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Oxidized LDL activates phospholipase A2 to supply fatty acids required for cholesterol esterification

2003; Elsevier BV; Volume: 44; Issue: 9 Linguagem: Inglês

10.1194/jlr.m300012-jlr200

1539-7262

Satoshi Akiba, Yukimasa Yoneda, Satoshi Ohno, Megumi Nemoto, Takashi Sato,

Fatty Acid Research and Health

We examined the roles of phospholipase A2 (PLA2) in oxidized LDL (oxLDL)-induced cholesteryl ester formation in macrophages. In [3H]oleic acid-labeled RAW264.7 cells and mouse peritoneal macrophages, oxLDL induced [3H]cholesteryl oleate formation with an increase in free [3H]oleic acid and a decrease in [3H]phosphatidylcholine. The changes in these lipids were suppressed by methyl arachidonyl fluorophosphonate (MAFP), a cytosolic PLA2 (cPLA2) inhibitor. However, MAFP had no effect on the ACAT activity or the binding and/or uptake of oxLDL. Stimulation with oxLDL in the presence of [3H]cholesterol increased [3H]cholesteryl ester bearing fatty acyl chains derived from cellular and/or exogenous (oxLDL) lipids. The formation of cholesteryl ester under this condition was also inhibited by MAFP, and the inhibitory effect was reversed by adding oleic acid. While oxLDL did not affect the activity or amounts of cPLA2, preincubation with oxLDL enhanced the release of oleic acid and arachidonic acid induced by ionomycin in RAW264.7 cells. 13(S)-hydroxyoctadecadienoic acid, but not 7-ketocholesterol, also enhanced ionomycin-induced oleic acid release.These results suggest that oxLDL induces cPLA2 activation, which contributes, at least in part, to the supply of fatty acids required for the cholesteryl esterification, probably through the acceleration by oxidized lipids of the catalytic action of cPLA2 in macrophages. We examined the roles of phospholipase A2 (PLA2) in oxidized LDL (oxLDL)-induced cholesteryl ester formation in macrophages. In [3H]oleic acid-labeled RAW264.7 cells and mouse peritoneal macrophages, oxLDL induced [3H]cholesteryl oleate formation with an increase in free [3H]oleic acid and a decrease in [3H]phosphatidylcholine. The changes in these lipids were suppressed by methyl arachidonyl fluorophosphonate (MAFP), a cytosolic PLA2 (cPLA2) inhibitor. However, MAFP had no effect on the ACAT activity or the binding and/or uptake of oxLDL. Stimulation with oxLDL in the presence of [3H]cholesterol increased [3H]cholesteryl ester bearing fatty acyl chains derived from cellular and/or exogenous (oxLDL) lipids. The formation of cholesteryl ester under this condition was also inhibited by MAFP, and the inhibitory effect was reversed by adding oleic acid. While oxLDL did not affect the activity or amounts of cPLA2, preincubation with oxLDL enhanced the release of oleic acid and arachidonic acid induced by ionomycin in RAW264.7 cells. 13(S)-hydroxyoctadecadienoic acid, but not 7-ketocholesterol, also enhanced ionomycin-induced oleic acid release. These results suggest that oxLDL induces cPLA2 activation, which contributes, at least in part, to the supply of fatty acids required for the cholesteryl esterification, probably through the acceleration by oxidized lipids of the catalytic action of cPLA2 in macrophages. The oxidation of LDL within the artery wall is widely accepted to participate in atherogenesis (1Steinberg D. Low density lipoprotein oxidation and its pathobiological significance.J. Biol. Chem. 1997; 272: 20963-20966Abstract Full Text Full Text PDF PubMed Scopus (1456) Google Scholar, 2Chisolm III, G.M. Hazen S.L. Fox P.L. Cathcart M.K. The oxidation of lipoproteins by monocytes-macrophages. Biochemical and biological mechanisms.J. Biol. Chem. 1999; 274: 25959-25962Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 3Ross R. Atherosclerosis—an inflammatory disease.N. Engl. J. Med. 1999; 340: 115-126Crossref PubMed Scopus (19277) Google Scholar, 4Lusis A.J. Atherosclerosis.Nature. 2000; 407: 233-241Crossref PubMed Scopus (4697) Google Scholar). The oxidized LDL (oxLDL) stimulates the transmigration of monocytes into the arterial intima, their differentiation into macrophages, and the transformation of macrophages into lipid-laden foam cells (3Ross R. Atherosclerosis—an inflammatory disease.N. Engl. J. Med. 1999; 340: 115-126Crossref PubMed Scopus (19277) Google Scholar, 4Lusis A.J. Atherosclerosis.Nature. 2000; 407: 233-241Crossref PubMed Scopus (4697) Google Scholar, 5de Winther M.P.J. Hofker M.H. Scavenging new insights into atherogenesis.J. Clin. Invest. 2000; 105: 1039-1041Crossref PubMed Scopus (56) Google Scholar). Similarly, oxLDL elicits the migration of smooth muscle cells from the media and their transformation into foam cells (3Ross R. Atherosclerosis—an inflammatory disease.N. Engl. J. Med. 1999; 340: 115-126Crossref PubMed Scopus (19277) Google Scholar, 4Lusis A.J. Atherosclerosis.Nature. 2000; 407: 233-241Crossref PubMed Scopus (4697) Google Scholar, 6Chisolm III, G.M. Chai Y. Regulation of cell growth by oxidized LDL.Free Radic. Biol. Med. 2000; 28: 1697-1707Crossref PubMed Scopus (78) Google Scholar). The accumulation of foam cells, resulting from these biological activities of oxLDL, leads to the formation of fatty streak lesions, which is a critical event in the early stages of atherosclerosis (3Ross R. Atherosclerosis—an inflammatory disease.N. Engl. J. Med. 1999; 340: 115-126Crossref PubMed Scopus (19277) Google Scholar, 4Lusis A.J. Atherosclerosis.Nature. 2000; 407: 233-241Crossref PubMed Scopus (4697) Google Scholar). In the advanced stages of atherosclerosis, the death and necrosis of foam cells facilitates the development of vulnerable atherosclerotic plaques with large lipid cores and very thin fibrous caps, the rupture of which leads to thrombus formation followed by clinical manifestations of coronary heart disease, such as myocardial infraction (3Ross R. Atherosclerosis—an inflammatory disease.N. Engl. J. Med. 1999; 340: 115-126Crossref PubMed Scopus (19277) Google Scholar, 4Lusis A.J. Atherosclerosis.Nature. 2000; 407: 233-241Crossref PubMed Scopus (4697) Google Scholar). Thus, the foam cell formation by oxLDL represents a key event in the development and progression of atherosclerosis. The formation of macrophage-derived foam cells is associated with the uptake of oxLDL particles by macrophages via several types of scavenger receptors, including two novel receptors (LOX-1 and SR-PSOX) identified recently (5de Winther M.P.J. Hofker M.H. Scavenging new insights into atherogenesis.J. Clin. 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The endocytosed oxLDL is delivered to lysosomes, and then cholesteryl esters in the oxLDL are hydrolyzed by lysosomal acid cholesteryl ester hydrolase to liberate free cholesterol, which moves to the cholesterol pool in the plasma membrane. The excessive free cholesterol is reesterified with fatty acyl-CoA by ACAT at the endoplasmic reticulum to form cholesteryl ester, which accumulates as cytoplasmic lipid droplets, thus leading to foam cell formation (10Chang T.Y. Chang C.C.Y. Cheng D. Acyl-coenzyme A:cholesterol acyltransferase.Annu. Rev. Biochem. 1997; 66: 613-638Crossref PubMed Scopus (440) Google Scholar, 11Brown A.J. Mander E.L. Gelissen I.C. Kritharides L. Dean R.T. Jessup W. Cholesterol and oxysterol metabolism and subcellular distribution in macrophage foam cells. Accumulation of oxidized esters in lysosomes.J. Lipid Res. 2000; 41: 226-237Abstract Full Text Full Text PDF PubMed Google Scholar, 12Tabas I. Cholesterol and phospholipid metabolism in macrophages.Biochim. Biophys. Acta. 2000; 1529: 164-174Crossref PubMed Scopus (118) Google Scholar). In the process of accumulation of cholesteryl ester, although ACAT utilizes free cholesterol supplied from the cholesterol pool as a substrate, little is known about the pathways involved in the supply of fatty acids required for the ACAT-catalyzed cholesterol esterification. A previous study showed that while human LDL contains cholesteryl linoleate more than cholesteryl oleate, foam cells in the fatty streak lesions contain predominantly cholesteryl oleate (13Smith E.B. The relationship between plasma and tissue lipids in human atherosclerosis.Adv. Lipid Res. 1974; 12: 1-49Crossref PubMed Google Scholar). Furthermore, stimulation of mouse peritoneal macrophages with acetylated LDL containing cholesteryl linoleate results in hydrolysis of the ester with an increase in cholesteryl oleate (14Brown M.S. Goldstein J.L. Lipoprotein metabolism in the macrophage: implications for cholesterol deposition in atherosclerosis.Annu. Rev. Biochem. 1983; 52: 223-261Crossref PubMed Google Scholar). These findings suggest that free fatty acids utilized for the reesterification are supplied from lipids other than cholesteryl esters derived from oxLDL. It has been reported that in [14C]oleic acid-labeled mouse and [3H]arachidonic acid-labeled rat peritoneal macrophages, uptake of acetylated LDL or liposomes containing cholesterol induces an increase in cholesteryl ester bearing radioactive fatty acyl chains with a decrease in radioactive phosphatidylcholine (15Nishikawa K. Sato Y. Arai H. Inoue K. Mobilization of acyl chains from endogenous cellular phospholipids into cholesteryl esters during foam-cell formation in mouse peritoneal macrophages.Biochim. Biophys. Acta. 1993; 1169: 257-263Crossref PubMed Scopus (16) Google Scholar, 16Pollaud C. Krause S. Lepert J.C. Orfila C. Seguelas M. Festal D. Decerprit J. 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Stimulated arachidonate metabolism during foam cell transformation of mouse peritoneal macrophages with oxidized low density lipoprotein.J. Clin. Invest. 1988; 81: 720-729Crossref PubMed Scopus (129) Google Scholar, 18Claus R. Fyrnys B. Deigner H.P. Wolf G. Oxidized low-density lipoprotein stimulates protein kinase C (PKC) and induces expression of PKC-isotypes via prostaglandin-H-synthase in P388D1 macrophage-like cells.Biochemistry. 1996; 35: 4911-4922Crossref PubMed Scopus (40) Google Scholar, 19Panini S.R. Yang L. Rusinol A.E. Sinensky M.S. Bonventre J.V. Leslie C.C. Arachidonate metabolism and the signaling pathway of induction of apoptosis by oxidized LDL/oxysterol.J. Lipid Res. 2001; 42: 1678-1686Abstract Full Text Full Text PDF PubMed Google Scholar). Among numerous types of PLA2s identified in mammalian cells and tissues (20Kudo I. Murakami M. Hara S. Inoue K. Mammalian non-pancreatic phospholipases A2.Biochim. Biophys. Acta. 1993; 1170: 217-231Crossref PubMed Scopus (371) Google Scholar, 21Six D.A. Dennis E.A. The expanding superfamily of phospholipases A2 enzymes. Classification and characterization.Biochim. Biophys. Acta. 2000; 1488: 1-19Crossref PubMed Scopus (1220) Google Scholar, 22Murakami M. Kudo I. Phospholipase A2.J. Biochem. 2002; 131: 285-292Crossref PubMed Scopus (436) Google Scholar), Ca2+-dependent cytosolic PLA2 (cPLA2), but not Ca2+-independent cytosolic PLA2 (iPLA2), participates in the oxLDL-induced arachidonic acid liberation in mouse peritoneal macrophages (19Panini S.R. Yang L. Rusinol A.E. Sinensky M.S. Bonventre J.V. Leslie C.C. Arachidonate metabolism and the signaling pathway of induction of apoptosis by oxidized LDL/oxysterol.J. Lipid Res. 2001; 42: 1678-1686Abstract Full Text Full Text PDF PubMed Google Scholar). Recently, we demonstrated that the oxLDL-induced formation of [3H]cholesteryl oleate is suppressed by methyl arachidonyl fluorophosphonate (MAFP), an inhibitor of cPLA2, in RAW264.7 macrophages (23Kitatani K. Nemoto M. Akiba S. Sato T. Stimulation by de novo-synthesized ceramide of phospholipase A2-dependent cholesterol esterification promoted by the uptake of oxidized low-density lipoprotein in macrophages.Cell. Signal. 2002; 14: 695-701Crossref PubMed Scopus (18) Google Scholar). These observations led us to assume that cPLA2 plays an important role in the release of fatty acids utilized for cholesterol esterification in response to oxLDL. However, our results (23Kitatani K. Nemoto M. Akiba S. Sato T. Stimulation by de novo-synthesized ceramide of phospholipase A2-dependent cholesterol esterification promoted by the uptake of oxidized low-density lipoprotein in macrophages.Cell. Signal. 2002; 14: 695-701Crossref PubMed Scopus (18) Google Scholar) also suggest the possible involvement of iPLA2 in the esterification, because MAFP is shown to inhibit the activity of iPLA2 in addition to that of cPLA2 (24Lio Y.C. Reynolds L.J. Balsinde J. Dennis E.A. Irreversible inhibition of Ca2+-independent phospholipase A2 by methyl arachidonyl fluorophosphonate.Biochim. Biophys. Acta. 1996; 1302: 55-60Crossref PubMed Scopus (209) Google Scholar). Furthermore, because the uptake by mouse peritoneal macrophages of liposomes containing cholesterol and phosphatidylcholine bearing a radioactive fatty acyl chain induces an increase in radioactive cholesteryl ester (15Nishikawa K. Sato Y. Arai H. Inoue K. Mobilization of acyl chains from endogenous cellular phospholipids into cholesteryl esters during foam-cell formation in mouse peritoneal macrophages.Biochim. Biophys. Acta. 1993; 1169: 257-263Crossref PubMed Scopus (16) Google Scholar), it is possible that fatty acids derived from phospholipids in oxLDL particles may be also utilized for the cholesterol esterification. Therefore, the present study was undertaken to further examine the contribution of cPLA2 to the cholesteryl ester formation and the regulation of cPLA2 activation associated with the uptake of oxLDL in RAW264.7 macrophages and mouse peritoneal macrophages. [3H]oleic acid (7 Ci/mmol), [3H]arachidonic acid (209 Ci/mmol), and [14C]oleoyl-CoA (56 mCi/mmol) were purchased from Amersham Pharmacia Biotech (Buckinghamshire, UK). [3H]cholesterol (51.2 Ci/mmol), 1-stearoyl-2-[3H]arachidonoyl-sn-glycero-3-phosphocholine (172 Ci/mmol), and 1,2-dipalmitoyl-sn-glycero-3-[choline-methyl-14C]phosphocholine (159 mCi/mmol) were from PerkinElmer Life Science (Boston, MA). 13(S)-hydroxyoctadecadienoic acid (13-HODE), 7-ketocholesterol, FBS, bromoenol lactone, and 1,1′-dioctadecyl-3,3,3′,3′-tetra-methylindocarbocyanine (DiI) perchlorate were from Sigma-Aldrich Fine Chemicals (St. Louis, MO). Human LDL (BT-903) was from Biomedical Technologies Inc. (Stoughton, MI), MAFP was from Cayman Chemical (Ann Arbor, MI), ionomycin was from Calbiochem (La Jolla, CA), triacsin C was from Kyowa Medex (Tokyo, Japan), and the anti-cPLA2 antibody was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). All other reagents were obtained from Wako Pure Chemical Industries (Osaka, Japan). RAW264.7 cells (Dainippon Pharmaceutical Co., Ltd.) were maintained in DMEM (Nissui Pharmaceutical Co., Ltd.) supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C under humidified air containing 5% CO2. Cells were plated in 35 mm culture dishes at 6 × 105 cells or in 100 mm culture dishes at 6 × 106 cells in DMEM containing 0.01% BSA for the following experiments. Resident peritoneal macrophages were isolated from female ddY mice (25–30 g, Japan SLC, Inc.). Peritoneal cells were suspended in DMEM containing 0.01% BSA, 100 U/ml penicillin, and 100 μg/ml streptomycin, and plated in 35 mm culture dishes at 1 × 106 cells or in 100 mm culture dishes at 6 × 106 cells. The cells were incubated at 37°C for 2 h under humidified air containing 5% CO2, and then washed with DMEM containing 0.01% BSA. The attached cells were placed in DMEM containing 0.01% BSA, and used as mouse peritoneal macrophages. Commercial human LDL, prepared from plasma from multiple donors, was dialyzed against PBS at 4°C. The LDL (2.5 mg protein/ml) was oxidized with 10 μM CuSO4 at 37°C for 3 h. The degree of oxidation of the oxLDL was evaluated by a thiobarbituric acid-reactive substances (TBARS) assay according to the method of Yagi (25Yagi K. A simple fluorometric assay for lipoperoxide in blood plasma.Biochem. Med. 1976; 15: 212-216Crossref PubMed Scopus (2053) Google Scholar). The oxLDL contained 10–15 nmol TBARS/mg protein, while native LDL (before oxidation) contained 0.6–1.2 nmol TBARS/mg protein. The oxLDL was further dialyzed against PBS containing 200 μM EDTA at 4°C, and used within 2 weeks. For the labeling with [3H]oleic acid, RAW264.7 cells or mouse peritoneal macrophages (35 mm dishes) were incubated with [3H]oleic acid (0.5 μCi/ml) at 37°C for 12 h or 18 h, respectively. After being washed three times with PBS containing 0.01% BSA, the labeled RAW264.7 cells or mouse peritoneal macrophages were further cultured in DMEM containing 0.01% BSA for 12 h or 6 h, respectively. The labeled cells were placed in fresh DMEM containing 0.01% BSA. For the labeling with [3H]arachidonic acid, RAW264.7 cells (35 mm dishes) were incubated with [3H]arachidonic acid (1 μCi/ml) at 37°C for 12 h. After being washed, the cells were placed in DMEM containing 0.01% BSA. The labeled cells were treated with various reagents, and then stimulated with oxLDL as described in the figure legends. When cells were stimulated with ionomycin after incubation with oxLDL, the oxLDL-stimulated cells were washed three times with DMEM containing 0.01% BSA, and then ionomycin was added. Lipids in the medium and cells were extracted and separated by TLC on a silica gel G plate with the following development systems: for the analysis of cholesteryl ester and free fatty acid, petroleum ether/diethyl ether/formic acid (100:25:2.5; v/v/v); and for the analysis of phosphatidylcholine, the combination of chloroform/methanol/7 M NH4OH (65:25:5.6; v/v/v) for the first dimension and chloroform/methanol/acetic acid/H2O (60:30:8:5; v/v/v/v) for the second. The area corresponding to cholesteryl ester, free fatty acid, phosphatidylcholine, or other lipids was scraped off, and the radioactivity was measured by liquid scintillation counting. The total radioactivity of the fractions recovered from the plate was usually in the range of 2.8 × 105 to 3.3 × 105 dpm in RAW264.7 cells and of 1.8 × 105 to 2.1 × 105 dpm in mouse peritoneal macrophages. The radioactivity of each lipid was corrected by adjusting the total radioactivity to 3.0 × 105 dpm in RAW264.7 cells and to 2.0 × 105 dpm in mouse peritoneal macrophages. These experimental conditions did not affect cell viability (more than 90%), as estimated by trypan blue dye exclusion. RAW264.7 cells (35 mm dishes) were treated with MAFP or triacsin C, and then incubated with [3H]oleic acid (0.5 μCi/ml). After the cells were washed three times with PBS containing 0.01% BSA, lipids in the cells were extracted and separated by TLC as described above. The area corresponding to phosphatidylcholine was scraped off, and the radioactivity was measured by liquid scintillation counting. RAW264.7 cells or mouse peritoneal macrophages (35 mm dishes) were treated with MAFP and then stimulated with oxLDL in the presence of [3H]cholesterol (1 μCi/ml) and unlabeled cholesterol (10 μM) as described in the figure legends. After the cells were washed three times with PBS containing 0.01% BSA, lipids in the cells were extracted and separated by TLC on a silica gel G plate with petroleum ether/diethyl ether/formic acid (100:25:2.5; v/v/v) as the developing system. The area corresponding to cholesteryl ester was scraped off, and the radioactivity was measured by liquid scintillation counting. For the cPLA2 assay, RAW264.7 cells (100 mm dishes) were treated with MAFP or stimulated with oxLDL. After being washed, the cells were collected and sonicated in buffer A [100 mM NaCl, 2 mM EGTA, 100 μM leupeptin, 100 μM p-(amidinophenyl)methanesulfonyl fluoride, 20 mM β-glycerophosphate, 1 mM Na3VO4, and 10 mM Tris-HCl, pH 7.4]. The lysate was centrifuged at 100,000 g for 30 min at 4°C. The cPLA2 activity in the resultant supernatant (cytosol fraction) was determined as described previously (26Akiba S. Hatazawa R. Ono K. Kitatani K. Hayama M. Sato T. Secretory phospholipase A2 mediates cooperative prostaglandin generation by growth factor and cytokine independently of preceding cytosolic phospholipase A2 expression in rat gastric epithelial cells.J. Biol. Chem. 2001; 276: 21854-21862Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Briefly, the supernatant was treated with 5 mM dithiothreitol at 37°C for 15 min. The reaction mixture (30 μg protein) was incubated with a mixture of 1-stearoyl-2-[3H]arachidonoyl-sn-glycero-3-phosphocholine and the unlabeled compound (125 Ci/mol, 2 μM) at 37°C for 15 min in the presence of 5 mM CaCl2 and 50 mM Tris-HCl (pH 7.4) in a final volume of 200 μl. For the iPLA2 assay (27Akiba S. Mizunaga S. Kume K. Hayama M. Sato T. Involvement of group VI Ca2+-independent phospholipase A2 in protein kinase C-dependent arachidonic acid liberation in zymosan-stimulated P388D1 cells.J. Biol. Chem. 1999; 274: 19906-19912Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), RAW264.7 cells (100 mm dishes) were treated with MAFP or bromoenol lactone and washed. The cells were collected and sonicated in a buffer [340 mM sucrose, 2 mM EGTA, 100 μM leupeptin, 100 μM p-(amidinophenyl)methanesulfonyl fluoride, 0.05% Triton X-100, and 10 mM HEPES, pH 7.4]. The lysate (50 μg protein) was treated with 5 mM dithiothreitol at 37°C for 15 min and then incubated with a mixture of 1,2-dipalmitoyl-sn-glycero-3-[choline-methyl-14C]phosphocholine and the unlabeled compound (10 Ci/mol, 50 μM) at 37°C for 1 h in the presence of 5 mM EDTA, 0.03% Triton X-100 and 50 mM HEPES (pH 7.4) in a final volume of 200 μl. After lipids were extracted and separated by TLC, the radioactivity of the [3H]arachidonic acid liberated and [14C]lysophosphatidylcholine generated were determined as described previously (26Akiba S. Hatazawa R. Ono K. Kitatani K. Hayama M. Sato T. Secretory phospholipase A2 mediates cooperative prostaglandin generation by growth factor and cytokine independently of preceding cytosolic phospholipase A2 expression in rat gastric epithelial cells.J. Biol. Chem. 2001; 276: 21854-21862Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 27Akiba S. Mizunaga S. Kume K. Hayama M. Sato T. Involvement of group VI Ca2+-independent phospholipase A2 in protein kinase C-dependent arachidonic acid liberation in zymosan-stimulated P388D1 cells.J. Biol. Chem. 1999; 274: 19906-19912Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), and the enzymatic activity was calculated. RAW264.7 cells (100 mm dishes) were stimulated with oxLDL and washed. The cells were collected and sonicated in buffer A. The lysate (20 μg protein) was solubilized and subjected to SDS-PAGE on a 7.5% gel. The proteins were transferred to a nitrocellulose membrane, and then antibodies against cPLA2 were applied. The bound antibodies were visualized using peroxidase-conjugated secondary antibodies and enhanced chemiluminescence Western blotting detection reagents (Amersham Pharmacia Biotech). RAW264.7 cells or mouse peritoneal macrophages (100 mm dishes) were incubated with cholesterol (10 μM) at 37°C for 12 h. After being washed, the cells were collected and sonicated in a buffer (10 mM EGTA and Tris-HCl, pH 7.7). The lysate was centrifuged at 100,000 g for 30 min at 4°C. The resultant pellet (membrane fraction) was resuspended in the buffer, and the protein concentrations in the sample were adjusted to 1 mg/ml. The sample (60 μl) was treated with MAFP at 37°C for 10 min, and then the ACAT activity in the mixture was determined by incubation with [14C]oleoyl-CoA (40 μM) at 37°C for 10 min in the presence of 1% BSA in a final volume of 100 μl. Lipids were extracted and separated by TLC on a silica gel G plate with petroleum ether/diethyl ether/formic acid (100:25:2.5; v/v/v) as the developing system. The radioactivity of the [14C]cholesteryl oleate formed was measured, and the enzymatic activity was calculated. The incubation of oxLDL (1 mg protein/ml) with DiI (30 μg DiI/mg protein of oxLDL, at 37°C for 3 h) and measurement of its fluorescence were performed as described elsewhere (28Stephan Z.F. Yurachek E.C. Rapid fluorometric assay of LDL receptor activity of DiI-labeled LDL.J. Lipid Res. 1993; 34: 325-330Abstract Full Text PDF PubMed Google Scholar, 29Teupser D. Thiery J. Walli A.K. Seidel D. Determination of LDL- and scavenger-receptor activity in adherent and non-adherent cultured cells with a new single-step fluorometric assay.Biochim. Biophys. Acta. 1996; 1303: 193-198Crossref PubMed Scopus (68) Google Scholar, 30Beppu M. Watanabe M. Sunohara M. Ohishi K. Mishima E. Kawachi H. Fujii M. Kikugawa K. Participation of the arachidonic acid cascade pathway in macrophage binding/uptake of oxidized low density lipoprotein.Biol. Pharm. Bull. 2002; 25: 710-717Crossref PubMed Scopus (13) Google Scholar). RAW264.7 cells or mouse peritoneal macrophages (1 × 106/35 mm dish) were treated with MAFP for 1 h, and then stimulated with DiI-labeled oxLDL (20 or 50 μg/ml) or unlabeled oxLDL (50 μg/ml) as described in the figure legends. After being washed three times with PBS, cells were lysed with 0.1 M NaOH (29Teupser D. Thiery J. Walli A.K. Seidel D. Determination of LDL- and scavenger-receptor activity in adherent and non-adherent cultured cells with a new single-step fluorometric assay.Biochim. Biophys. Acta. 1996; 1303: 193-198Crossref PubMed Scopus (68) Google Scholar), and the solution was neutralized with 0.1 M HCl. The mixture was sonicated, and the protein concentrations in the sample were adjusted to 80 μg/ml (RAW264.7 cells) or 25 μg/ml (peritoneal macrophages). Fluorescence intensity in the sample was measured with a spectrofluorometer (F-2000, Hitachi), with excitation at 524 nm and emission at 567 nm (30Beppu M. Watanabe M. Sunohara M. Ohishi K. Mishima E. Kawachi H. Fujii M. Kikugawa K. Participation of the arachidonic acid cascade pathway in macrophage binding/uptake of oxidized low density lipoprotein.Biol. Pharm. Bull. 2002; 25: 710-717Crossref PubMed Scopus (13) Google Scholar). Values are expressed as the mean ± SEM of 3–5 separate experiments. Student's t-test was used to analyze the significance of differences between two conditions. P < 0.05 was considered statistically significant. As shown in Fig. 1A, stimulation of [3H]oleic acid-labeled RAW264.7 macrophages with oxLDL (50 μg/ml) induced the formation of [3H]cholesteryl oleate time dependently. During the course of the formation, free [3H]oleic acid increased, with a maximum observed 3 h after the stimulation (Fig. 1B), and [3H]oleoyl phosphatidylcholine decreased (Fig. 1C). These findings seem to indicate that the oxLDL-induced production of cholesteryl oleate occurs in parallel with the release of oleic acid resulting from PLA2-mediated hydrolysis of phosphatidylcholine. Therefore, to examine the possible involvement of PLA2 in the cholesteryl oleate formation, the effects of MAFP, a cPLA2 inhibitor, were tested as shown in Fig. 2. Pretreatment with MAFP (2–10 μM) suppressed oxLDL-induced cholesteryl oleate formation as well as oleic acid release in a dose-dependent manner (Fig. 2A, B). Under the conditions, cPLA2 activity in the MAFP-pretreated cells was inhibited dose dependently (Fig. 2C). When [3H]arachidonic acid-labeled RAW264.7 cells were stimulated with oxLDL (50 μg/ml), an increase in cholesteryl arachidonate (3,783 dpm; basal level, 714 dpm; the mean of two separate experiments) was observed and suppressed by MAFP (10 μM) (1,366 dpm; basal level, 634 dpm). Because MAFP is known to inhibit the activity of iPLA2 in addition to that of cPLA2 (24Lio Y.C. Reynolds L.J. Balsinde J. Dennis E.A. Irreversible inhibition of Ca2+-independent phospholipase A2 by methyl arachidonyl fluorophosphonate.Biochim. Biophys. Acta. 1996; 1302: 55-60Crossref PubMed Scopus (209) Google Scholar), we tested this possibility. As shown in Fig. 3C, iPLA2 activity in MAFP- (10 μM) pretreated RAW264.7 cells was also inhibited. Therefore, to clarify whether the suppression by MAFP of oxLDL-induced oleic acid release and cholesteryl oleate formation results from inhibition of iPLA2 activity, the effects of bromoenol lactone, an iPLA2 inhibitor, were also examined. However, Fig. 3 showed that bromoenol lactone (2–10 μM) did not affect oxLDL-induced cholesteryl oleate formation or oleic acid release despite inhibiting iPLA2 activity. Furthermore, under conditions where [3H]oleic acid-labeled RAW264.7 cells were treated with manoalide (2 or 5 μM), an inhibitor for secretory PLA2, the oxLDL- (50 μg/ml) induced formation of cholesteryl oleate (9,625 dpm; basal level, 2,473 dpm; the mean of two separate experiments) was not affected by 2 μM (11,282 dpm;