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• W1970783006 abstract "Lysophosphatidylcholine (lyso-PC) is a product of phosphatidylcholine hydrolysis by phospholipase A2 (PLA2) and is present in cell membranes, oxidized lipoproteins, and atherosclerotic tissues. It has the ability to alter endothelial functions and is regarded as a causal agent in atherogenesis. In this study, the modulation of arachidonate release by lyso-PC in human umbilical vein endothelial cells was examined. Incubation of endothelial cells with lyso-PC resulted in an enhanced release of arachidonate in a time- and concentration-dependent manner. Maximum arachidonate release was observed at 10 min of incubation with 50 μmlyso-PC. Lyso-PC species containing palmitoyl (C16:0) or stearoyl (C18:0) groups elicited the enhancement of arachidonate release, while other lysolipids such as lysophosphatidylethanolamine, lysophosphatidylserine, lysophosphatidylinositol, or lysophosphatidate were relatively ineffective. Lyso-PC-induced arachidonate release was decreased by treatment of cells with PLA2 inhibitors such as para-bromophenacyl bromide and arachidonoyl trifluoromethyl ketone. Furthermore, arachidonate release was attenuated in cells grown in the presence of antisense oligodeoxynucleotides that specifically bind cytosolic PLA2 mRNA. Treatment of cells with lyso-PC resulted in a translocation of PLA2 activity from the cytosolic to the membrane fractions of cells. Lyso-PC induced a rapid influx of Ca2+ from the medium into the cells, with a simultaneous enhancement of protein kinase C (PKC) activity in the membrane fractions. The lyso-PC-induced arachidonate release was attenuated when cells were preincubated with specific inhibitors of PKC (staurosporine and Ro31–8220) or a specific inhibitor of mitogen-activated protein kinase/extracellular regulated kinase kinase (PD098059). Taken together, the results of this study show that lyso-PC caused the elevation of cellular Ca2+ and the activation of PKC, which stimulated cytosolic PLA2 in an indirect manner and resulted in an enhanced release of arachidonate. Lysophosphatidylcholine (lyso-PC) is a product of phosphatidylcholine hydrolysis by phospholipase A2 (PLA2) and is present in cell membranes, oxidized lipoproteins, and atherosclerotic tissues. It has the ability to alter endothelial functions and is regarded as a causal agent in atherogenesis. In this study, the modulation of arachidonate release by lyso-PC in human umbilical vein endothelial cells was examined. Incubation of endothelial cells with lyso-PC resulted in an enhanced release of arachidonate in a time- and concentration-dependent manner. Maximum arachidonate release was observed at 10 min of incubation with 50 μmlyso-PC. Lyso-PC species containing palmitoyl (C16:0) or stearoyl (C18:0) groups elicited the enhancement of arachidonate release, while other lysolipids such as lysophosphatidylethanolamine, lysophosphatidylserine, lysophosphatidylinositol, or lysophosphatidate were relatively ineffective. Lyso-PC-induced arachidonate release was decreased by treatment of cells with PLA2 inhibitors such as para-bromophenacyl bromide and arachidonoyl trifluoromethyl ketone. Furthermore, arachidonate release was attenuated in cells grown in the presence of antisense oligodeoxynucleotides that specifically bind cytosolic PLA2 mRNA. Treatment of cells with lyso-PC resulted in a translocation of PLA2 activity from the cytosolic to the membrane fractions of cells. Lyso-PC induced a rapid influx of Ca2+ from the medium into the cells, with a simultaneous enhancement of protein kinase C (PKC) activity in the membrane fractions. The lyso-PC-induced arachidonate release was attenuated when cells were preincubated with specific inhibitors of PKC (staurosporine and Ro31–8220) or a specific inhibitor of mitogen-activated protein kinase/extracellular regulated kinase kinase (PD098059). Taken together, the results of this study show that lyso-PC caused the elevation of cellular Ca2+ and the activation of PKC, which stimulated cytosolic PLA2 in an indirect manner and resulted in an enhanced release of arachidonate. The release of arachidonate from phospholipids is the rate-limiting step in the synthesis of eicosanoids via the arachidonate cascade (1Smith W.L. Biochem. J. 1989; 259: 315-324Crossref PubMed Scopus (776) Google Scholar). Arachidonate and its metabolites possess diverse biological properties, many of which are related to vascular homeostasis (1Smith W.L. Biochem. J. 1989; 259: 315-324Crossref PubMed Scopus (776) Google Scholar). In endothelial cells, arachidonate is converted to prostacyclin, a potent vasodilator and platelet antiaggregator (2Moncada S. Vane J.R. Pharmacol. Rev. 1979; 30: 293-331Google Scholar). Although different mechanisms have been proposed for the release of arachidonate in mammalian cells, the hydrolysis of the acyl chain at the sn-2 position of glycerophospholipids by phospholipase A2(PLA2) 1The abbreviations used are: PLA2, phospholipase A2; sPLA2, secretory phospholipase A2; cPLA2, cytosolic phospholipase A2; pBPB, para-bromophenacyl bromide; AACOCF3, arachidonoyl trifluoromethyl ketone; PKC, protein kinase C; MAPK, mitogen-activated protein kinase; lyso-PC, lysophosphatidylcholine. is regarded as the primary pathway for this reaction (1Smith W.L. Biochem. J. 1989; 259: 315-324Crossref PubMed Scopus (776) Google Scholar, 3Clark J.D. Schievella A.R. Nalefski E.A. Lin L.-L. J. Lipid Mediat. Cell Signal. 1995; 12: 83-117Crossref PubMed Scopus (425) Google Scholar). In mammalian cells, several forms of PLA2 have been identified. Those that have been purified and well characterized include the “type II” 14-kDa secretory PLA2 (sPLA2) and the “type IV” 85-kDa cytosolic PLA2 (cPLA2) (for reviews, see Refs. 3Clark J.D. Schievella A.R. Nalefski E.A. Lin L.-L. J. Lipid Mediat. Cell Signal. 1995; 12: 83-117Crossref PubMed Scopus (425) Google Scholar, 4Mayer R.J. Marshall L.A. FASEB J. 1993; 7: 339-348Crossref PubMed Scopus (446) Google Scholar, 5Dennis E.A. Trends Biochem. Sci. 1997; 22: 1-2Abstract Full Text PDF PubMed Scopus (758) Google Scholar). These two isoforms are products of distinct genes (5Dennis E.A. Trends Biochem. Sci. 1997; 22: 1-2Abstract Full Text PDF PubMed Scopus (758) Google Scholar) and have different properties. The cPLA2preferentially hydrolyzes phospholipid substrates containing arachidonate at the sn-2 position (6Clark J.D. Lin L.-L. Kriz R.W. Ramesha C.S. Sultzman L.A. Lin A.Y. Milona N. Knopf J.L. Cell. 1991; 65: 1043-1051Abstract Full Text PDF PubMed Scopus (1465) Google Scholar), while sPLA2 does not exhibit any preference with respect to substrate acyl composition. The sPLA2 requires millimolar concentrations of Ca2+ for maximum activity, while cPLA2 contains a calcium-dependent lipid binding domain and requires submicromolar levels for translocation to cellular membranes (6Clark J.D. Lin L.-L. Kriz R.W. Ramesha C.S. Sultzman L.A. Lin A.Y. Milona N. Knopf J.L. Cell. 1991; 65: 1043-1051Abstract Full Text PDF PubMed Scopus (1465) Google Scholar, 7Nalefski E.A. Sultzman L.A. Martin D.M. Kriz R.W. Towler P.S. Knopf J.L. Clark J.D. J. Biol. Chem. 1994; 269: 18239-18249Abstract Full Text PDF PubMed Google Scholar). In stimulated cells, cPLA2activity is enhanced by phosphorylation at serine 505 by mitogen-activated protein kinase (MAPK) (3Clark J.D. Schievella A.R. Nalefski E.A. Lin L.-L. J. Lipid Mediat. Cell Signal. 1995; 12: 83-117Crossref PubMed Scopus (425) Google Scholar, 8Lin L.-L. Wartmann M. Lin A.Y. Knopf J.L. Seth A. Davis R.J. Cell. 1993; 72: 269-278Abstract Full Text PDF PubMed Scopus (1659) Google Scholar). Protein kinase C (PKC) also appears to play a role in the regulation of PLA2activity, although PKC is not thought to directly phosphorylate cPLA2 in vivo (3Clark J.D. Schievella A.R. Nalefski E.A. Lin L.-L. J. Lipid Mediat. Cell Signal. 1995; 12: 83-117Crossref PubMed Scopus (425) Google Scholar, 9Xing M. Firestein B.L. Shen G.H. Insel P.A. J. Clin. Invest. 1997; 99: 805-814Crossref PubMed Scopus (76) Google Scholar). Both isoforms are found in human endothelial cells and have been implicated in arachidonate release and prostacyclin production (10Murakami M. Kudo I. Inoue K. J. Biol. Chem. 1993; 268: 839-844Abstract Full Text PDF PubMed Google Scholar, 11Roshak A. Sathe G. Marshall L.A. J. Biol. Chem. 1994; 269: 25999-26005Abstract Full Text PDF PubMed Google Scholar, 12Lin L.-L. Lin A.Y. Knopf J.L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6147-6151Crossref PubMed Scopus (519) Google Scholar, 13Bartoli F. Lin H.-K. Ghomashchi F. Gelb M.H. Jain M.K. Apitz-Castro R. J. Biol. Chem. 1994; 269: 15625-15630Abstract Full Text PDF PubMed Google Scholar). Lysophosphatidylcholine (lyso-PC) is a product of phosphatidylcholine hydrolysis by PLA2. This lysophospholipid possesses detergent properties at high concentrations (14Weltzien H.U. Biochim. Biophys. Acta. 1979; 559: 259-287Crossref PubMed Scopus (505) Google Scholar) but is quickly metabolized or reacylated within cells (15Chilton F.H. Murphy R.C. J. Biol. Chem. 1986; 261: 7771-7777Abstract Full Text PDF PubMed Google Scholar, 16Sugiura T. Masuzawa Y. Nakagawa Y. Waku K. J. Biol. Chem. 1987; 262: 1199-1205Abstract Full Text PDF PubMed Google Scholar). Lyso-PC is a normal constituent of blood plasma (17Phillips G.B. Proc. Natl. Acad. Sci. U. S. A. 1957; 43: 566-570Crossref PubMed Google Scholar), vascular tissue (18Portman O.W. Alexander M. J. Lipid Res. 1969; 10: 158-165Abstract Full Text PDF PubMed Google Scholar), and lipoproteins (19Steinbrecher U.P. Parthasarathy S. Leake D.S. Witztum J.L. Steinberg D. Proc. Natl. Acad. Sci. U. S. A. 1984; 83: 3883-3887Crossref Scopus (1419) Google Scholar, 20Liu S.-Y. Lu X. Choy S. Dembinski T.C. Hatch G.M. Mymin D. Shen X. Angel A. Choy P.C. Man R.Y.K. Cardiovasc. Res. 1994; 28: 1476-1481Crossref PubMed Scopus (70) Google Scholar), but its levels are greatly elevated in hyperlipidemia (21Rodriguez F. López I.M. Jover E. J. Med. (Westbury). 1987; 18: 153-163PubMed Google Scholar), atherosclerotic tissue (18Portman O.W. Alexander M. J. Lipid Res. 1969; 10: 158-165Abstract Full Text PDF PubMed Google Scholar), oxidized lipoproteins (19Steinbrecher U.P. Parthasarathy S. Leake D.S. Witztum J.L. Steinberg D. Proc. Natl. Acad. Sci. U. S. A. 1984; 83: 3883-3887Crossref Scopus (1419) Google Scholar, 20Liu S.-Y. Lu X. Choy S. Dembinski T.C. Hatch G.M. Mymin D. Shen X. Angel A. Choy P.C. Man R.Y.K. Cardiovasc. Res. 1994; 28: 1476-1481Crossref PubMed Scopus (70) Google Scholar), and ischemic hearts (22Kinnaird A.A.A. Choy P.C. Man R.Y.K. Lipids. 1988; 23: 32-35Crossref PubMed Scopus (44) Google Scholar). A growing body of evidence has implicated lyso-PC in the pathogenesis of cardiovascular diseases. For example, lyso-PC in oxidized low density lipoproteins impairs vascular relaxation (20Liu S.-Y. Lu X. Choy S. Dembinski T.C. Hatch G.M. Mymin D. Shen X. Angel A. Choy P.C. Man R.Y.K. Cardiovasc. Res. 1994; 28: 1476-1481Crossref PubMed Scopus (70) Google Scholar, 23Murohara T. Kugiyama K. Ohgushi M. Sugiyama S. Ohta Y. Yasue H. Am. J. Physiol. 1994; 267: H2441-H2449PubMed Google Scholar, 24Chen L. Liang B. Froese D.E. Liu S. Wong J.T. Tran K. Hatch G.M. Mymin D. Kroeger E.A. Man R.Y.K. Choy P.C. J. Lipid Res. 1997; 38: 192-200Google Scholar) and induces mitogenesis of macrophages (25Sakai M. Miyazaki A. Hakamata H. Sasaki T. Yui S. Yamazaki M. Shichiri M. Horiuchi S. J. Biol. Chem. 1994; 269: 31430-31435Abstract Full Text PDF PubMed Google Scholar). Lyso-PC is chemotactic for monocytes (26Quinn M.T. Parthasarathy S. Steinberg D. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2805-2809Crossref PubMed Scopus (585) Google Scholar) and T lymphocytes (27McMurray H.F. Parthasarathy S. Steinberg D. J. Clin. Invest. 1993; 92: 1004-1008Crossref PubMed Scopus (223) Google Scholar). In endothelial cells, lyso-PC can induce the expression of genes for various growth factors (28Kume N. Gimbrone Jr., M.A. J. Clin. Invest. 1994; 93: 907-911Crossref PubMed Scopus (299) Google Scholar, 29Nakano T. Raines E.W. Abraham J.A. Klagsbrun M. Ross R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1069-1073Crossref PubMed Scopus (123) Google Scholar) and cellular adhesion molecules (30Kume N. Cybulsky M.I. Gimbrone Jr., M.A. J. Clin. Invest. 1992; 90: 1138-1144Crossref PubMed Scopus (724) Google Scholar,31Ochi H. Kume N. Nishi E. Kita T. Circ. Res. 1995; 77: 530-535Crossref PubMed Scopus (30) Google Scholar). The perturbation of vascular endothelial function and recruitment of various cell types to sites of lesion have been implicated as early events in atherogenesis (32Ross R. Nature. 1993; 362: 801-809Crossref PubMed Scopus (10004) Google Scholar, 33Rubanyi G.M. J. Cardiovasc. Pharmacol. 1993; 22: S1-S14Crossref PubMed Scopus (500) Google Scholar). Thus, given its many biological properties, lyso-PC has been postulated to be an important causal agent in inflammation and atherosclerosis (34Vadas P. Pruzanski W. Lab. Invest. 1986; 55: 391-404PubMed Google Scholar, 35Steinberg D.S. Parthasarathy S. Carew T.E. Khoo J.C. N. Engl. J. Med. 1989; 320: 915-924Crossref PubMed Google Scholar). The interactions among phosphatidylcholine, fatty acids, lyso-PC, and sPLA2 have been examined in in vitro kinetic studies (36Bent E.D. Bell J.D. Biochim. Biophys. Acta. 1995; 1254: 349-360Crossref PubMed Scopus (21) Google Scholar). An abrupt increase in PLA2 activity after an initial lag period was observed in these studies. This pattern of activity was attributed to the accumulation of fatty acid and lysophospholipids, which together altered the organization of substrate vesicles (36Bent E.D. Bell J.D. Biochim. Biophys. Acta. 1995; 1254: 349-360Crossref PubMed Scopus (21) Google Scholar). In light of the many biological effects of lyso-PC, we hypothesize that it can modulate PLA2 in intact cells. In the present study, the effects of lyso-PC on the release of arachidonate in endothelial cells was examined. The involvement of Ca2+, PKC, and MAPK in the modulation of PLA2activity was examined. Medium 199 with Hanks' salt andl-glutamine, heat-inactivated fetal calf serum, and other standard culture reagents were obtained from Life Technologies, Inc. Type I collagenase was obtained from Worthington. Endothelial cell growth supplement was obtained from Collaborative Biomedical Products (Bedford, MA). Phorbol 12-myristate 13-acetate, staurosporine, para-bromophenacyl bromide, and all other chemicals were purchased from Sigma. PD098059 was a product of Calbiochem. [5,6,8,11,12,14,15-3H]arachidonate (230.5 Ci/mmol) was obtained from NEN Life Science Products, and 1-stearoyl-2-[1-14C]arachidonoyl-l-3-phosphatidylcholine (55 mCi/mmol) was obtained from Amersham Corp. Arachidonoyl trifluoromethyl ketone (AACOCF3) and H89 were obtained from Biomol Inc. (Plymouth Meeting, PA). Ro31–8220 was a gift from Roche Research Center (Welwyn Garden City, Hertfordshire, United Kingdom). Lysophospholipids and all lipid standards were obtained from Serdary Research Laboratory (London, Ontario, Canada). Thin layer chromatography plates (silica gel G) were products of Fisher. Anti-cPLA2 polyclonal antibody was a generous gift from Drs. J. L. Knopf and L-L. Lin of the Genetics Institute (Boston, MA). Anti-human sPLA2 monoclonal antibody was a product of Upstate Biotechnology Inc. (Lake Placid, NY). Endothelial cells were harvested from human umbilical veins using Type I collagenase as described previously (37Jaffe E.A. Jaffe E.A. Biology of Endothelial Cells. Martinus, Nijhoff, Boston1984: 1-13Google Scholar,38Chan A.C. Tran K. Lipids. 1990; 25: 17-21Crossref PubMed Scopus (50) Google Scholar). The cells were grown in flasks or culture dishes pretreated with 0.2% gelatin, in medium 199 (pH 7.4) supplemented with 25 mm HEPES, 30 μg/ml endothelial cell growth supplement, 90 μg/ml heparin, 10% fetal calf serum, 100 units/ml penicillin, 100 μg/ml streptomycin, and 1.25 μg/ml Fungizone. The cells were subcultured at a 1:3 ratio using 0.05% trypsin to free the cells from the culture ware. Near-confluent cell monolayers from the third passage were used for all experiments. Cells were radiolabeled as described previously (39Tran K. Wong J.T. Lee E. Chan A.C. Choy P.C. Biochem. J. 1996; 319: 385-391Crossref PubMed Scopus (41) Google Scholar). Cell monolayers grown to near-confluence in 35-mm culture dishes were incubated for 20 h with 1 μCi/ml [3H]arachidonate in medium 199 containing 10% fetal calf serum. The cells were washed three times with HEPES-buffered saline (140 mm NaCl, 4 mm KCl, 5.5 mm glucose, 10 mm HEPES, 1.5 mmCaCl2, and 1.0 mm MgCl2, pH 7.4) containing 0.025% (w/v) essentially fatty acid-free bovine serum albumin. Aliquots of lysophospholipids were dissolved in chloroform/methanol (2:1, v/v). The solvent was evaporated under N2, and the lysophospholipid samples were then resuspended in HEPES-buffered saline containing bovine serum albumin. The arachidonate released from cells was determined as described previously (39Tran K. Wong J.T. Lee E. Chan A.C. Choy P.C. Biochem. J. 1996; 319: 385-391Crossref PubMed Scopus (41) Google Scholar). Briefly, the lysophospholipid was added to the cell culture and incubated for the prescribed period. The buffer was then removed and acidified with 50 μl of glacial acetic acid. A 0.8-ml aliquot was used for lipid extraction in a solvent mixture consisting of chloroform/methanol/water (4:3:2, by volume). Oleic acid was added as an internal fatty acid standard. The free fatty acid fraction in the organic phase was resolved by thin layer chromatography in a solvent system consisting of hexane/diethyl ether/acetic acid (70:30:1, v/v). The fatty acid fraction was visualized by iodine vapor, and its radioactivity was determined by liquid scintillation counting. Endothelial cells were cultured on 60-mm plates and incubated with medium 199 containing 100 nm [14C]lyso-PC (57 nCi/nmol) for 15 min. The medium was removed, and the cells were incubated for 15 min with medium 199 (control) or medium 199 containing 10 μmlyso-PC (a 100-fold excess of nonradioactive lyso-PC). The media were subsequently removed, and the cells were dislodged from the culture dish in HEPES-buffered saline. Samples were taken for protein determination or scintillation counting. Immunoblotting analysis of cPLA2 or sPLA2 was performed as described previously (39Tran K. Wong J.T. Lee E. Chan A.C. Choy P.C. Biochem. J. 1996; 319: 385-391Crossref PubMed Scopus (41) Google Scholar). Cell lysates containing approximately 50 μg of protein were subjected to sodium dodecylsulfate, 7.5% polyacrylamide gel electrophoresis. The protein fractions from the gels were transferred to nitrocellulose membranes and then allowed to react with a polyclonal anti-cPLA2 antibody or with an anti-sPLA2antibody. The nitrocellulose membranes were then exposed to a goat anti-rabbit antibody that was coupled to horseradish peroxidase. The cPLA2 or sPLA2 bands were detected on film using a Western blotting detection reagent kit (from Amersham), which yields a fluorescent compound via a reaction catalyzed by the peroxidase. The antisense oligonucleotides for group II PLA2 (ASsA2, 5′-GAT CCT CTG CCA CCC ACA CC-3′) (40Barbour S.E. Dennis E.A. J. Biol. Chem. 1993; 268: 21875-21882Abstract Full Text PDF PubMed Google Scholar) and for cPLA2 (AScA2, 5′-GTA AGG ATC TAT AAA TGA CAT-3′) (11Roshak A. Sathe G. Marshall L.A. J. Biol. Chem. 1994; 269: 25999-26005Abstract Full Text PDF PubMed Google Scholar) with phosphorothioate linkages were synthesized by the University Core DNA Services, University of Calgary (Alberta, Canada). Complementary sense oligomers were used as controls. Seventy-two hours prior to challenge with lyso-PC, the cells were incubated with medium containing 10 μmoligonucleotides. The cells were supplied with fresh medium containing 10 μm oligonucleotides at 24-h intervals thereafter. The presence of oligonucleotides did not affect cell viability or arachidonate labeling. Cells were lysed by sonication in a buffer containing 50 mm Tris-HCl (pH 8.0), 1 mm EDTA, 10 μm leupeptin, 10 μm aprotinin, 20 mm NaF, and 10 mmNa2HPO4. Cell lysates were centrifuged at 100,000 × g for 60 min. The supernatant was designated as the cytosolic fraction, while the pellet was designated as the membrane fraction and resuspended in the buffer described above. PLA2 activity in the subcellular fractions was determined by the hydrolysis of 1-stearoyl-2-[1-14C]arachidonoyl-sn-glycero-3-phosphocholine to yield free radiolabeled arachidonate. The assay mixture contained 50 mm Tris-HCl (pH 8.0), 1.5 mm CaCl2, 0.9 nmol of 1-stearoyl-2-[1-14C]arachidonoyl-sn-glycero-3-phosphocholine (100,000 dpm/assay), and approximately 10 μg of protein in a final volume of 100 μl. The reaction mixtures were incubated at 37 °C for 30 min, and the reactions were terminated by the addition of 1.5 ml of chloroform/methanol (2:1, by volume). Total lipid was extracted, and the radioactivity of the arachidonate released was determined as described above. The amounts of protein in the samples were determined by the bicinchoninic acid method (41Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (18713) Google Scholar). Changes in cytosolic free Ca2+ were monitored using the fluorescent Ca2+ indicator fura-2 as described previously (42Liu K. Massaeli H. Pierce G.N. J. Biol. Chem. 1993; 268: 4145-4151Abstract Full Text PDF PubMed Google Scholar). Briefly, monolayers grown on microscope coverslips were incubated in medium with 5 μm fura-2/AM for 30 min. Fura-2/AM is permeable to cells, and once inside the cells the compound is hydrolyzed by endogenous esterases to yield the cell-impermeable fura-2. The cells on the coverslip were transferred into a cuvette, rinsed with HEPES-buffered saline containing 0.025% bovine serum albumin, and immersed in the same buffer. Fluorescent signals were monitored on a SPEX fluorescence spectrophotometer at the excitation and emission wavelengths of 340 and 380 nm, respectively. Cells were then challenged with lyso-PC or A23187 for 10 min, and the ratio of the fluorescence at the two wavelengths was monitored as an indicator of changes in cytosolic Ca2+ levels. The isosbestic (cross-over) point of fura-2 remained constant during lyso-PC treatment. Cells were sonicated in buffer B (50 mm Tris-HCl, pH 7.5, 5 mm EDTA, 10 mm EGTA, 0.25 m sucrose, 0.3% β-mercaptoethanol, 10 μm benzamidine, 1 mmPMSF, 10 μg/ml leupeptin, and 10 μg/ml aprotinin) and were centrifuged at 1500 × g for 10 min. The supernatants were subjected to ultracentrifugation at 100,000 × gfor 60 min to obtain the soluble and membrane fractions. Approximately 15–30 μg of protein from these fractions were used to determine PKC activity using a PKC assay kit (Amersham), which is based on the incorporation of 32P from [γ-32P]ATP into a PKC-specific substrate peptide. The data were analyzed using a two-tailed independent Student's t test. The level of statistical significance was defined as p < 0.05. To determine the effect of lyso-PC on arachidonate release, human umbilical vein endothelial cells were labeled with [3H]arachidonate in medium 199 containing 10% fetal calf serum for 20 h. The cells were rinsed, and then incubated with HEPES-buffered saline containing 0.025% bovine serum albumin and 0 or 50 μm lyso-PC for various time periods (Fig.1 A). Lyso-PC elicited a time-dependent arachidonate release, which reached a maximum at 10 min of incubation, after which arachidonate release was slightly diminished. A nominal amount of bovine serum albumin was required to bind the arachidonate that is released into the buffer. The optimal concentration of lyso-PC for the induction of arachidonate release was determined at bovine serum albumin concentrations ranging from 0.025 to 0.1% (w/v) (4–16 μm albumin). The effect of lyso-PC on arachidonate release was affected by the albumin concentration (Fig. 1 B). Higher concentrations of lyso-PC was required to elicit a stimulation of arachidonate release at higher albumin concentrations. For example, at 0.025% albumin, the maximal stimulation of arachidonate release was observed at 50 μmlyso-PC. Lyso-PC at this concentration has been found to be nonlethal to endothelial cells (43Su Z. Ling Q. Guo Z.G. Cardioscience. 1995; 6: 31-37PubMed Google Scholar), and we confirmed cell viability under the incubation conditions by the exclusion of trypan blue dye. Hence, these conditions were routinely used in subsequent experiments. Initial experiments on the effect of lyso-PC on arachidonate release were performed using lyso-PC derived from egg lecithin. Since egg lysolecithin contains mainly saturated acyl species, we tested the ability of palmitoyl (C16:0)- and stearoyl (C18:0)-lyso-PC to stimulate arachidonate release. Fig. 2 shows that lyso-PC containing palmitoyl and stearoyl chains induced a high release of arachidonate. To determine if the stimulation of arachidonate release is specific to lyso-PC or if it is a property common to all lysolipids, we tested the effect of other lysophospholipids such as lysophosphatidylethanolamine, lysophosphatidylserine, lysophosphatidylinositol, and lysophosphatidate on arachidonate release. As shown in Fig. 2, lysolipids with head groups other than choline were minimally effective in the stimulation of arachidonate release. Based on these results, lyso-PC containing a palmitoyl (C16:0) chain was used in subsequent experiments. Lyso-PC is an amphiphilic molecule and can incorporate into lipid membranes. Thus, we performed binding studies as described under “Experimental Procedures” to determine the nature of the association of lyso-PC with the endothelial cells. Cells were labeled with [14C]lyso-PC (100 nm, 57 nCi/nmol), followed by incubation with control medium (without lyso-PC) or medium containing 10 μm nonradioactive lyso-PC. The majority of radioactivity remained associated with cells in the presence of excess lyso-PC (results not shown), indicating that the association of lyso-PC with cells is mainly nonspecific. To determine whether the release of arachidonate is mediated by PLA2, we examined the effects of the PLA2 inhibitors para-bromophenacyl bromide (pBPB) and arachidonoyl trifluoromethyl ketone (AACOCF3), the latter of which specifically inhibits the cPLA2 (44Street I.P. Lin H.K. Laliberté F. Ghomashchi F. Wang Z. Perrier H. Tremblay N.M. Huang Z. Weech P.K. Gelb M.H. Biochemistry. 1993; 32: 5935-5940Crossref PubMed Scopus (419) Google Scholar). As shown in TableI, arachidonate release was significantly inhibited in those cells that were preincubated with these inhibitors prior to challenge with lyso-PC. The inhibition of arachidonate release by up to 62% by AACOCF3 indicates that the cPLA2 may be involved in the arachidonate release induced by lyso-PC. However, sPLA2 is also present in endothelial cells and may also participate in arachidonate release (10Murakami M. Kudo I. Inoue K. J. Biol. Chem. 1993; 268: 839-844Abstract Full Text PDF PubMed Google Scholar).Table IInhibition of lyso-PC-induced arachidonate release by PLA 2inhibitorsTreatmentArachidonate releaseInhibitiondpm × 10−3/dish%Control5.98 ± 0.4050 μm lyso-PC31.08 ± 2.35AACOCF3 + lyso-PC 1 μm19.11 ± 0.601-ap < 0.05.39 25 μm11.93 ± 1.551-ap < 0.05.62pBPB + lyso-PC 5 μm22.09 ± 1.791-ap < 0.05.29 25 μm16.44 ± 2.611-ap < 0.05.471-a p < 0.05. Open table in a new tab To further delineate the type of PLA2 that was involved in the lyso-PC-induced arachidonate release, we used antisense oligonucleotides toward cPLA2 and sPLA2. These oligonucleotides were designed to bind specifically to the respective mRNAs and prevent the translation and synthesis of the enzyme protein (11Roshak A. Sathe G. Marshall L.A. J. Biol. Chem. 1994; 269: 25999-26005Abstract Full Text PDF PubMed Google Scholar, 40Barbour S.E. Dennis E.A. J. Biol. Chem. 1993; 268: 21875-21882Abstract Full Text PDF PubMed Google Scholar). Complementary sense oligonucleotides were used as negative controls. Cells were grown in the presence of sense or antisense oligonucleotides to either PLA2 isoform for 3 days prior to challenge with lyso-PC. Treatment of the cells with either sense or antisense oligonucleotides did not alter the total incorporation of [3H]arachidonate. However, lyso-PC-induced arachidonate release was significantly attenuated in cells grown in the presence of antisense oligonucleotides for cPLA2, compared with cells grown without oligonucleotides or with sense oligonucleotides (Fig. 3). The level of cPLA2 protein after the treatment with antisense cPLA2 oligonucleotides was determined by immunoblotting analysis with a polyclonal antibody for cPLA2. The level of cPLA2 protein was decreased (40% reduction) by the antisense oligonucleotide treatment (Fig.4 A). In cells treated with antisense sPLA2 oligonucleotides, the lyso-PC-induced arachidonate release was not significantly affected (Fig. 3), despite a 35% decrease in the sPLA2 protein level in those cells (Fig. 4 B).Figure 4Immunoblots of cPLA2 and sPLA2 in cells grown in the presence of sense or antisense oligonucleotides to cPLA2. Cells were cultured in the absence or presence of 10 μm sense or antisense oligonucleotides to cPLA2 or sPLA2 for 72 h. The levels of cPLA2 (A) or sPLA2(B) in the cell lysates were quantitated by immunoblotting as described under “Experimental Procedures.” A, control, cells cultured without oligonucleotides;ScA2, cells cultured in the presence of sense oligonucleotides for cPLA2; AScA2, cells cultured in the presence of antisense oligonucleotides for cPLA2. B, control, cells cultured without oligonucleotides; SsA2, cells cultured in the presence of sense oligonucleotides for sPLA2;ASsA2, cells cultured in the presence of antisense oligonucleotides for sPLA2.View Large" @default.
• W1970783006 created "2016-06-24" @default.
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• W1970783006 date "1998-03-01" @default.
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• W1970783006 title "Lysophosphatidylcholine Stimulates the Release of Arachidonic Acid in Human Endothelial Cells" @default.
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