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• W2347079605 abstract "Lysophosphatidylcholine (lyso-PC) and arachidonate are products of phosphatidylcholine hydrolysis by phospholipase A2. In this study, the modulation of arachidonate release by exogenous lyso-PC in rat heart myoblastic H9c2 cells was examined. Incubation of H9c2 cells with lyso-PC resulted in an enhanced release of arachidonate in both a time- and dose-dependent fashion. Lyso-PC species containing palmitoyl (C16:0) or stearoyl (C18:0) groups evoked the highest amount of arachidonate release, while other lysophospholipid species were relatively ineffective. Cells treated with phospholipase A2 inhibitors resulted in the attenuation of the enhanced arachidonate release in the presence of lyso-PC. Lyso-PC caused the translocation of phospholipase A2 from the cytosol to the membrane fraction and induced an increase in Ca2+ flux from the medium into the cells. Nimodipine, a specific Ca2+-channel blocker, partially attenuated the lyso-PC-induced rise in intracellular Ca2+. Concurrent with Ca2+ influx, lyso-PC caused an enhancement of protein kinase C activity. The lyso-PC-induced arachidonate release was attenuated when cells were preincubated with specific protein kinase C and mitogen activated protein kinase kinase inhibitors. Taken together, these results strongly indicate that the lyso-PC-induced increases in levels of intracellular calcium and stimulation of protein kinase C lead to the activation of cytosolic phospholipase A2 which results in the enhancement of arachidonate release in H9c2 cells.—Golfman, L. S., N. J. Haughey, J. T. Wong, J. Y. Jiang, D. Lee, J. D. Geiger, and P. C. Choy. Lysophosphatidylcholine induces arachidonic acid release and calcium overload in cardiac myoblastic H9c2 cells. J. Lipid Res. 1999. 40: 1818–1826. Lysophosphatidylcholine (lyso-PC) and arachidonate are products of phosphatidylcholine hydrolysis by phospholipase A2. In this study, the modulation of arachidonate release by exogenous lyso-PC in rat heart myoblastic H9c2 cells was examined. Incubation of H9c2 cells with lyso-PC resulted in an enhanced release of arachidonate in both a time- and dose-dependent fashion. Lyso-PC species containing palmitoyl (C16:0) or stearoyl (C18:0) groups evoked the highest amount of arachidonate release, while other lysophospholipid species were relatively ineffective. Cells treated with phospholipase A2 inhibitors resulted in the attenuation of the enhanced arachidonate release in the presence of lyso-PC. Lyso-PC caused the translocation of phospholipase A2 from the cytosol to the membrane fraction and induced an increase in Ca2+ flux from the medium into the cells. Nimodipine, a specific Ca2+-channel blocker, partially attenuated the lyso-PC-induced rise in intracellular Ca2+. Concurrent with Ca2+ influx, lyso-PC caused an enhancement of protein kinase C activity. The lyso-PC-induced arachidonate release was attenuated when cells were preincubated with specific protein kinase C and mitogen activated protein kinase kinase inhibitors. Taken together, these results strongly indicate that the lyso-PC-induced increases in levels of intracellular calcium and stimulation of protein kinase C lead to the activation of cytosolic phospholipase A2 which results in the enhancement of arachidonate release in H9c2 cells.—Golfman, L. S., N. J. Haughey, J. T. Wong, J. Y. Jiang, D. Lee, J. D. Geiger, and P. C. Choy. Lysophosphatidylcholine induces arachidonic acid release and calcium overload in cardiac myoblastic H9c2 cells. J. Lipid Res. 1999. 40: 1818–1826. The principal pathway for the hydrolysis of phosphatidylcholine is via the action of phospholipase A2 (1Waite M. Phospholipases.in: Vance D.E. Vance J.E. Biochemistry of Lipids, Lipoproteins and Membranes. Elsevier Science Publishers, Amsterdam1991: 269-296Google Scholar, 2Mayer R.J. Marshal L.A. New insights on mammalian phospholipase A2(s): comparison of arachidonoyl-selective and non-selective enzymes.FASEB. J. 1993; 7: 339-348Google Scholar). In mammalian tissues, several major isoforms of phospholipase A2 have been identified. Each form appears to differ from the others in the primary structure, subcellular localization, calcium requirement for activation, and substrate specificity (reviewed in 2–6). Two forms of calcium-dependent phospholipase A2 are prominent in mammalian cells; the type II 14-kDa secretory form (s-form) (7Kudo I. Murakami M. Hara S. Inoue K. Mammalian non-pancreatic phospholipase A2.Biochim. Biophys. Acta. 1993; 1170: 217-231Google Scholar) and the type IV, 85–110 kDa cytosolic form (c-form) (3Leslie C.C. Properties and regulation of cytosolic phospholipase A2.J. Biol. Chem. 1997; 272: 16709-16712Google Scholar, 6Clark J.D. Schievella A.R. Nalefski E.A. Lin L-L Cytosolic phospholipase A2.J. Lipid. Mediat. Cell Signal. 1995; 12: 83-117Google Scholar, 8Sharp J.D. White D.L. Chiou X.G. Goodson T. Gamboa G.C. McClure D. Bugett S. Hoskins J. Skatrud P.L. Sportman J.R. Becker G.W. Kang L.H. Roberts E.F. Kramer R.M. Molecular cloning and expression of human Ca2+-sensitive cytosolic phospholipase A2.J. Biol. Chem. 1991; 266: 14850-14853Google Scholar, 9Dennis E.A. The growing phospholipase A2 superfamily of signal transduction enzymes.Trends Biochem. Sci. 1997; 22: 1-2Google Scholar). In addition to both isoforms of phospholipase A2, a calcium-insensitive 40-kDa phospholipase A2 isoform is present in cardiovascular tissues (9Dennis E.A. The growing phospholipase A2 superfamily of signal transduction enzymes.Trends Biochem. Sci. 1997; 22: 1-2Google Scholar). The c-phospholipase A2 requires that Ca2+ increase to the micromolar range for translocation of the enzyme to the phospholipid-containing membrane, and possesses a high specificity for the arachidonoyl group at the sn-2 position of the phospholipid molecule (9Dennis E.A. The growing phospholipase A2 superfamily of signal transduction enzymes.Trends Biochem. Sci. 1997; 22: 1-2Google Scholar). The s-phospholipase A2 requires that Ca2+ increase to millimolar levels for maximum activity and shows little or no selectivity towards the chain length, number of unsaturated bonds of the acyl group at the sn-2 position (9Dennis E.A. The growing phospholipase A2 superfamily of signal transduction enzymes.Trends Biochem. Sci. 1997; 22: 1-2Google Scholar). The c-phospholipase A2 activity is regulated directly by phosphorylation at serine505 via the mitogen-activated protein kinase (MAPK) (6Clark J.D. Schievella A.R. Nalefski E.A. Lin L-L Cytosolic phospholipase A2.J. Lipid. Mediat. Cell Signal. 1995; 12: 83-117Google Scholar, 9Dennis E.A. The growing phospholipase A2 superfamily of signal transduction enzymes.Trends Biochem. Sci. 1997; 22: 1-2Google Scholar), and indirectly by protein kinase C, but the role of protein kinase C in direct phosphorylation of c-phospholipase A2 in vivo is unclear at present (4Balsinde J. Dennis E.A. Function and inhibition of intracellular calcium-independent phospholipase A2.J. Biol. Chem. 1997; 272: 16069-16072Google Scholar, 5Dennis E.A. Diversity of group types, regulation, and function of phospholipase A2.J. Biol. Chem. 1994; 269: 13057-13060Google Scholar, 6Clark J.D. Schievella A.R. Nalefski E.A. Lin L-L Cytosolic phospholipase A2.J. Lipid. Mediat. Cell Signal. 1995; 12: 83-117Google Scholar). The hydrolysis of phosphatidylcholine by the action of phospholipase A2 results in the production of lysophosphatidylcholine (lyso-PC) and a fatty acid. Due to its amphipathic property, lyso-PC is cytolytic at high concentrations and its intracellular levels are therefore under rigid control (10Weltzien H.U. Cytolytic and membrane-perturbing properties of lysophosphatidylcholine.Biochim. Biophys. Acta. 1979; 559: 259-287Google Scholar). The majority of lyso-PC formed in the tissue is rapidly metabolized or reacylated under normal physiological conditions (11Giffin M.D. Arthur G. Choy P.C. Man R.Y.K. Lysophosphatidylcholine metabolism and cardiac arrhythmias.Can. J. Physiol. Pharmacol. 1988; 66: 185-189Google Scholar, 12Choy P. Arthur G. Phosphatidylcholine biosynthesis from lysophosphatidylcholine.Phosphatidylcholine Metabolism. CRC Press, Boca Raton, FL1989Google Scholar, 13Choy P. Skrzypczak M. Lee D. Jay F.T. Acyl-GPC and alkenyl/alkyl-GPC:acyl-CoA acyltransferases.Biochim. Biophys. Acta. 1997; 1348: 124-133Google Scholar). In ischemic myocardium, lyso-PC accumulates (14Sobel B.E. Corr P.B. Robinson A.K. Goldstein R.A. Witkowski F.X. Klein M.S. Accumulation of lysophosphoglycerides with arrhythmogenic properties in ischemic myocardium.J. Clin. Invest. 1978; 62: 546-553Google Scholar, 15Man R.Y.K. Slater T.L. Pelletier M.P.J. Choy P.C. Alterations of phospholipids in ischemic canine myocardium during acute arrythmias.Lipids. 1983; 18: 677-681Google Scholar, 16Datorre S.D. Creer M.H. Pogwizd S.M. Corr P.B. Amphipathic lipid metabolites and their relation to arrhythmogenesis in the ischemic heart.J. Mol. Cell. Cardiol. 1991; 23 (Suppl. I): 11-22Google Scholar) and leads to electrophysiological and mechanical dysfunction of the heart (17Hoque A.N.E. Haist J.V. Karmazyn M. Na+-H+ exchange inhibition protects against mechanical, ultrastructural, and biochemical impairment induced by low concentrations of lysophosphatidylcholine in isolated rat hearts.Circ. Res. 1997; 80: 95-102Google Scholar, 18Woodley S.L. Ikenouchi H. Barry W.H. Lysophosphatidylcholine increases cytosolic calcium in ventricular myocytes by direct action on the sarcolemma.J. Mol. Cell. Cardiol. 1991; 23: 671-680Google Scholar). A relationship between high lyso-PC concentrations and calcium overloading in cardiac tissues has been postulated (19Ver Donck L. Verellen G. Geerts V. Borgers M. Lysophosphatidylcholine-induced Ca2+-overload in isolated cardiomyocytes and effect of cytoprotective drugs.J. Mol. Cell. Cardiol. 1992; 24: 977-988Google Scholar, 20Yu L. Netticadan T. Xu Y-J. Panagia V. Dhalla N.S. Mechanisms of lysophosphatidylcholine-induced increase in intracellular calcium in rat cardiomyocytes.J. Pharmacol. Exp. Ther. 1998; 286: 1-8Google Scholar, 21Golfman L.S. Hata T. Netticadan T. Panagia V. Dhalla N.S. Modification of cardiac sarcolemmal Na+-Ca2+ exchange activity by lysophosphatidylcholine and palmitoylcarnitine.Cardiovasc. Pathobiol. 1998; 2: 181-185Google Scholar), but the mechanism for this phenomenon has been subjected to much debate (20Yu L. Netticadan T. Xu Y-J. Panagia V. Dhalla N.S. Mechanisms of lysophosphatidylcholine-induced increase in intracellular calcium in rat cardiomyocytes.J. Pharmacol. Exp. Ther. 1998; 286: 1-8Google Scholar, 21Golfman L.S. Hata T. Netticadan T. Panagia V. Dhalla N.S. Modification of cardiac sarcolemmal Na+-Ca2+ exchange activity by lysophosphatidylcholine and palmitoylcarnitine.Cardiovasc. Pathobiol. 1998; 2: 181-185Google Scholar, 22Ahumada G.C. Bergmann S.R. Carlson E. Corr P.B. Sobel B.E. Augmentation of cyclic AMP content induced by lysophosphatidylcholine in rabbit hearts.Cardiovasc. Res. 1979; 13: 377-382Google Scholar, 23Watanabe A.M. Besch Jr., H.R. Cyclic adenosine monophosphate modulation of slow calcium influx channels in guinea pig hearts.Circ. Res. 1974; 35: 316-318Google Scholar). In plasma, the concentration of lyso-PC is normally low (24Phillips G.B. The isolation of lysolecithin from human serum.Proc. Natl. Acad. Sci. USA. 1957; 43: 566-570Google Scholar) but high amounts of lyso-PC are found in atherogenic lipoproteins such as the oxidatively modified low density lipoprotein and β-very low density lipoprotein (25Portman O.W. Alexander M. Lysophosphatidylcholine concentrations and metabolism in aortic intima plus inner media: effect of nutritionally induced atherosclerosis.J. Lipid Res. 1969; 10: 158-165Google Scholar, 26Steinberg D. Parthasarathy S. Carew T.E. Khoo J.C. Beyond cholesterol: modifications of low-density lipoprotein that increase its atherogenicity.N. Engl. J. Med. 1989; 320: 915-924Google Scholar). In the last decade, the accumulation of lyso-PC in atherosclerotic and inflammatory lesions of vascular vessels has been reported (14Sobel B.E. Corr P.B. Robinson A.K. Goldstein R.A. Witkowski F.X. Klein M.S. Accumulation of lysophosphoglycerides with arrhythmogenic properties in ischemic myocardium.J. Clin. Invest. 1978; 62: 546-553Google Scholar, 27Witztum J.L. Steinberg D. Role of oxidized low-density lipoprotein in atherogenesis.J. Clin. Invest. 1991; 88: 1785-1792Google Scholar). Furthermore, lyso-PC induces the expression of mononuclear leukocyte adhesion molecules (28Kume K. Cybulsky M.I. Gimbrone Jr., M.A. Lysophosphatidylcholine, a component of atherogenic lipoproteins, induces mononuclear leukocyte adhesion molecules in cultured human and rabbit arterial endothelial cells.J. Clin. Invest. 1992; 90: 1138-1144Google Scholar), gene expression of potent smooth muscle growth factors in monocytes and in cultured human endothelial cells (29Kume N. Gimbrone M.A. Lysophosphatidylcholine tracscriptionally induces growth factor gene expression in cultured human endothelial cells.J. Clin. Invest. 1994; 93: 907-911Google Scholar), modulates smooth muscle contractility (30Saito T. Wolf A. Menon N.K. Saeed M. Bing R.J. Lysolecithins as endothelium-dependent vascular smooth muscle relaxants that differ from endothelium-derived relaxing factor.Proc. Natl. Acad. Sci. USA. 1988; 85: 8246-8250Google Scholar), and acts as a chemotactic factor for human T lymphocytes (31McMurray H.F. Parthasarathy S. Steinberg D. Oxidatively modified low density lipoprotein is a chemoattractant for human T lymphocytes.J. Clin. Invest. 1993; 92: 1004-1008Google Scholar) and monocytes (32Quinn M.T. Parthasarathy S. Steinberg D. Lysophosphatidylcholine: a chemotactic factor for human monocytes and its potential role in atherogenesis.Proc. Natl. Acad. Sci. USA. 1988; 85: 2805-2809Google Scholar). It is clear that lyso-PC produced in the plasma may be an important signal molecule that impairs endothelium-dependent relaxation of blood vessels (27Witztum J.L. Steinberg D. Role of oxidized low-density lipoprotein in atherogenesis.J. Clin. Invest. 1991; 88: 1785-1792Google Scholar). In addition to lyso-PC, free fatty acids are produced from the hydrolysis of phosphatidylcholine by phospholipase A2. The release of arachidonic acid from phospholipids is regarded as an important step for the biosynthesis of eicosanoids (33Dennis E.A. The regulation of eicosanoid production: role of phospholipases and inhibitors.Biol. Technol. 1987; 5: 1294-1299Google Scholar). In most mammalian tissues including the cardiac tissue, arachidonate is converted to prostacyclin, a potent vasodilator which would attenuate the impairment of endothelium-dependent relaxation of blood vessels produced by lyso-PC. In view of the antagonistic effect of lyso-PC and arachidonic acid on endothelium-dependent relaxation of blood vessels, the primary objective of this study was to determine the effects of lyso-PC on arachidonic acid release in non-vascular cells. The H9c2 cell line was selected as a model for this study because it was derived from the rat heart but retained many of the properties of the skeletal muscle (34Kimes B.W. Brandt B.L. Properties of a clonal muscle cell line from rat heart.Exp. Cell Res. 1976; 98: 367-381Google Scholar). Secondary objectives of this study were to determine the involvement of Ca2+, protein kinase C, and mitogen-activated protein kinase in the modulation of c-phospholipase A2 activity. Dulbecco's modified Eagle's medium and Dulbecco's phosphate-buffered saline were obtained from Sigma Chemical Company (St. Louis, MO). Heat-inactivated newborn calf serum and trypsin were obtained from Life Technologies, Inc. Staurosporine, H-89, arachidonyl trifluoromethyl ketone (AACOCF3), PD098059, and bisindolylmaleimide I were products of Biomol Inc. (Plymouth Meeting, PA). Ro31-8220 was purchased from Calbiochem-Novabiochem Corp. (La Jolla, CA) and para-bromophenacyl bromide and all other chemicals were reagent grade and were purchased from Sigma. [5,6,8,9,11,12,14,15-3H]arachidonic acid (210 Ci/mmol), adenosine 5′-[γ-32P]triphosphate (3,000 Ci/mmol), 1-stearoyl-2-[1-14C]arachidonyl-l-3-phosphatidylcholine (55 mCi/mmol) and 1-[1-14C]palmitoyl-l-lyso-3-phosphatidylcholine (55 mCi/mmol) were all obtained from Amersham Corp. Lysophospholipids and all lipid standards were purchased from Serdary Research Laboratory (London, Ontario, Canada). Thin-layer chromatography plates (silica gel G) were the product of Fisher Scientific. Rat heart myoblast (H9c2) cells were obtained from ATCC and cultured according to the company's guidelines. The cells were grown in flasks or culture dishes in Dulbecco's modified Eagle's medium supplemented with 10% bovine serum albumin, 100 units/ml penicillin, 100 μg/ml streptomycin, and 1.25 μg/ml fungizone. Cells 85–90% confluent were used for all subsequent experiments. Cells were radiolabeled (35Wong J.T. Tran K. Pierce G.N. Chan A.C. O K. Choy P.C. Lysophosphatidylcholine stimulates the release of arachidonic acid in human endothelial cells.J. Biol. Chem. 1998; 273: 6830-6836Google Scholar) in 35-mm culture dishes and were incubated for 16–20 h with 1 μCi/ml [3H]arachidonate in Dulbecco's modified Eagle's medium containing 1% bovine serum albumin. The cells were washed 3 times with HEPES-buffered saline containing 140 mm NaCl, 4 mm KCl, 5.5 mm glucose, 10 mm HEPES, 1.5 mm CaCl2, and 1.0 mm MgCl2, pH 7.4, and 0.1% (w/v) essentially fatty acid-free bovine serum albumin. Aliquots of lysophospholipids were dissolved in chloroform–methanol 2:1 (v/v), evaporated under N2, and the lysophospholipid samples were resuspended in HEPES-buffered saline containing bovine serum albumin. The binding of lysophospholipid to H9c2 cells was studied in the following manner. Cells were cultured on 60-mm plates with Dulbecco's modified Eagle's medium containing 100 nm [14C]lyso-PC (55 nCi/nmol) for 15 min. Subsequently, the medium was removed and cells were incubated for another 15 min with the same medium containing 10 μm non-radioactive lysophospholipid (100 times excess) or 0.1% bovine serum albumin. After the second incubation, the cells were dislodged from the culture dish in HEPES-buffered saline. The labeled lysophospholipid content in each dish was determined by scintillation counting. The arachidonate released from the cells was determined as described previously (36Lin L-L. Wartmann M. Lin A.Y. Knopf J.L. Seth A. Davis R.J. cPhospholipase A2 is phosphorylated and activated by MAP kinase.Cell. 1993; 72: 269-278Google 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. The fatty acid fraction was visualized by iodine vapor, and its radioactivity was determined by liquid scintillation counting. 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 mm Na2HPO4. Cell lysates were centrifuged at 100,000 g for 1 h. The supernatant was designated as the cytosolic fraction, while the pellet was designated as the membrane fraction and was resuspended in the buffer described above. Phospholipase A2 activity in the subcellular fractions was determined by the hydrolysis of 1-stearoyl-2-[1-14C] arachidonyl-sn-glycero-3-phosphocholine to yield free radiolabeled arachidonate (35Wong J.T. Tran K. Pierce G.N. Chan A.C. O K. Choy P.C. Lysophosphatidylcholine stimulates the release of arachidonic acid in human endothelial cells.J. Biol. Chem. 1998; 273: 6830-6836Google Scholar). The assay mixture contained 50 mm Tris-HCl (pH 8.0), 1.5 mm CaCl2, 0.9 nmol of 1-stearoyl-2-[1-14C]arachidonyl-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. Total lipid was extracted and the radioactivity of arachidonate released was determined as described above. The amounts of protein in samples were determined by the bicinchoninic acid method. H9c2 cells were plated on poly-d-lysine-coated 35-mm glass cover slips 3–4 days prior to experimentation. [Ca2+]i levels were determined using the Ca2+-specific fluorescent probe Fura-2/AM. Cells were incubated for 40 min at 25°C followed by 10 min at 37°C in HEPES-buffered saline buffer containing 1.2 mm Ca2+, 0.1% BSA, and 2 μm Fura-2/AM. The cover slips containing Fura-2-loaded cells were placed in a PDMI-2 open perfusion micro-incubator (Medical Microsystems Corp., Greenvale, NY) set at 37°C. Cells were superfused at a rate of 2 ml/min with HEPES-buffered saline during baseline measurements. After 5 min, flow was stopped and buffer was removed and replaced with HEPES-buffered saline containing lyso-PC (25–150 μm). Cells were alternately excited at 340 and 380 nm, and emission was recorded at 510 nm with a video-based Universal Imaging System (EMPIX, Mississauga, ON). Rmax/Rmin ratios were converted to nanomolar [Ca2+]i according to the method of Grynkiewicz, Poenie, and Tsien (37Grynkiewicz G. Poenie M. Tsien R.Y. A new generation of Ca2+ indicators with greatly improved fluorescence properties.J. Biol. Chem. 1985; 260: 3440-3450Google Scholar). Images were acquired every 15 sec during baseline measurements and every 5 sec after lyso-PC additions by real-time averaging of 16 frames of each wavelength that included a background reference subtraction from each of the acquired images. Increases in [Ca2+]i represent average cytosolic concentrations determined by subtracting [Ca2+]i levels at 5, 10, and 15 min after lyso-PC applications from baseline [Ca2+]i. All cells in the visual field were monitored for time periods up to 30 min. 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 mm PMSF, 10 μg/ml leupeptin, and 10 μg/ml aprotinin) and were centrifuged at 1,500 g for 10 min. The supernatants were subjected to ultracentrifugation at 100,000 g for 1 h to obtain soluble and membrane fractions. Approximately 15–30 μg of protein from these fractions was 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 with a two-tailed independent Student's t test or, where appropriate, a one-way analysis of variance (ANOVA) followed by Duncan's new multiple range post hoc test to detect individual differences. The level of significance was determined at P < 0.05. All values represent mean ± standard error of the mean. The effect of lyso-PC on arachidonate release in H9c2 cells was examined. Cells were prelabeled with [3H]arachidonate and then incubated with HEPES-buffered saline containing 0.1% bovine serum albumin and 0 or 150 μm lyso-PC for various time periods (Fig. 1A). Lyso-PC elicited a time-dependent enhancement of arachidonate release, which reached a maximum at 15 min of incubation. The enhancement of arachidonate release was slightly diminished after the first 15 min. The presence of 100 μm lyso-PC resulted in optimal arachidonate release (Fig. 1B) irrespective of the bovine serum albumin concentration (0.025–0.1% w/v) (4–16 μm). Hence, 100 μm lyso-PC in a medium containing 0.1% bovine serum albumin was used in all subsequent studies. Cell viability was confirmed by trypan blue exclusion that showed minimal dye infiltration under the conditions described above. Lyso-PC is an amphiphilic molecule which can be incorporated into lipid membranes. Thus, we performed binding studies to determine the nature of the association of lyso-PC with the H9c2 cells. These cells were labeled with 1-[1-14C]palmitoyl-lyso-PC (100 nm, 55 nCi/nmol) for 15 min, and a considerable amount (45 ± 7%) of radioactivity was found to be associated with cells after the incubation. When lipids were extracted from these cells and analyzed by thin-layer chromatography, the majority of the radioactivity (>90%) in the lipid extract was found in the lyso-PC fraction. Subsequently, these cells were incubated for another 15 min in the control medium (without lyso-PC) or medium containing 10 μm non-radioactive lyso-PC. The majority of the radioactivity (70–76%) associated with the cells was not removed by either treatment. However, more than two-thirds (67–74%) of the radioactivity was removed from these cells by incubation with medium containing 0.1% albumin. Taken together, our data indicate that the binding of lyso-PC to cells was nonspecific and was not significantly metabolized within 15 min of incubation. As egg lysolecithin contains mainly saturated species of lyso-PC, the ability of myristoyl-(C14:0), palmitoyl (C16:0)-, stearoyl (C18:0)-lyso-PC as well as several unsaturated lyso-PC species (i.e., C18:1, C18:2, and C18:3) to stimulate arachidonate release were investigated. As depicted in Fig. 2, lyso-PC containing palmitoyl and stearoyl chains caused the highest amount of arachidonate release by these cells. The specificity of other lysophospholipids for the stimulation of arachidonate release in H9c2 cells was also examined. Cells were incubated with 100 μm of lysophosphatidylethanolamine, lysophosphatidylserine, lysophosphatidylinositol, or lysophosphatidate under identical conditions and the arachidonic acid released was determined. As indicated in Fig. 2, lysophospholipids with head groups other than choline were minimally effective in the stimulation of arachidonate release. Based on these results, lyso-PC containing palmitoyl (C16:0) chain was used in subsequent experiments. In order to determine whether the enhanced release of arachidonate is mediated via phospholipase A2, the effect of lyso-PC on arachidonate release in H9c2 cells was studied in the presence of arachidonoyl trifluoromethyl ketone (AACOCF3), a specific inhibitor of c-phospholipase A2 (6Clark J.D. Schievella A.R. Nalefski E.A. Lin L-L Cytosolic phospholipase A2.J. Lipid. Mediat. Cell Signal. 1995; 12: 83-117Google Scholar, 9Dennis E.A. The growing phospholipase A2 superfamily of signal transduction enzymes.Trends Biochem. Sci. 1997; 22: 1-2Google Scholar). As shown in Table 1, cells pre-incubated with AACOCF3 at concentrations of 0.5, 5.0, and 25 μm significantly reduced arachidonate release when compared to control cells. Our results indicate that c-phospholipase A2 is a major phospholipase involved in lyso-PC-induced arachidonate release in the H9c2 cells.TABLE 1.Effect of arachidonoyl trifluoromethyl ketone (AACOCF3) on arachidonate release in H9c2 cellsTreatmentArachidonate ReleaseInhibitiondpm/dish (×10−2)%Control10 ± 1.1Lyso-PC132 ± 2.3AACOCF3 + Lyso-PC0.5 μm75 ± 2.2aP < 0.05.435 μm69 ± 1.9aP < 0.05.4825 μm54 ± 2.4aP < 0.05.59Cells were prelabeled with [3H]arachidonate and incubated with the indicated concentrations of AACOCF3 for 2 min prior to challenge with 120 μm lyso-PC for 15 min. The release of arachidonate into the medium was determined. Results are expressed as mean ± standard error of the mean from 5 separate experiments.a P < 0.05. Open table in a new tab Cells were prelabeled with [3H]arachidonate and incubated with the indicated concentrations of AACOCF3 for 2 min prior to challenge with 120 μm lyso-PC for 15 min. The release of arachidonate into the medium was determined. Results are expressed as mean ± standard error of the mean from 5 separate experiments. To determine the mechanism of enzyme activation by lyso-PC, the enzyme activities in the cystosolic and membrane fractions were assayed. Direct addition of lyso-PC to the in vitro assay of phospholipase A2 activity did not cause any significant changes in enzyme activity (data not shown). When enzyme activity was assayed in subcellular fractions prepared from cells incubated with lyso-PC, enzyme activity was decreased by 40% in cytosolic fractions and increased by 47% in membrane fractions (Table 2). These results are consistent with lyso-PC-induced translocation of the enzyme from the cytosol to the membrane where it is in its most active form (6Clark J.D. Schievella A.R. Nalefski E.A. Lin L-L Cytosolic phospholipase A2.J. Lipid. Mediat. Cell Signal. 1995; 12: 83-117Google Scholar).TABLE 2.Effect of lyso-PC on c-phospholipase A2 activity in H9c2 cellsc-Phospholipase A2 ActivityTreatmentCytosolMembranepmol/min/mgControl (without lyso-PC)8.2 ± 0.11.2 ± 0.1Lyso-PC (100 μm)4.9 ± 0.2aP < 0.05.1.8 ± 0.1aP < 0.05.Cells were treated with or without 100 μm lyso-PC in HEPES-buffered saline containing 0.1% bovine serum albumin. Cells were lysed and phospholipase A2 activity was assayed in the cytosolic and membrane fractions. Results are expressed as mean ± standard error of the mean from 4 separate experiments.a P < 0.05. Open table in a new tab Cells were treated with or without 100 μm lyso-PC in HEPES-buffered saline containing 0.1% bovine serum albumin. Cells were lysed and phospholipase A2 activity was assayed in the cytosolic and membrane fractions. Results are expressed as mean ± standard error of the mean from 4 separate experiments. Enzyme phosphorylation has been shown to play a major role in the regulation of phospholipase A2 activity in a number of cell types (6Clark J.D. Schievella A.R. Nalefski E.A. Lin L-L Cytosolic phospholipase A2.J. Lipid. Mediat. Cell Signal. 1995; 12: 83-117Google Scholar, 38Qui Z-H. Leslie C.C. Protein kinase C-dependent and -i" @default.
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