspase-mediated breakdown of the enzyme (48, 52). It has been implied from these studies that the decrease in cPLA2 represents a novel mechanism by which inflammatory processes are regulated when cells are undergoing apoptosis. The present study suggests that even when cPLA2 levels are decreased in cells, it may not be strictly correct to assume that inflammatory capacity is attenuated, since other enzymes regulate AA levels. Our study also reveals that cPLA2 levels within mast cells are regulated by cytokines. In the presence of optimum levels of SCF or IL-3, BMMC proliferate in culture. Under these conditions, cPLA2 expression is maintained at high levels. During cytokine depletion, there is a decrease in cPLA2 expression. These changes support the observation of Anderson et al. (51) suggesting that cPLA2 is linked to cell proliferation.

Recent studies also suggest that inhibitors of cyclooxygenase or 5-lipoxygenase increase free AA levels within cells (41, 53). These inhibitors also induce apoptosis of cancer cells (53-61). Thus, changes in cyclooxygenase activity and AA levels are closely linked to apoptosis. In the present study, BMMC undergoing apoptosis produced more prostanoids than control cells. LTB4 levels remained the same, regardless of the apoptotic status of the cells, demonstrating that decreased capacity to form eicosanoids does not account for the accumulation of AA in mast cells undergoing apoptosis induced by cytokine withdrawal.

When AA is provided to mammalian cells, it is rapidly incorporated into various glycerolipid pools through both CoA-dependent and CoA-independent acylation reactions (17, 62, 63). Initially, AA is converted to AA-CoA by arachidonoyl-CoA synthetase. AA-CoA is then transferred to 1-acyl-2-lyso-sn-glycero-3-phospholipids by CoA-dependent acyltransferase. Various studies have shown that when this incorporation process is inhibited with Triacsin C, there is accumulation of AA products within cells and in the culture media (64, 65). Importantly, inhibition of this process has been associated with apoptosis of some cells (66). The present study suggests that this initial incorporation of AA into mast cells is not altered when these cells are undergoing apoptosis. Thus, a decrease in AA incorporation does not account for the increase in AA levels within mast cells or in culture media.

In mast cells, AA that is initially incorporated into 1-acyl-linked glycerophospholipids is slowly remodeled from these initial pools into 1-ether-linked glycerophospholipid pools. Our studies suggest that the remodeling of arachidonate from 1-acyl-linked to 1-ether-linked phospholipids is orchestrated by CoA-IT (7, 24). We recently demonstrated that treating cancer cell lines with CoA-IT inhibitors led to accumulation of intracellular AA (41, 67-69). Importantly, this inhibition of CoA-IT also resulted in the inhibition of cell cycle progression within breast cancer cells. The present study underscores the role of CoA-IT in AA metabolism. When mast cells were placed in cytokine-depleted media, the mole quantity of AA in PE was significantly reduced, concomitant with a decrease in CoA-IT activity in microsomal pellets. Therefore, a decrease in CoA-IT is responsible for the accumulation of AA in mast cells undergoing apoptosis. Since a decrease in CoA-IT activity results in arachidonate accumulation in PC, hydrolysis of PC by PLA2 could also potentially account for the increase in free AA levels (Fig. 8c). Because no changes were observed in lysophospholipids levels during apoptosis, and since specific activity measurements suggest that most free AA is generated from PE (7), it is likely that hydrolysis of PC plays only a minor role in free AA accumulation. However, during BMMC activation, when there is an increase in lysophospholipid levels and release of AA from all major phospholipid pools, hydrolysis of PC by PLA2 may contribute to free AA release (7, 63, 70).

Various pathways have been described for the incorporation of AA into glycerophospholipids, depending upon the concentration of AA to which cells are exposed (71). Exposure of cells to low AA concentrations results in AA incorporation into primarily 1-acyl-linked phospholipid molecular species through a high affinity, low capacity enzymatic pathway. In contrast, exposure of cells to high levels of AA will result in the incorporation of AA into neutral lipid fractions (mainly TG) and the formation of 1,2-diarachidonoyl-sn-glycero-3-phosphocholine by a low affinity, high capacity pathway (42, 71). Our data show that there is an increase in AA mass in the neutral lipid fractions of mast cells undergoing apoptosis. In addition, the AA mass in 1-acyl-linked PC species also increased in mast cells undergoing apoptosis, such that this becomes the single largest phospholipid subclass instead of 1-alk-1-enyl-2-AA-GPE. These data suggest that there is induction of the low affinity, high capacity pathway of AA metabolism in mast cells undergoing apoptosis. The addition of exogenous AA increased cellular [3H]AA incorporation into TG in a dose-dependent manner. Exogenous AA also induced free [3H]AA accumulation in the cell culture media. However, much higher levels of exogenous AA were needed to induce these changes, because exogenous AA is bound to serum albumin and is distributed over a larger volume than cell-associated AA. Overall, the increase of AA in neutral lipid fractions via de novo synthesis (Fig. 8e) and in PC subclasses (Fig. 8a) suggests that BMMC undergoing apoptosis have been exposed to high concentrations of free AA.

In addition to the accumulation of AA in some pools of lipids, another consequence of the build up of high levels of AA within and outside mast cells is the formation of oxidized products. High levels of AA may induce the formation of proapoptotic molecules such as ceramides, while fatty acids may affect various apoptotic proteins (53, 69, 72, 73). An important role for lipid peroxidation in apoptosis is further shown by our data showing a significant increase in lipid peroxidation only in mast cells that are undergoing apoptosis (Fig. 8f). Since lipid peroxidation has been linked to apoptosis (38), our present study suggests that mast cells may be undergoing apoptosis as a result of the formation of reactive lipid peroxides.

Overall, this study implies that perturbation of cellular arachidonate metabolism is a critical process in BMMC that are undergoing apoptosis. Importantly, AA accumulation is a predictive indicator of apoptosis in BMMC after cytokine removal. The major enzyme activities that change when mast cells are undergoing apoptosis are cPLA2 and CoA-IT. It is likely that there is a link between these two activities, since they exhibit similar biological properties. First, our data suggest that a decrease in cPLA2 is accompanied by a decrease in CoA-IT activity during apoptosis of mast cells. Second, both cPLA2 and CoA-IT are selective for arachidonate (19, 74). Third, inhibitors of cPLA2 induce apoptosis of cells, as do inhibitors of CoA-IT (51, 68). Fourth, inhibitors of CoA-IT prevent AA release and eicosanoid biosynthesis, as do inhibitors of cPLA2 (75). Fifth, cytokine treatment of BMMC (present study) and neutrophils results in an increase in both CoA-IT and cPLA2 activity (76). Sixth, both activities are heat labile and are not influenced by sulfhydryl reducing agents (77). Finally, Reynolds et al. (78) reported that cPLA2 has weak transacylase activity. However, even with these similarities, there are some striking differences between CoA-IT and cPLA2. Whereas CoA-IT does not require calcium for activation and is mainly membrane-bound, the major cPLA2 isotypes require calcium for activation and reside mainly in the cytosol. However, cPLA2 translocates to perinuclear membranes during cell activation, and a recently cloned cPLA2 isotype (cPLA2gamma ) is reported to be calcium-independent and to be mainly membrane-bound (79). Since CoA-IT has not been cloned, further studies will be required to show whether these two activities cooperate within cells in regulating AA metabolism. In addition to the possible link between cPLA2 and CoA-IT, the present studies also raise important implications concerning the role of AA in apoptosis. Further investigations will be required to determine the signal transduction processes that link AA accumulation, lipid peroxidation, and changes in arachidonate pools to apoptosis of BMMC.


    ACKNOWLEDGEMENTS

We are grateful for expert technical assistance from Chad Marion, Brooke Barham, and Michelle Edens. We thank Drs. A. Trimboli and F. Chilton for helpful suggestions.


    FOOTNOTES

* This work was supported in part by National Institutes of Health Grant AI 24985 SI (to F. A. N.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom all correspondence should be addressed: Dept. of Internal Medicine, Section on Pulmonary and Critical Care Medicine, Wake Forest University School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157-1054. Tel.: 336-716-9923; Fax: 336-716-7277; E-mail: afonteh@wfubmc.edu.

§ Present address: US EPA, 86 TW Alexander Dr., MD 82, Research Triangle Park, NC 27111.

Published, JBC Papers in Press, October 5, 2000, DOI 10.1074/jbc.M006551200


    ABBREVIATIONS

The abbreviations used are: AA, arachidonic acid; PLA2, phospholipase A2; cPLA2, cytosolic phospholipase A2; LTB4 and LTC4, leukotriene B4 and C4, respectively; PGD2, prostaglandin D2; HSA, human serum albumin; NICI-GC/MS, negative ion chemical ionization gas chromatography/mass spectrometry; GPC, glycero-3-phosphocholine; GPE, glycero-3-phosphoethanolamine; [2H8]AA, octadeuterated AA; [2H3]AA, tritradeuterated AA; LA, linoleic acid; OA, oleic acid; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; TG, triglyceride; BMMC, murine bone marrow-derived mast cell(s); PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; HPLC, high pressure liquid chromatography; SCF, stem cell factor; IL, interleukin; FITC, fluorescein isothiocyanate; MDA, malondialdehyde; NL, neutral lipids; CoA-IT, CoA-independent transacylase; TXB2, thromboxane B2.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


1. Costa, J. J., Weller, P. F., and Galli, S. J. (1997) JAMA 278, 1815-1822[Abstract]
2. Galli, S. J., Gordon, J. R., and Wershil, B. K. (1991) Curr. Opin. Immunol. 3, 865-872[Medline] [Order article via Infotrieve]
3. Gordon, J. R., Burd, P. R., and Galli, S. J. (1990) Immunol. Today 11, 458-464[CrossRef][Medline] [Order article via Infotrieve]
4. Metcalfe, D. D., Baram, D., and Mekori, Y. A. (1997) Physiol Rev. 77, 1033-1079[Abstract/Free Full Text]
5. Wasserman, S. I. (1994) Am. J. Respir. Crit. Care Med. 150, S39-S41[Medline] [Order article via Infotrieve]
6. Chilton, F. H., and Lichtenstein, L. M. (1990) Chem. Immunol. 49, 173-205[Medline] [Order article via Infotrieve]
7. Fonteh, A. N., and Chilton, F. H. (1993) J. Immunol. 150, 563-570[Abstract/Free Full Text]
8. Bingham, C. O., III, Murakami, M., Fujishima, H., Hunt, J. E., Austen, K. F., and Arm, J. P. (1996) J. Biol. Chem. 271, 25936-25944[Abstract/Free Full Text]
9. Murakami, M., Matsumoto, R., Urade, Y., Austen, K. F., and Arm, J. P. (1995) J. Biol. Chem. 270, 3239-3246[Abstract/Free Full Text]
10. Murakami, M., Shimbara, S., Kambe, T., Kuwata, H., Winstead, M. V., Tischfield, J. A., and Kudo, I. (1998) J. Biol. Chem. 273, 14411-14423[Abstract/Free Full Text]
11. Murakami, M., Nakatani, Y., Atsumi, G., Inoue, K., and Kudo, I. (1997) Crit Rev. Immunol. 17, 225-283[Medline] [Order article via Infotrieve]
12. Dennis, E. A. (1997) Trends Biochem. Sci. 22, 1-2[CrossRef][Medline] [Order article via Infotrieve]
13. Cupillard, L., Koumanov, K., Mattei, M. G., Lazdunski, M., and Lambeau, G. (1997) J. Biol. Chem. 272, 15745-15752[Abstract/Free Full Text]
14. Valentin, E., Ghomashchi, F., Gelb, M. H., Lazdunski, M., and Lambeau, G. (1999) J. Biol. Chem. 274, 31195-31202[Abstract/Free Full Text]
15. Valentin, E., Ghomashchi, F., Gelb, M. H., Lazdunski, M., and Lambeau, G. (2000) J. Biol. Chem. 275, 7492-7496[Abstract/Free Full Text]
16. MacDonald, J. I., and Sprecher, H. (1989) Biochim. Biophys. Acta 1004, 151-157[Medline] [Order article via Infotrieve]
17. Chilton, F. H., Fonteh, A. N., Surette, M. E., Triggiani, M., and Winkler, J. D. (1996) Biochim. Biophys. Acta 1299, 1-15[Medline] [Order article via Infotrieve]
18. Kramer, R. M., Pritzker, C. R., and Deykin, D. (1984) J. Biol. Chem. 259, 2403-2406[Abstract/Free Full Text]
19. Yamashita, A., Sugiura, T., and Waku, K. (1997) J. Biochem. (Tokyo) 122, 1-16[Abstract]
20. Lands, W. E., Inoue, M., Sugiura, Y., and Okuyama, H. (1982) J. Biol. Chem. 257, 14968-14972[Abstract/Free Full Text]
21. Wilson, D. B., Prescott, S. M., and Majerus, P. W. (1982) J. Biol. Chem. 257, 3510-3515[Abstract/Free Full Text]
22. Sugiura, T., Katayama, O., Fukui, J., Nakagawa, Y., and Waku, K. (1984) FEBS Lett. 165, 273-276[CrossRef][Medline] [Order article via Infotrieve]
23. Chilton, F. H., and Murphy, R. C. (1986) J. Biol. Chem. 261, 7771-7777[Abstract/Free Full Text]
24. Fonteh, A. N., and Chilton, F. H. (1992) J. Immunol. 148, 1784-1791[Abstract/Free Full Text]
25. Kramer, R. M., and Deykin, D. (1983) J. Biol. Chem. 258, 13806-13811[Abstract/Free Full Text]
26. Dvorak, A. M., Seder, R. A., Paul, W. E., Morgan, E. S., and Galli, S. J. (1994) Am. J. Pathol. 144, 160-170[Abstract]
27. Mekori, Y. A., Oh, C. K., and Metcalfe, D. D. (1993) J. Immunol. 151, 3775-3784[Abstract/Free Full Text]
28. Tsai, M., Tam, S. Y., and Galli, S. J. (1993) Eur. J. Immunol. 23, 867-872[Medline] [Order article via Infotrieve]
29. Tsujimura, T., Hashimoto, K., Morii, E., Tunio, G. M., Tsujino, K., Kondo, T., Kanakura, Y., and Kitamura, Y. (1997) Am. J. Pathol. 151, 1043-1051[Abstract]
30. Gerard-Monnier, D., Erdelmeier, I., Regnard, K., Moze-Henry, N., Yadan, J. C., and Chaudiere, J. (1998) Chem. Res. Toxicol. 11, 1176-1183[CrossRef][Medline] [Order article via Infotrieve]
31. Bligh, E. A., and Dyer, W. T. (1959) Can. J. Physiol Pharmacol. 37, 911-917
32. Fonteh, A. N., Atsumi, G., LaPorte, T., and Chilton, F. H. (2000) J. Immunol. 165, 2773-2782[Abstract/Free Full Text]
33. Chilton, F. H. (1990) Methods Enzymol. 187, 157-167[Medline] [Order article via Infotrieve]
34. Fonteh, A. N. (1999) Methods Mol. Biol. 120, 77-89[Medline] [Order article via Infotrieve]
35. Nakagawa, Y., and Horrocks, L. A. (1983) J. Lipid Res. 24, 1268-1275[Abstract]
36. Fonteh, A. N., Samet, J. M., and Chilton, F. H. (1995) J. Clin. Invest. 96, 1432-1439[Medline] [Order article via Infotrieve]
37. Schor, N. F., Tyurina, Y. Y., Fabisiak, J. P., Tyurin, V. A., Lazo, J. S., and Kagan, V. E. (1999) Brain Res. 831, 125-130[CrossRef][Medline] [Order article via Infotrieve]
38. Das, U. N. (1999) Prostaglandins Leukotrienes Essent. Fatty Acids 61, 157-163[CrossRef][Medline] [Order article via Infotrieve]
39. Chen, Q., Galleano, M., and Cederbaum, A. I. (1998) Alcohol Clin. Exp. Res. 22, 782-784[Medline] [Order article via Infotrieve]
40. Fabisiak, J. P., Tyurin, V. A., Tyurina, Y. Y., Sedlov, A., Lazo, J. S., and Kagan, V. E. (2000) Biochemistry 39, 127-138[CrossRef][Medline] [Order article via Infotrieve]
41. Trimboli, A. J., Waite, B. M., Atsumi, G., Fonteh, A. N., Namen, A. M., Clay, C. E., Kute, T. E., High, K. P., Willingham, M. C., and Chilton, F. H. (1999) Cancer Res. 59, 6171-6177[Abstract/Free Full Text]
42. Johnson, M. M., Vaughn, B., Triggiani, M., Swan, D. D., Fonteh, A. N., and Chilton, F. H. (1999) Am. J. Respir. Cell Mol. Biol. 21, 253-258[Abstract/Free Full Text]
43. Triggiani, M., Oriente, A., Seeds, M. C., Bass, D. A., Marone, G., and Chilton, F. H. (1995) J. Exp. Med. 182, 1181-1190[Abstract]
44. Fonteh, A. N., Bass, D. A., Marshall, L. A., Seeds, M., Samet, J. M., and Chilton, F. H. (1994) J. Immunol. 152, 5438-5446[Abstract/Free Full Text]
45. Reddy, S. T., Winstead, M. V., Tischfield, J. A., and Herschman, H. R. (1997) J. Biol. Chem. 272, 13591-13596[Abstract/Free Full Text]
46. Fujishima, H., Sanchez Mejia, R. O., Bingham, C. O., III, Lam, B. K., Sapirstein, A., Bonventre, J. V., Austen, K. F., and Arm, J. P. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 4803-4807[Abstract/Free Full Text]
47. Nemenoff, R. A., Winitz, S., Qian, N. X., Van, P., V, Johnson, G. L., and Heasley, L. E. (1993) J. Biol. Chem. 268, 1960-1964[Abstract/Free Full Text]
48. Atsumi, G., Tajima, M., Hadano, A., Nakatani, Y., Murakami, M., and Kudo, I. (1998) J. Biol. Chem. 273, 13870-13877[Abstract/Free Full Text]
49. Enari, M., Hug, H., Hayakawa, M., Ito, F., Nishimura, Y., and Nagata, S. (1996) Eur. J. Biochem. 236, 533-538[Abstract]
50. Jaattela, M., Benedict, M., Tewari, M., Shayman, J. A., and Dixit, V. M. (1995) Oncogene 10, 2297-2305[Medline] [Order article via Infotrieve]
51. Anderson, K. M., Roshak, A., Winkler, J. D., McCord, M., and Marshall, L. A. (1997) J. Biol. Chem. 272, 30504-30511[Abstract/Free Full Text]
52. Adam-Klages, S., Schwandner, R., Luschen, S., Ussat, S., Kreder, D., and Kronke, M. (1998) J. Immunol. 161, 5687-5694[Abstract/Free Full Text]
53. Chan, T. A., Morin, P. J., Vogelstein, B., and Kinzler, K. W. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 681-686[Abstract/Free Full Text]
54. Elder, D. J., Halton, D. E., Hague, A., and Paraskeva, C. (1997) Clin. Cancer Res. 3, 1679-1683[Abstract]
55. Elder, D. J., and Paraskeva, C. (1998) Nat. Med. 4, 392-393[Medline] [Order article via Infotrieve]
56. Hara, A., Yoshimi, N., Niwa, M., Ino, N., and Mori, H. (1997) Jpn. J. Cancer Res. 88, 600-604[Medline] [Order article via Infotrieve]
57. Kamitani, H., Geller, M., and Eling, T. (1998) J. Biol. Chem. 273, 21569-21577[Abstract/Free Full Text]
58. Lim, J. T., Piazza, G. A., Han, E. K., Delohery, T. M., Li, H., Finn, T. S., Buttyan, R., Yamamoto, H., Sperl, G. J., Brendel, K., Gross, P. H., Pamukcu, R., and Weinstein, I. B. (1999) Biochem. Pharmacol. 58, 1097-1107[CrossRef][Medline] [Order article via Infotrieve]
59. Sawaoka, H., Kawano, S., Tsuji, S., Tsujii, M., Murata, H., and Hori, M. (1998) J. Clin. Gastroenterol. 27 Suppl. 1, 47-52[CrossRef][Medline] [Order article via Infotrieve]
60. Sheng, H., Shao, J., Morrow, J. D., Beauchamp, R. D., and Dubois, R. N. (1998) Cancer Res. 58, 362-366[Abstract]
61. Subbaramaiah, K., Zakim, D., Weksler, B. B., and Dannenberg, A. J. (1997) Proc. Soc. Exp. Biol. Med. 216, 201-210[Abstract]
62. MacDonald, J. I., and Sprecher, H. (1991) Biochim. Biophys. Acta 1084, 105-121[Medline] [Order article via Infotrieve]
63. Ramanadham, S., Hsu, F. F., Bohrer, A., Ma, Z., and Turk, J. (1999) J. Biol. Chem. 274, 13915-13927[Abstract/Free Full Text]
64. Hundley, T. R., Marshall, L. A., Hubbard, W. C., and MacGlashan, D. W., Jr. (1998) J. Pharmacol. Exp. Ther. 284, 847-857[Abstract/Free Full Text]
65. Oh-ishi, S., Yamaki, K., Abe, M., Tomoda, H., and Omura, S. (1992) Jpn. J. Pharmacol. 59, 417-418[Medline] [Order article via Infotrieve]
66. Tomoda, H., Igarashi, K., Cyong, J. C., and Omura, S. (1991) J. Biol. Chem. 266, 4214-4219[Abstract/Free Full Text]
67. Winkler, J. D., Eris, T., Sung, C. M., Chabot-Fletcher, M., Mayer, R. J., Surette, M. E., and Chilton, F. H. (1996) J. Pharmacol. Exp. Ther. 279, 956-966[Abstract]
68. Surette, M. E., Winkler, J. D., Fonteh, A. N., and Chilton, F. H. (1996) Biochemistry 35, 9187-9196[CrossRef][Medline] [Order article via Infotrieve]
69. Surette, M. E., Fonteh, A. N., Bernatchez, C., and Chilton, F. H. (1999) Carcinogenesis 20, 757-763[Abstract/Free Full Text]
70. Nakamura, T., Fonteh, A. N., Hubbard, W. C., Triggiani, M., Inagaki, N., Ishizaka, T., and Chilton, F. H. (1991) Biochim. Biophys. Acta 1085, 191-200[Medline] [Order article via Infotrieve]
71. Chilton, F. H., and Murphy, R. C. (1987) Biochem. Cell Biol. Commun. 145, 1126-1133
72. Das, U. N., Begin, M. E., Ells, G., Huang, Y. S., and Horrobin, D. F. (1987) Biochem. Cell Biol. Commun. 145, 15-24
73. Morisaki, N., Sprecher, H., Milo, G. E., and Cornwell, D. G. (1982) Lipids 17, 893-899[Medline] [Order article via Infotrieve]
74. Lin, L. L., Lin, A. Y., and Knopf, J. L. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 6147-6151[Abstract]
75. Winkler, J. D., Fonteh, A. N., Sung, C. M., Huang, L., Chabot-Fletcher, M., Marshall, L. A., and Chilton, F. H. (1995) Adv. Prostaglandin Thromboxane Leukotriene Res. 23, 89-91[Medline] [Order article via Infotrieve]
76. Winkler, J. D., Sung, C. M., Huang, L., and Chilton, F. H. (1994) Biochim. Biophys. Acta 1215, 133-140[Medline] [Order article via Infotrieve]
77. Winkler, J. D., McCarte-Roshak, A., Huang, L., Sung, C. M., Bolognese, B., and Marshall, L. A. (1994) J. Lipid Mediat. Cell Signal. 10, 315-330[Medline] [Order article via Infotrieve]
78. Reynolds, L. J., Hughes, L. L., Louis, A. I., Kramer, R. M., and Dennis, E. A. (1993) Biochim. Biophys. Acta 1167, 272-280[Medline] [Order article via Infotrieve]
79. Underwood, K. W., Song, C., Kriz, R. W., Chang, X. J., Knopf, J. L., and Lin, L. L. (1998) J. Biol. Chem. 273, 21926-21932[Abstract/Free Full Text]


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