Long-chain acyl-CoA synthetase 4 modulates prostaglandin E2 release from human arterial smooth muscle cells

Long-chain acyl-CoA synthetases (ACSLs) catalyze the thioesterification of long-chain FAs into their acyl-CoA derivatives. Purified ACSL4 is an arachidonic acid (20:4)-preferring ACSL isoform, and ACSL4 is therefore a probable regulator of lipid mediator production in intact cells. Eicosanoids play important roles in vascular homeostasis and disease, yet the role of ACSL4 in vascular cells is largely unknown. In the present study, the ACSL4 splice variant expressed in human arterial smooth muscle cells (SMCs) was identified as variant 1. To investigate the function of ACSL4 in SMCs, ACSL4 variant 1 was overexpressed, knocked-down by small interfering RNA, or its enzymatic activity acutely inhibited in these cells. Overexpression of ACSL4 resulted in a markedly increased synthesis of arachidonoyl-CoA, increased 20:4 incorporation into phosphatidylethanolamine, phosphatidylinositol, and triacylglycerol, and reduced cellular levels of unesterified 20:4. Accordingly, secretion of prostaglandin E2 (PGE2) was blunted in ACSL4-overexpressing SMCs compared with controls. Conversely, acute pharmacological inhibition of ACSL4 activity resulted in increased release of PGE2. However, long-term downregulation of ACSL4 resulted in markedly reduced PGE2 secretion. Thus, ACSL4 modulates PGE2 release from human SMCs. ACSL4 may regulate a number of processes dependent on the release of arachidonic acid-derived lipid mediators in the arterial wall. Long-chain acyl-CoA synthetases (ACSLs) catalyze the thioesterification of long-chain FAs into their acyl-CoA derivatives. Purified ACSL4 is an arachidonic acid (20:4)-preferring ACSL isoform, and ACSL4 is therefore a probable regulator of lipid mediator production in intact cells. Eicosanoids play important roles in vascular homeostasis and disease, yet the role of ACSL4 in vascular cells is largely unknown. In the present study, the ACSL4 splice variant expressed in human arterial smooth muscle cells (SMCs) was identified as variant 1. To investigate the function of ACSL4 in SMCs, ACSL4 variant 1 was overexpressed, knocked-down by small interfering RNA, or its enzymatic activity acutely inhibited in these cells. Overexpression of ACSL4 resulted in a markedly increased synthesis of arachidonoyl-CoA, increased 20:4 incorporation into phosphatidylethanolamine, phosphatidylinositol, and triacylglycerol, and reduced cellular levels of unesterified 20:4. Accordingly, secretion of prostaglandin E2 (PGE2) was blunted in ACSL4-overexpressing SMCs compared with controls. Conversely, acute pharmacological inhibition of ACSL4 activity resulted in increased release of PGE2. However, long-term downregulation of ACSL4 resulted in markedly reduced PGE2 secretion. Thus, ACSL4 modulates PGE2 release from human SMCs. ACSL4 may regulate a number of processes dependent on the release of arachidonic acid-derived lipid mediators in the arterial wall. Arachidonic acid (20:4) is an omega-6 FA with a plethora of effects in vascular cells due to its processing by cyclooxygenases (COX-1 and COX-2), lipoxygenases, or cytochrome P450 pathways into lipid mediators with diverse biological activities, such as prostaglandin E2 (PGE2), prostacyclin, leukotrienes, and thromboxanes (1Natarajan R. Nadler J.L. Lipid inflammatory mediators in diabetic vascular disease.Arterioscler. Thromb. Vasc. Biol. 2004; 24: 1542-1548Crossref PubMed Scopus (191) Google Scholar, 2Smyth E.M. Grosser T. Wang M. Yu Y. FitzGerald G.A. Prostanoids in health and disease.J. Lipid Res. 2009; 50: 423-428Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar). These lipid mediators are crucial regulators of vascular homeostasis, but also play important roles in vascular disease, including atherosclerosis. For example, studies in mouse models have demonstrated that prostacyclin exerts athero-protective effects, whereas PGE2, thromboxane A2, and leukotriene B4 promote atherosclerosis (3Wang M. Song W.L. Cheng Y. Fitzgerald G.A. Microsomal prostaglandin E synthase-1 inhibition in cardiovascular inflammatory disease.J. Intern. Med. 2008; 263: 500-505Crossref PubMed Scopus (55) Google Scholar, 4Bäck M. Hansson G.K. Leukotriene receptors in atherosclerosis.Ann. Med. 2006; 38: 493-502Crossref PubMed Scopus (91) Google Scholar, 5Kobayashi T. Tahara Y. Matsumoto M. Iguchi M. Sano H. Murayama T. Arai H. Oida H. Yurugi-Kobayashi T. Yamashita J.K. Roles of thromboxane A(2) and prostacyclin in the development of atherosclerosis in apoE-deficient mice.J. Clin. Invest. 2004; 114: 784-794Crossref PubMed Scopus (336) Google Scholar, 6Wang M. Zukas A.M. Hui Y. Ricciotti E. Puré E. FitzGerald G.A. Deletion of microsomal prostaglandin E synthase-1 augments prostacyclin and retards atherogenesis.Proc. Natl. Acad. Sci. USA. 2006; 103: 14507-14512Crossref PubMed Scopus (174) Google Scholar). Smooth muscle cells (SMCs) contribute to atherosclerosis through increased accumulation in the developing lesion, and later by forming a fibrous cap covering advanced lesions (7Doran A.C. Meller N. McNamara C.A. Role of smooth muscle cells in the initiation and early progression of atherosclerosis.Arterioscler. Thromb. Vasc. Biol. 2008; 28: 812-819Crossref PubMed Scopus (629) Google Scholar), and are believed to be an important source of 20:4-derived lipid mediators in the vascular wall. As in other cells, release of 20:4-derived lipid mediators from human arterial SMCs is dependent on liberation of 20:4 from membrane phospholipids by activation of phospholipase A2 through the action of, e.g., growth factors (8Graves L.M. Bornfeldt K.E. Sidhu J.S. Argast G.M. Raines E.W. Ross R. Leslie C.C. Krebs E.G. Platelet-derived growth factor stimulates protein kinase A through a mitogen-activated protein kinase-dependent pathway in human arterial smooth muscle cells.J. Biol. Chem. 1996; 271: 505-511Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar) and cytokines. Free 20:4 is then converted by COX-1/COX-2, or lipoxygenases to downstream lipid mediators, including PGE2, a principal prostaglandin species secreted from human SMCs (9Libby P. Warner S.J. Friedman G.B. Interleukin 1: a mitogen for human vascular smooth muscle cells that induces the release of growth-inhibitory prostanoids.J. Clin. Invest. 1988; 81: 487-498Crossref PubMed Scopus (417) Google Scholar, 10Bornfeldt K.E. Campbell J.S. Koyama H. Argast G.M. Leslie C.C. Raines E.W. Krebs E.G. Ross R. The mitogen-activated protein kinase pathway can mediate growth inhibition and proliferation in smooth muscle cells. Dependence on the availability of downstream targets.J. Clin. Invest. 1997; 100: 875-885Crossref PubMed Scopus (144) Google Scholar). However, little is known about the upstream events regulating 20:4 incorporation into phospholipids in SMCs. Incorporation of free 20:4 into phospholipids requires thioesterification of free 20:4 by enzymes belonging to the group of long-chain acyl-CoA synthetases. These enzymes produce acyl-CoAs from FAs >12 carbons in length. There are five long-chain acyl-CoA synthetase (ACSL) isoforms in humans and rodents: ACSL1, ACSL3, ACSL4, ACSL5, and ACSL6 (11Coleman R.A. Lewin T.M. Van Horn C.G. Gonzalez-Baró M.R. Do long-chain acyl-CoA synthetases regulate fatty acid entry into synthetic versus degradative pathways?.J. Nutr. 2002; 132: 2123-2126Crossref PubMed Scopus (248) Google Scholar, 12Lewin T.M. Kim J.H. Granger D.A. Vance J.E. Coleman R.A. Acyl-CoA synthetase isoforms 1, 4, and 5 are present in different subcellular membranes in rat liver and can be inhibited independently.J. Biol. Chem. 2001; 276: 24674-24679Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 13Watkins P.A. Maiguel D. Jia Z. Pevsner J. Evidence for 26 distinct acyl-coenzyme A synthetase genes in the human genome.J. Lipid Res. 2007; 48: 2736-2750Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). On the basis of data generated with purified or recombinant ACSL enzymes, these isoforms have different FA preferences and different tissue expression. Recombinant ACSL4 exhibits a high preference for 20:4 and omega-3 FAs (fish oils), and lower activity with saturated, mono-, di-, and trisaturated 12–18-carbon FAs (14Kang M.J. Fujino T. Sasano H. Minekura H. Yabuki N. Nagura H. Iijima H. Yamamoto T.T. A novel arachidonate-preferring acyl-CoA synthetase is present in steroidogenic cells of the rat adrenal, ovary, and testis.Proc. Natl. Acad. Sci. USA. 1997; 94: 2880-2884Crossref PubMed Scopus (207) Google Scholar). In humans, ACSL4 undergoes alternative splicing, producing a shorter isoform (variant 1) and a longer isoform (variant 2) containing an additional 41-amino acid N-terminal tail (15Meloni I. Muscettola M. Raynaud M. Longo I. Bruttini M. Moizard M.P. Gomot M. Chelly J. des Portes V. Fryns J.P. FACL4, encoding fatty acid-CoA ligase 4, is mutated in nonspecific X-linked mental retardation.Nat. Genet. 2002; 30: 436-440Crossref PubMed Scopus (124) Google Scholar, 16Mashek D.G. Bornfeldt K.E. Coleman R.A. Berger J. Bernlohr D.A. Black P. DiRusso C.C. Farber S.A. Guo W. Hashimoto N. Revised nomenclature for the mammalian long-chain acyl-CoA synthetase gene family.J. Lipid Res. 2004; 45: 1958-1961Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). We have previously shown that ACSL4 is one of the ACSL isoforms expressed in human arterial SMCs (17Askari B. Kanter J.E. Sherrid A.M. Golej D.L. Bender A.T. Liu J. Hsueh W.A. Beavo J.A. Coleman R.A. Bornfeldt K.E. Rosiglitazone inhibits acyl-CoA synthetase activity and fatty acid partitioning to diacylglycerol and triacylglycerol via a peroxisome proliferator-activated receptor-gamma-independent mechanism in human arterial smooth muscle cells and macrophages.Diabetes. 2007; 56: 1143-1152Crossref PubMed Scopus (56) Google Scholar). These cells also express ACSL1, ACSL3, and ACSL5 (17Askari B. Kanter J.E. Sherrid A.M. Golej D.L. Bender A.T. Liu J. Hsueh W.A. Beavo J.A. Coleman R.A. Bornfeldt K.E. Rosiglitazone inhibits acyl-CoA synthetase activity and fatty acid partitioning to diacylglycerol and triacylglycerol via a peroxisome proliferator-activated receptor-gamma-independent mechanism in human arterial smooth muscle cells and macrophages.Diabetes. 2007; 56: 1143-1152Crossref PubMed Scopus (56) Google Scholar). In the present study, we therefore asked if ACSL4 modulates 20:4 bioavailability and plays a role in PGE2 production in these cells. Our results demonstrate that human arterial SMCs express ACSL4 variant 1, which allows important incorporation of 20:4 into phospholipids and, in turn, modulates PGE2 release. These observations suggest that SMC ACSL4 might play an important role in vascular biology and pathology. Normal human newborn aortic SMCs were isolated by an explant method, as previously described (18Suzuki L.A. Poot M. Gerrity R.G. Bornfeldt K.E. Diabetes accelerates smooth muscle accumulation in lesions of atherosclerosis: lack of direct growth-promoting effects of high glucose levels.Diabetes. 2001; 50: 851-860Crossref PubMed Scopus (175) Google Scholar). SMCs used for experiments were maintained in DMEM 5 mM glucose with 10% FBS. Cells were infected with retrovirus for ACSL1 or ACSL4 overexpression at passage 6, and were used for experiments through passage 10. In a few experiments, immortalized primary human aortic SMCs (19Perez-Reyes N. Halbert C.L. Smith P.P. Benditt E.P. McDougall J.K. Immortalization of primary human smooth muscle cells.Proc. Natl. Acad. Sci. USA. 1992; 89: 1224-1228Crossref PubMed Scopus (98) Google Scholar) were used. Unless otherwise noted, subconfluent SMCs were quiesced for 24–48 h in DMEM 5 mM glucose plus 0.5% human plasma-derived serum before experiments. SMCs were infected with retroviral vectors for ACSL overexpression 10 different times over a period of three years with similar results. ACSL4 siRNA experiments were done in two independent infections with similar results. FAs oleic acid (18:1), palmitic acid (16:0), or arachidonic acid (20:4) were added to the cells prebound to 0.5% FA-free BSA at a BSA:FA molar ratio of <1:3. All experiments using human tissues were reviewed and approved by the Institutional Review Board at the University of Washington. Phoenix amphotropic cells (Orbigen; San Diego, CA) were maintained in DMEM 25 mM glucose, 10% FBS, nonessential amino acids, 100 U/ml penicillin, and 100 mg/ml streptomycin. After transfection, 1 μg/ml puromycin was added in order to select for successfully transfected cells. Human ACSL4 has two splice variants, variant 1 (NM_004458) and the longer variant 2 (NM_022977). To determine which ACSL4 variant is expressed in SMCs, real-time quantitative PCR (qPCR) primers were designed (Applied Biosystems Primer Express 2.0 software), and ordered from Operon (Huntsville, AL). Primers were used to detect ACSL4 variant 2 or both variants 1 and 2 (Table 1). Total RNA was extracted from SMCs, using the RNeasy Mini Kit (Qiagen; Valencia, CA), and used for semi-quantitative or real-time qPCR, as described below. Real-time PCR was also used to measure mRNA levels of other ACSL isoforms and FA transport protein (FATP) isoforms, at least some of which have acyl-CoA synthetase activity (Table 1).TABLE 1Primers for real-time qPCR and semi-quantitative RT-PCRPrimerPrimer SequenceAmplicon LengthReal-time qPCR ACSL1-F5′-AACAGACGGAAGCCCAAGC-3′102 bp ACSL1-R5′-TCGGTGAGTGACCATTGCTC-3′ ACSL3-F5′-CCCCTGAAACTGGTCTGGTG-3′73 bp ACSL3-R5′-TCCGCCTGGTAATGTGTTTTAA-3′ ACSL4-F variant 25′-GGCGTACTTTATTGTCGGCTTC-3′75 bpNM_022977 ACSL4-R variant 25′-TACAGCCAAGGCAGTTCAATCTTAG-3′ ACSL4-F5′-GCTTCCTATCTGATTACCAGTGTTGA-3′96 bp ACSL4-R5′-GTCCACATAAATGATATGTTTAACACAACT-3′ ACSL5-F5′-CCCCATGTCCACTTCAGTCAT-3′84 bp ACSL5-R5′-GTGCATTCTGTTTGACCATAAGCT-3′ FATP1-F5′-CTGCCCTTAAATGAGGCAGTCT-3′64 bp FATP1-R5′-AACAGCTTCAGAGGGCGAAG -3′ FATP3-F5′-TACCTGCCCCTCACAACTGC-3′70 bp FATP3-R5′-GTGGAAGTTCTCAGATTCGAAGG-3′ FATP4-F5′-TTCTGTGAAAGTCTCATGTCCAAGT-3′69 bp FATP4-R5′-TCTCAGCCTGGGAACCAGAG-3′ 18S-F5′-CATTAAATCAGTTATGGTTCCTTTGG-3′88 bp 18S-R5′-CCCGTCGGCATGTATTAGCT-3′RT-PCR ACSL1-F5′-TGCAGCACTCACCACCTTC-3′582 bp ACSL1-R5′-TAGGCATCCATGACAACTA-3′ ACSL4-F5′-CCGACCTAAGGGAGTGATGA-3′169 bp ACSL4-R5′-CCTGCAGCCATAGGTAAAGC-3′ β-actin-F5′-ATCTGGCACCACACCTTCTACAATGAGCTGCG-3′852 bp β-actin-R5′-CGTCATACTCCTGCTTGCTGATCCACATCTGC-3′qPCR, quantitative PCR. Open table in a new tab qPCR, quantitative PCR. Human cDNA ACSL4 variant 1 (NM_004458) and human ACSL1 variant 2 (NM_001995.2) clones were obtained in plasmid cytomegalovirus expression vectors from OriGene (Rockville, MD). The retroviral pBM-IRES-PURO (pBM) vector, which contains a puromycin resistance element (20Garton K.J. Ferri N. Raines E.W. Efficient expression of exogenous genes in primary vascular cells using IRES-based retroviral vectors.Biotechniques. 2002; 32 (832, 834 passim): 830Crossref PubMed Scopus (41) Google Scholar), was used to generate vectors for stable overexpression of ACSL4 and ACSL1. Briefly, the plasmid cytomegalovirus vectors were used to transform XL-1 Blue Supercompetent cells (Stratagene; Cedar Creek, TX). The ACSL sequences were amplified using cloning primers with a Kozak sequence added to the beginning of the 5′ primer (Invitrogen). The PCR products were gel purified and ligated into the pGEM-T Easy Vector, a TOPO vector (Promega; Madison, WI), and the ACSL sequences were then excised and ligated into the dephosphorylated pBM vector. A pBM-eGFP (enhanced green fluorescent protein) plasmid was used as a positive control. All vectors were sequenced to verify correct directionality by using an ABI 3730XL high-throughput capillary DNA analyzer. Phoenix amphotropic cells (70–80% confluent cultures) were transfected with the empty pBM vector, pBM-eGFP, pBM-ACSL4, or pBM-ACSL1 plasmids by CaCl2 transfection, according to Orbigen's instructions. The next day, the cells were passaged into media containing 1 μg/ml puromycin for positive selection, and then maintained in media containing puromycin until virus collection at ∼90% confluency. The retrovirus-containing media, collected in fresh medium during a 24 h period, were removed from the Phoenix cells and filtered through a 0.45 μm syringe filter, and 4 mg/ml polybrene (Sigma; St. Louis, MO) was added. Retrovirus was incubated with the SMCs for 16–18 h. The SMCs were then treated with puromycin at 5 μg/ml for 36–48 h until all nonvirus-treated control SMCs were dead. Immortalized human primary aortic SMCs were transduced for stable expression of ACSL4 siRNAs, using HuSHTM 29 mer constructs (OriGene Technologies, Inc.; Rockville, MD). Four different ACSL4 siRNA constructs in the pRS plasmid, negative control pRS plasmid, and a scrambled negative control siRNA in the pRS plasmid (OriGene) were used for generation of SMCs stably expressing these constructs following transfection of Phoenix amphotropic cells, and subsequent transduction of SMCs, as described above. Initial experiments revealed that the ACSL4 siRNA construct TI359914 (CGCTATCTCCTCAGACACACCGATTCATG) resulted in the most significant (60–70%) downregulation of ACSL4, and this construct, together with the two controls, was used for subsequent experiments. Another construct, TI359913 (GGCTCATGTGCTAGAACTGACAGCAGAGA), resulted in a less-marked (∼30%) reduction of ACSL4, and was used as an additional control in some experiments. Following SMC infection with these constructs, transduced cells were selected by puromycin incubation, as described above. Total RNA (250 ng) was reverse-transcribed into cDNA using random hexamers and 0.2 U/ml Omniscript reverse transcriptase (Invitrogen). The mixture was incubated at 37°C for 60 min. The cDNA template was then amplified with PCR using a GeneAmp PCR system (Applied Biosystems) with a primer concentration of 400 nM. ACSL primers were designed using Primer3 (21Rozen S. Skaletsky H. Primer3 on the WWW for general users and for biologist programmers.Methods Mol. Biol. 2000; 132: 365-386Crossref PubMed Google Scholar). β-actin primers were from Clontech Labs, Inc. (Mountain View, CA). The PCR products were separated by gel electrophoresis in 2% agarose gels and visualized with ethidium bromide. RNA was treated with RNase Free DNase I (Stratagene). Real-time qPCR was performed on an Mx4000 Multiplex QPCR System (Stratagene). RNA samples (20 ng) were loaded in triplicate and run in a 10 μl reaction using SYBR Green PCR Master Mix. Each reaction contained 5 μl 2× Master Mix, 400 nM of each primer, 0.5 units of StrataScript RT, and 0.5 units of RNase Block and was run with PCR cycling conditions of 48°C for 30 min, 95°C for 10 min, and 40 cycles of 95°C for 15 s and 60°C for 1 min. Dissociation curves were run to confirm specificity of all PCR amplicons. For standard curves, PCR amplicons were used at 1:4 serial dilutions. Results were then converted to copy number and normalized to total RNA levels, or were normalized to 18S. Total RNA and PCR amplicons were quantitated on an Mx4000 Multiplex QPCR System using the RiboGreen RNA Quantitation Kit (Molecular Probes; Eugene, OR) and standards from the manufacturer. SMCs in 10-cm dishes were harvested in Western lysis buffer (25 mM Tris-HCl, pH 7.4, 2 mM EDTA, 10 mM Na2SO4, 150 mM NaCl, 50 mM NaF, 1% Triton-X, 5 mM benzamidine, 10 mg/ml aprotinin, 20 mg/ml leupeptin, and 5 mg/ml pepstatin). Protein concentrations were determined using a modified BCA protein assay (Thermo Scientific; Rockford, IL). Cell lysates (60 μg) were then resolved on SDS-PAGE gels and electro-transferred to polyvinylidene difluoride membranes overnight. Membranes were blocked in 5% milk in TBS/0.1% Tween-20 for 1 h at room temperature, and then incubated with primary antibody overnight at 4°C. A polyclonal ACSL1 antibody (Aviva Systems Biology; San Diego, CA) was used at a dilution of 1:500 in 5% milk in PBS. A polyclonal anti-rat ACSL4 antibody was generously provided by Dr. Rosalind Coleman (University of North Carolina, Chapel Hill, NC), and used at a 1:10,000 dilution, as described previously (17Askari B. Kanter J.E. Sherrid A.M. Golej D.L. Bender A.T. Liu J. Hsueh W.A. Beavo J.A. Coleman R.A. Bornfeldt K.E. Rosiglitazone inhibits acyl-CoA synthetase activity and fatty acid partitioning to diacylglycerol and triacylglycerol via a peroxisome proliferator-activated receptor-gamma-independent mechanism in human arterial smooth muscle cells and macrophages.Diabetes. 2007; 56: 1143-1152Crossref PubMed Scopus (56) Google Scholar). After primary antibody incubation, membranes were incubated with secondary antibody (anti-rabbit-HRP at a 1:5,000 dilution) and developed with chemoluminescent reagent. To verify equal loading, membranes were stripped and reprobed with a monoclonal β-actin antibody (Sigma) at a 1:15,000 dilution, followed by anti-mouse-HRP at a 1:15,000 dilution. ACSL activity was determined as previously described (17Askari B. Kanter J.E. Sherrid A.M. Golej D.L. Bender A.T. Liu J. Hsueh W.A. Beavo J.A. Coleman R.A. Bornfeldt K.E. Rosiglitazone inhibits acyl-CoA synthetase activity and fatty acid partitioning to diacylglycerol and triacylglycerol via a peroxisome proliferator-activated receptor-gamma-independent mechanism in human arterial smooth muscle cells and macrophages.Diabetes. 2007; 56: 1143-1152Crossref PubMed Scopus (56) Google Scholar). Briefly, cell lysates (50–200 μg) were incubated for 20 min at 37°C in a reaction mixture containing 175 mM Tris (pH 7.4), 8 mM MgCl2, 5 mM DTT, 10 mM ATP, 2 mM CoA with 50 mM [9,10(n)-3H]18:1 (1 μCi), [5,6,8,9,11,12,14,15-3H]20:4, or [9,10(n)-3H]16:0 (GE Healthcare Life Sciences; Piscataway, NJ). After extraction, the radioactivity of the lower aqueous phase was used to calculate ACSL enzymatic activity. The results were corrected for blanks (samples without cell lysates added and samples analyzed in the absence of CoA or ATP) and for protein content. All reactions were confirmed to occur within the linear range. Long-chain fatty acyl-CoAs were quantified by LC-ESI-MS/MS, as described by Haynes et al. (22Haynes C.A. Allegood J.C. Sims K. Wang E.W. Sullards M.C. Merrill Jr, A.H. Quantitation of fatty acyl-coenzyme As in mammalian cells by liquid chromatography-electrospray ionization tandem mass spectrometry.J. Lipid Res. 2008; 49: 1113-1125Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Briefly, cells were homogenized in 25 mM phosphate buffer (pH 4.9), and fatty acyl-CoAs were extracted (23Golovko M.Y. Murphy E.J. An improved method for tissue long-chain acyl-CoA extraction and analysis.J. Lipid Res. 2004; 45: 1777-1782Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). For LC/ESI/tandem MS experiments, an Agilent 6410 triple quadruple MS system equipped with an Agilent 1200 LC system and an ESI source was utilized. The neutral loss of m/z 507 is the most-intense product ion for each compound. Acyl-CoA species were detected by their characteristic LC retention time in the multiple reaction monitoring mode following ESI. A known amount of C17:0 acyl-CoA was added into the biological samples to quantify C16:0 (palmitoyl-CoA), C18:0 (stearoyl-CoA), C18:1 (oleoyl-CoA), 20:4 (arachidonoyl-CoA), and C22:6 (docosahexaenoyl-CoA) by comparing the relative peak areas in the reconstructed ion chromatogram in the multiple reaction monitoring mode. The production of acid-soluble metabolites was used as an index of the β-oxidation of FAs. SMCs were incubated in DMEM + 0.5% FA-free BSA with 50 μM carnitine and 0.1 μCi [1-14C]18:1 (50–62 mCi/mmol), 0.1 μCi [1-14C]16:0 (50–62 mCi/mmol), or 0.5 μCi [5,6,8,9,11,12,14,15-3H]20:4 (150–230 Ci/mmol, all from GE Healthcare Life Sciences) per well in 6-well plates. After 24 h, 800 μl of the medium was harvested on ice, and 200 μl of ice-cold 70% perchloric acid was added in order to precipitate BSA:FA complexes. The samples were centrifuged for 10 min at 14,000 g, and the radioactivity of the supernatant was determined by liquid scintillation (24Lewin T.M. Wang S. Nagle C.A. Van Horn C.G. Coleman R.A. Mitochondrial glycerol-3-phosphate acyltransferase-1 directs the metabolic fate of exogenous fatty acids in hepatocytes.Am. J. Physiol. Endocrinol. Metab. 2005; 288: E835-844Crossref PubMed Scopus (49) Google Scholar). The cells were then harvested in 1 M NaCl for protein analysis. For analysis of triacylglycerol (TAG) mass in SMCs, cells in 10-cm dishes were treated with DMEM, 0.5% FA-free BSA, 50 μM carnitine, and 10–70 μM 18:1, 16:0, or 20:4 for 24–48 h. Lipids were extracted using a modified Bligh and Dyer method (25Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42878) Google Scholar). A colorimetric TAG kit (Sigma) was used according to the manufacturer's instructions. Cells were plated and treated as described for the acid-soluble metabolite assay. After 5–10 min or 24 h, the cells were placed on ice, washed with PBS, and then harvested with 0.5 ml 1 M NaCl per well. For analysis of FA incorporation into neutral lipids, cell lysates from two wells on a 6-well plate were pooled. Lipids were extracted by using a modified Bligh and Dyer method (25Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42878) Google Scholar), and were loaded, together with mono-, di-, and triglyceride standards (NuChek Prep), onto unmodified Silica Gel G TLC plates (Sigma) preheated at 60°C for 15 min. The lipids were separated for 45–60 min, using a mobile phase of hexane-diethyl ether-glacial acetic acid (105:45:3). Lipid spots were visualized by iodine vapor. An Amersham Biosciences Storm 860 PhosphorImager was then used to detect and quantify the radioactivity in each lipid spot, or the radioactivity was detected by liquid scintillation following scraping of the spots. For analysis of [3H]20:4 incorporation into phospholipids, SMCs were treated with 0.1 μCi/ml [3H]20:4 (0.5 nM) for 5–15 min and then harvested with 1 M NaCl. Lipids were extracted as described above. Incorporation of [3H]20:4 into the phospholipid pool was first examined by TLC, as described for neutral lipid analysis. To analyze incorporation into specific phospholipids by HPLC, samples were resuspended in 2 ml CH3Cl-acetic acid (100:1) and were then run through Sep-pak columns (Sep-pak vac 3 cc, 500 mg, silica cartridges; Waters Corporation, Milford, MA). The phospholipids were eluted by step-wise addition of 2 ml CH3Cl-acetic acid (100:1) two times, 2 ml methanol-CH3Cl (2:1) three times, and 2 ml methanol-CH3Cl-H2O (2:1:0.8) three times. The eluted phospholipid solution was condensed and resuspended in 50 μl HPLC-grade methanol and run through reverse-phase HPLC (Shimadzu Scientific Instruments; Columbia, MD). Phospholipids in each 1 ml fraction were analyzed by determining the incorporation of [3H]20:4 by liquid scintillation, as described previously (26Askari B. Carroll M.A. Capparelli M. Kramer F. Gerrity R.G. Bornfeldt K.E. Oleate and linoleate enhance the growth-promoting effects of insulin-like growth factor-I through a phospholipase D-dependent pathway in arterial smooth muscle cells.J. Biol. Chem. 2002; 277: 36338-36344Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). The phospholipids were identified by comparison to elution times of phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidylcholine (PC) standards (Avanti Polar Lipids; Alabaster, AL), detected by ultraviolet monitoring at 206 nm. To determine the endogenous relative distribution of FAs in PE and PC, human SMCs, maintained in 10% FBS, were harvested, and lipids were extracted by the Bligh and Dyer method (25Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42878) Google Scholar) and then processed as described by Hamilton and Comai (27Hamilton J.G. Comai K. Separation of neutral lipids and free fatty acids by high-performance liquid chromatography using low wavelength ultraviolet detection.J. Lipid Res. 1984; 25: 1142-1148Abstract Full Text PDF PubMed Google Scholar). In short, the extracted lipids were dried under nitrogen, and then resolubilized in CH3Cl-acetic acid at a ratio of 100:1 and separated on Sep-pak columns (Waters) equilibrated with hexane-methyltertiarybutylether (96:4), as described above. The samples were eluted in three fractions. Fraction 1, consisting primarily of neutral lipids and free FAs (27Hamilton J.G. Comai K. Separation of neutral lipids and free fatty acids by high-performance liquid chromatography using low wavelength ultraviolet detection.J. Lipid Res. 1984; 25: 1142-1148Abstract Full Text PDF PubMed Google Scholar), was eluted in 2 + 2 + 12 ml CH3Cl-acetic acid (100:1); fraction 2, enriched in PE (27Hamilton J.G. Comai K. Separation of neutral lipids and free fatty acids by high-performance liquid chromatography using low wavelength ultraviolet detection.J. Lipid Res. 1984; 25: 1142-1148Abstract Full Text PDF PubMed Google Scholar), was eluted by 5 ml methanol-CH3Cl (2:1); and fraction 3, enriched in PC (27Hamilton J.G. Comai K. Separation of neutral lipids and free fatty acids by high-performance liquid chromatography using low wavelength ultraviolet detection.J. Lipid Res. 1984; 25: 1142-1148Abstract Full Text PDF PubMed Google Scholar), was eluted in 5 ml methanol-CH3Cl-H2O (2:1:0.8). Fractions 2 and 3 were dried separately under nitrogen and used for analysis of FA composition. FAs were methylated under acidic conditions (25 μl of H2SO4 into 975 μl of methanol) for 60 min at 80°C, followed by a hexane-H2O extraction (200:1,500). The hexane layer was collected after freeze-separation and loaded onto a GC-mass spectrometer (Agilent 6890-5973; Agilent Technologies, Foster City, CA). The GC-MS spectr