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• W1997298990 abstract "Lysophosphatidic acid is a multifunctional phospholipid mediator and elicits a variety of biological responses in vitro and in vivo. Evidence is accumulating that lysophosphatidic acid plays important physiological roles in diverse mammalian tissues and cells. In the present study, we first examined whether lysophosphatidic acid is present in human saliva. We found that a significant amount of lysophosphatidic acid is present in the saliva (0.785 nmol/22276). The predominant fatty acyl moiety of lysophosphatidic acid was 18:1n-9 + n-7 followed by 18:0 and 16:0. A small amount of lysoplasmanic acid, an alkyl ether-linked analog of lysophosphatidic acid, was also detected in the saliva (0.104 nmol/ml). We found that physiologically relevant concentrations of lysophosphatidic acid induced accelerated growth of cells of mouth, pharynx, and esophagus origin in vitro. Lysophosphatidic acid also induced rapid increases in the intracellular free Ca2+ concentrations in these cells. We obtained evidence that lysophosphatidic acid receptor mRNAs are actually present in these cells.These results strongly suggest that lysophosphatidic acid is involved in wound healing in the upper digestive organs such as the mouth, pharynx, and esophagus. Lysophosphatidic acid is a multifunctional phospholipid mediator and elicits a variety of biological responses in vitro and in vivo. Evidence is accumulating that lysophosphatidic acid plays important physiological roles in diverse mammalian tissues and cells. In the present study, we first examined whether lysophosphatidic acid is present in human saliva. We found that a significant amount of lysophosphatidic acid is present in the saliva (0.785 nmol/22276). The predominant fatty acyl moiety of lysophosphatidic acid was 18:1n-9 + n-7 followed by 18:0 and 16:0. A small amount of lysoplasmanic acid, an alkyl ether-linked analog of lysophosphatidic acid, was also detected in the saliva (0.104 nmol/ml). We found that physiologically relevant concentrations of lysophosphatidic acid induced accelerated growth of cells of mouth, pharynx, and esophagus origin in vitro. Lysophosphatidic acid also induced rapid increases in the intracellular free Ca2+ concentrations in these cells. We obtained evidence that lysophosphatidic acid receptor mRNAs are actually present in these cells. These results strongly suggest that lysophosphatidic acid is involved in wound healing in the upper digestive organs such as the mouth, pharynx, and esophagus. Lysophosphatidic acid (acyl LPA) is a common intermediate in the biosynthesis of glycerolipids in diverse mammalian tissues and cells. In addition to the role as a metabolic intermediate, acyl LPA is known to act as a potent lipid mediator. Acyl LPA has been shown to induce a variety of biological responses in vitro and in vivo, such as hypertension (1Tokumura A. Fukuzawa K. Tsukatani H. Effects of synthetic and natural lysophosphatidic acids on the arterial blood pressure of different animal species.Lipids. 1978; 13: 572-574Google Scholar), smooth muscle contraction (2Tokumura A. Fukuzawa K. Tsukatani H. Contractile actions of lysophosphatidic acids with a chemically-defined fatty acyl group on longitudinal muscle from guinea-pig ileum.J. Pharm. Pharmacol. 1982; 34: 514-516Google Scholar), platelet activation (3Schumacher K.A. Classen H.G. Spath M. Platelet aggregation evoked in vitro and in vivo by phosphatidic acids and lysoderivatives: identity with substances in aged serum (DAS).Thromb. Haemost. 1979; 42: 631-640Google Scholar, 4Gerrard J.M. Kindom S.E. Peterson D.A. Peller J. Krantz K.E. White J.G. Lysophosphatidic acid. Influence on platelet aggregation and intracellular calcium flux.Am. J. Pathol. 1979; 96: 423-438Google Scholar, 5Tokumura A. Fukuzawa K. Isobe J. Tsukatani H. Lysophosphatidic acid-induced aggregation of human and feline platelets: structure-activity relationship.Biochem. Biophys. Res. Commun. 1981; 99: 391-398Google Scholar), cell proliferation (6van Corven E.J. Groenink A. Jalink K. Eichholtz T. Moolenaar W.H. Lysophosphatidate-induced cell proliferation: identification and dissection of signaling pathways mediated by G proteins.Cell. 1989; 59: 45-54Google Scholar, 7Tokumura A. Iimori M. Nishioka Y. Kitahara M. Sakashita M. Tanaka S. Lysophosphatidic acids induce proliferation of cultured vascular smooth muscle cells from rat aorta.Am. J. Physiol. 1994; 267: C204-C210Google Scholar), wound healing (8Balazs L. Okolicany J. Ferrebee M. Tolley B. Tigyi G. Topical application of the phospholipid growth factor lysophosphatidic acid promotes wound healing in vivo.Am. J. Physiol. Regul. Integr. Comp. Physiol. 2001; 280: R466-R472Google Scholar), neurite retraction (9Tigyi G. Miledi R. Lysophosphatidates bound to serum albumin activate membrane currents in Xenopus oocytes and neurite retraction in PC 12 pheochromocytoma cells.J. Biol. Chem. 1992; 267: 21360-21367Google Scholar, 10Jalink K. Eichholtz T. Postma F.R. van Corven E.J. Moolenaar W.H. Lysophosphatidic acid induces neuronal shape changes via a novel, receptor-mediated signaling pathway: similarity to thrombin action.Cell Growth Differ. 1993; 4: 247-255Google Scholar), cell survival (11Levine J.S. Koh J.S. Triaca V. Lieberthal W. Lysophosphatidic acid: a novel growth and survival factor for renal proximal tubular cells.Am. J. Physiol. 1997; 273: F575-F585Google Scholar, 12Weiner J.A. Chun J. Schwann cell survival mediated by the signaling phospholipid lysophosphatidic acid.Proc. Natl. Acad. Sci. USA. 1999; 96: 5233-5238Google Scholar, 13Koh J.S. Lieberthal W. Heydrick S. Levine J.S. Lysophosphatidic acid is a major serum noncytokine survival factor for murine macrophages which acts via the phosphatidylinositol 3-kinase signaling pathway.J. Clin. Invest. 1998; 102: 716-727Google Scholar), accelerated development of embryos (14Kobayashi T. Yamano S. Murayama S. Ishikawa H. Tokumura A. Aono T. Effect of lysophosphatidic acid on the preimplantation development of mouse embryos.FEBS Lett. 1994; 351: 38-40Google Scholar), stimulation of ovum transport in oviducts (15Kunikata K. Yamano S. Tokumura A. Aono T. Effect of lysophosphatidic acid on the ovum transport in mouse oviducts.Life Sci. 1999; 65: 833-840Google Scholar), regulation of endothelial permeability (16Schulze C. Smales C. Rubin L.L. Staddon J.M. Lysophosphatidic acid increases tight junction permeability in cultured brain endothelial cells.J. Neurochem. 1997; 68: 991-1000Google Scholar), haptotactic migration of monocytes (17Zhou D. Luini W. Bernasconi S. Diomede L. Salmona M. Mantovani A. Sozzani S. Phosphatidic acid and lysophosphatidic acid induce haptotactic migration of human monocytes.J. Biol. Chem. 1995; 270: 25549-25556Google Scholar), and tumor cell invasion (18Imamura F. Horai T. Mukai M. Shinki K. Sawada M. Akedo H. Induction of in vitro tumor cell invasion of cellular monolayers by lysophosphatidic acid or phospholipase D.Biochem. Biophys. Res. Commun. 1993; 193: 497-503Google Scholar). Lysoplasmanic acid (alkyl LPA) is also known to exhibit biological activities similar to those of acyl LPA (19Simon M.F. Chap H. Douste-Blazy L. Human platelet aggregation induced by 1-alkyl-lysophosphatidic acid and its analogs: a new group of phospholipid mediators?.Biochem. Biophys. Res. Commun. 1982; 108: 1743-1750Google Scholar, 20Tokumura A. Yoshida J. Maruyama T. Fukuzawa K. Tsukatani H. Platelet aggregation induced by ether-linked phospholipids. 1. Inhibitory actions of bovine serum albumin and structural analogues of platelet activating factor.Thromb. Res. 1987; 46: 51-63Google Scholar, 21Sugiura T. Tokumura A. Gregory L. Nouchi T. Weintraub S.T. Hanahan D.J. Biochemical characterization of the interaction of lipid phosphoric acids with human platelets: comparison with platelet-activating factor.Arch. Biochem. Biophys. 1994; 311: 358-368Google Scholar, 22Sugiura T. Nakane S. Kishimoto S. Waku K. Yoshioka Y. Tokumura A. Hanahan D.J. Occurrence of lysophosphatidic acid and its alkyl ether- linked analog in rat brain and comparison of their biological activities toward cultured neural cells.Biochim. Biophys. Acta. 1999; 1440: 194-204Google Scholar). Evidence is accumulating that LPAs play important physiological and pathophysiological roles in various biological systems in mammals (23Jalink K. Hordijk P.L. Moolenaar W.H. Growth factor-like effects of lysophosphatidic acid, a novel lipid mediator.Biochim. Biophys. Acta. 1994; 1198: 185-196Google Scholar, 24Moolenaar W.H. Lysophosphatidic acid, a multifunctional phospholipid messenger.J. Biol. Chem. 1995; 270: 12949-12952Google Scholar, 25Tokumura A. A family of phospholipid autacoids: occurrence, metabolism and bioactions.Prog. Lipid Res. 1995; 34: 151-184Google Scholar, 26Durieux M.E. Lysophosphatidate Signaling: Cellular Effects and Molecular Mechanisms. Spring-Verlag, Heidelberg1995Google Scholar, 27Goetzl E.J. An S. Diversity of cellular receptors and functions for the lysophospholipid growth factors lysophosphatidic acid and sphingosine 1- phosphate.FASEB J. 1998; 12: 1589-1598Google Scholar, 28Tigyi G.J. Liliom K. Fischer D. Guo Z. Phospholipid growth factors: identification and mechanism of action.in: Rubin R.P. Laychock S. Lipid Second Messengers. CRC Press, Boca Raton, FL.1998: 51-81Google Scholar, 29Contos J.J. Ishii I. Chun J. Lysophosphatidic acid receptors.Mol. Pharmacol. 2000; 58: 1188-1196Google Scholar). The detailed mechanism of action of LPAs remained uncertain until recently, although it had long been assumed that there exist specific binding sites for LPAs. Recently, several investigators have provided direct evidence that LPAs interact with specific receptors expressed in various mammalian tissues and cells, thereby eliciting biological responses (29Contos J.J. Ishii I. Chun J. Lysophosphatidic acid receptors.Mol. Pharmacol. 2000; 58: 1188-1196Google Scholar). Thus far, three separate types of LPA receptors have been identified: LPA1 (encoded by the Edg2 gene), LPA2 (encoded by the Edg4 gene), and LPA3 (encoded by the Edg7 gene) (30Hecht J.H. Weiner J.A. Post S.R. Chun J. Ventricular zone gene-1 (vzg-1) encodes a lysophosphatidic acid receptor expressed in neurogenic regions of the developing cerebral cortex.J. Cell Biol. 1996; 135: 1071-1083Google Scholar, 31An S. Dickens M.A. Bleu T. Hallmark O.G. Goetzl E.J. Molecular cloning of the human Edg2 protein and its identification as a functional cellular receptor for lysophosphatidic acid.Biochem. Biophys. Res. Commun. 1997; 231: 619-622Google Scholar, 32An S. Bleu T. Hallmark O.G. Goetzl E.J. Characterization of a novel subtype of human G protein-coupled receptor for lysophosphatidic acid.J. Biol. Chem. 1998; 273: 7906-7910Google Scholar, 33Bandoh K. Aoki J. Hosono H. Kobayashi S. Kobayashi T. Murakami-Murofushi K. Tsujimoto M. Arai H. Inoue K. Molecular cloning and characterization of a novel human G-protein-coupled receptor, EDG7, for lysophosphatidic acid.J. Biol. Chem. 1999; 274: 27776-27785Google Scholar). These LPA receptors are seven transmembrane, G protein-coupled receptors, and there are homologies between these receptors and the sphingosine-1-phosphate receptors. A number of investigators have already studied the intracellular signaling pathways for LPAs; LPAs have been shown to induce actin rearrangement, inhibition of adenylyl cyclase, activation of phospholipase C, activation of mitogen-activated protein kinase, stimulation of serum response elements, and DNA synthesis (23Jalink K. Hordijk P.L. Moolenaar W.H. Growth factor-like effects of lysophosphatidic acid, a novel lipid mediator.Biochim. Biophys. Acta. 1994; 1198: 185-196Google Scholar, 24Moolenaar W.H. Lysophosphatidic acid, a multifunctional phospholipid messenger.J. Biol. Chem. 1995; 270: 12949-12952Google Scholar, 25Tokumura A. A family of phospholipid autacoids: occurrence, metabolism and bioactions.Prog. Lipid Res. 1995; 34: 151-184Google Scholar, 26Durieux M.E. Lysophosphatidate Signaling: Cellular Effects and Molecular Mechanisms. Spring-Verlag, Heidelberg1995Google Scholar, 27Goetzl E.J. An S. Diversity of cellular receptors and functions for the lysophospholipid growth factors lysophosphatidic acid and sphingosine 1- phosphate.FASEB J. 1998; 12: 1589-1598Google Scholar, 28Tigyi G.J. Liliom K. Fischer D. Guo Z. Phospholipid growth factors: identification and mechanism of action.in: Rubin R.P. Laychock S. Lipid Second Messengers. CRC Press, Boca Raton, FL.1998: 51-81Google Scholar, 29Contos J.J. Ishii I. Chun J. Lysophosphatidic acid receptors.Mol. Pharmacol. 2000; 58: 1188-1196Google Scholar). Despite increasing information concerning the biological activities and the mechanism of action of LPAs, available information concerning the actual level of LPAs in mammalian tissues and fluids is still limited. Previously, we reported that substantial amounts of acyl LPA as well as small amounts of alkyl LPA are present in rat brain (22Sugiura T. Nakane S. Kishimoto S. Waku K. Yoshioka Y. Tokumura A. Hanahan D.J. Occurrence of lysophosphatidic acid and its alkyl ether- linked analog in rat brain and comparison of their biological activities toward cultured neural cells.Biochim. Biophys. Acta. 1999; 1440: 194-204Google Scholar) and hen egg yolk and white (34Nakane S. Tokumura T. Waku K. Sugiura T. Hen egg yolk and white contain high amounts of lysophosphatidic acids, growth factor-like lipids: Distinct molecular species compositions.Lipids. 2001; 36: 413-419Google Scholar). Das and Hajra (35Das A.K. Hajra A.K. Quantification, characterization and fatty acid composition of lysophosphatidic acid in different rat tissues.Lipids. 1989; 24: 329-333Google Scholar) also demonstrated the occurrence of acyl LPA in various rat organs. Furthermore, several investigators have provided evidence that acyl LPA is present in significant amounts in blood plasma (36Tokumura A. Harada K. Fukuzawa K. Tsukatani H. Involvement of lysophospholipase D in the production of lysophosphatidic acid in rat plasma.Biochim. Biophys. Acta. 1986; 875: 31-38Google Scholar, 37Tokumura A. Fujimoto H. Yoshimoto O. Nishioka Y. Miyake M. Fukuzawa K. Production of lysophosphatidic acid by lysophospholipase D in incubated plasma of spontaneously hypertensive rats and Wistar Kyoto rats.Life Sci. 1999; 65: 245-253Google Scholar, 38Baker D.L. Umstot E.S. Desiderio D.M. Tigyi G.J. Quantitative analysis of lysophosphatidic acid in human blood fractions.Ann. N. Y. Acad. Sci. 2000; 905: 267-269Google Scholar, 39Baker D.L. Desiderio D.M. Miller D.D. Tolley B. Tigyi G.J. Direct quantitative analysis of lysophosphatidic acid molecular species by stable isotope dilution electrospray ionization liquid chromatography-mass spectrometry.Anal. Biochem. 2001; 292: 287-295Google Scholar), serum (38Baker D.L. Umstot E.S. Desiderio D.M. Tigyi G.J. Quantitative analysis of lysophosphatidic acid in human blood fractions.Ann. N. Y. Acad. Sci. 2000; 905: 267-269Google Scholar, 39Baker D.L. Desiderio D.M. Miller D.D. Tolley B. Tigyi G.J. Direct quantitative analysis of lysophosphatidic acid molecular species by stable isotope dilution electrospray ionization liquid chromatography-mass spectrometry.Anal. Biochem. 2001; 292: 287-295Google Scholar, 40Eichholts T. Jalink K. Fahrenfort I. Moolenaar W.H. The bioactive phospholipid lysophosphatidic acid is released from activated platelets.Biochem. J. 1993; 291: 677-680Google Scholar) and follicular fluids (41Tokumura A. Miyake M. Nishioka Y. Yamano S. Aono T. Fukuzawa K. Production of lysophosphatidic acids by lysophospholipase D in human follicular fluids of in vitro fertilization patients.Biol. Reprod. 1999; 61: 195-199Google Scholar). It is apparent, however, that sufficient amounts of analytical data have not hitherto been accumulated regarding the levels of LPAs. In order to better understand the physiological and pathophysiological roles of LPAs in living animals, it is essential to determine the exact levels of LPAs in various mammalian tissues and fluids under various conditions. It is also important to determine the subclass composition as well as molecular species composition of naturally occurring LPAs, because certain species of LPA preferentially interact with certain receptor subtypes (42Bandoh K. Aoki J. Taira A. Tsujimoto M. Arai H. Inoue K. Lysophosphatidic acid (LPA) receptors of the EDG family are differentially activated by LPA species. Structure-activity relationship of cloned LPA receptors.FEBS Lett. 2000; 478: 159-165Google Scholar). In this study, we focused on the saliva. The reasons are as follows: 1) the epithelial cells of the mouth, pharynx and esophagus are in direct contact with saliva, and 2) epithelia of these organs are often injured microscopically or macroscopically by ingested materials. We found that human saliva contains a relatively high amount of acyl LPA and a small amount of alkyl LPA. We also found that acyl LPA stimulates the growth of cells of mouth, pharynx and esophagus origin in vitro at physiologically relevant concentrations. The possible physiological and pathophysiological implications of the occurrence of LPAs in the saliva are discussed. Arachidonic acid, heptadecanoic acid, and the sn-glycero-3-phosphocholine cadmium chloride complex were obtained from Sigma (St. Louis, MO). Phospholipase D (Streptomyces chromofuscus) was purchased from Boehringer Mannheim GmbH (Mannheim, Germany). Dicyclohexylcarbodiimide was obtained from Wako Pure Chem. Ind. (Osaka, Japan). Dimethylaminopyridine was from the Aldrich Chemical Co. (Milwaukee, WI). 1(2)-Heptadecanoyl-sn-glycero-3-phosphate [acyl LPA (17:0)] and 1(2)-octadecyl-sn-glycero-3-phosphate [acyl LPA (18:1n-9)] were prepared according to previously described methods (22Sugiura T. Nakane S. Kishimoto S. Waku K. Yoshioka Y. Tokumura A. Hanahan D.J. Occurrence of lysophosphatidic acid and its alkyl ether- linked analog in rat brain and comparison of their biological activities toward cultured neural cells.Biochim. Biophys. Acta. 1999; 1440: 194-204Google Scholar). All other chemicals were of reagent grade. EC-GI-10 cells (esophagus sqamous cell carcinoma) were obtained from Riken Gene Bank (Saitama, Japan). Detroit 562 cells (pharynx sqamous cells carcinoma) and SCC-9 cells (tongue sqamous cell carcinoma) were obtained from the American Type Culture Collection (Rockville, MD). Saliva was obtained from six young healthy volunteers. Saliva (80 ml), obtained from individual donors, was then mixed with 100 ml of chloroform and 200 ml of methanol and shaken vigorously. Butylated hydroxytoluene (final 0.05%) was added to avoid lipid peroxidation and acyl LPA (17:0) (10 nmol) was added as an internal standard. To make two phases, chloroform (100 ml) and water (100 ml) were added as previously described (the final ratio of chloroform-methanol-water, 2:2:1.8, v/v/v). After vigorous shaking, the mixture was left to stand at 4°C for 12 h. The mixture was then centrifuged to allow phase separation. We confirmed that LPAs were exclusively recovered from the upper phase but not from the lower phase (22Sugiura T. Nakane S. Kishimoto S. Waku K. Yoshioka Y. Tokumura A. Hanahan D.J. Occurrence of lysophosphatidic acid and its alkyl ether- linked analog in rat brain and comparison of their biological activities toward cultured neural cells.Biochim. Biophys. Acta. 1999; 1440: 194-204Google Scholar); the upper phase was carefully transferred to glass tubes and washed with chloroform. A total of 200 ml of chloroform and a total of 1.6 ml of 12 M HCl were then added to the upper phases. After vigorous shaking and centrifugation, the lower phase was carefully aspirated. The upper phase was washed twice with chloroform, and the lower phases were taken and combined with the initial lower phase. The combined lower phase was evaporated to dryness under vacuum and the residue was dissolved in chloroform-methanol (1:2, v/v). The LPAs were purified by TLC using chloroform-acetone-methanol-acetic acid-water (4.5:2:1:1.3:0.5, v/v/v/v) as the solvent system in a developing tank sealed with nitrogen gas. The areas corresponding to the LPAs were scraped off the TLC plates into a glass tube. The LPAs were extracted from the silica gel by a modified method of Bligh and Dyer where HCl was added (the final concentration of HCl in the upper phase, 0.07 M) prior to the phase separation. LPAs were further purified by TLC using chloroform-methanol-25% ammonia (65:35:5, v/v) as the solvent system and then by TLC using chloroform-acetone-methanol-acetic acid-water (4.5:2:1:1.3:0.5, v/v/v/v) as the solvent system (22Sugiura T. Nakane S. Kishimoto S. Waku K. Yoshioka Y. Tokumura A. Hanahan D.J. Occurrence of lysophosphatidic acid and its alkyl ether- linked analog in rat brain and comparison of their biological activities toward cultured neural cells.Biochim. Biophys. Acta. 1999; 1440: 194-204Google Scholar). The LPAs (250–280 nmol), purified from human saliva, were treated with 0.2 M NaOH in 90% methanol or 90% methanol alone (control) for 20 min, as previously described (22Sugiura T. Nakane S. Kishimoto S. Waku K. Yoshioka Y. Tokumura A. Hanahan D.J. Occurrence of lysophosphatidic acid and its alkyl ether- linked analog in rat brain and comparison of their biological activities toward cultured neural cells.Biochim. Biophys. Acta. 1999; 1440: 194-204Google Scholar, 34Nakane S. Tokumura T. Waku K. Sugiura T. Hen egg yolk and white contain high amounts of lysophosphatidic acids, growth factor-like lipids: Distinct molecular species compositions.Lipids. 2001; 36: 413-419Google Scholar). The LPAs remaining were then extracted by a modified method of Bligh and Dyer (43Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Google Scholar), where HCl was added to the extraction mixture prior to the phase separation to acidify the upper phase. The lower phase was transferred to another tube, and the upper phase was washed twice with chloroform. The combined lower phase was evaporated to dryness. The amounts of the LPAs (alkali-stable or total) were determined by measuring the lipid phosphorus (22Sugiura T. Nakane S. Kishimoto S. Waku K. Yoshioka Y. Tokumura A. Hanahan D.J. Occurrence of lysophosphatidic acid and its alkyl ether- linked analog in rat brain and comparison of their biological activities toward cultured neural cells.Biochim. Biophys. Acta. 1999; 1440: 194-204Google Scholar, 34Nakane S. Tokumura T. Waku K. Sugiura T. Hen egg yolk and white contain high amounts of lysophosphatidic acids, growth factor-like lipids: Distinct molecular species compositions.Lipids. 2001; 36: 413-419Google Scholar). The fatty acyl moiety of the acyl LPA was converted to fatty acid methyl esters by treating the purified acyl LPA with 0.5 M methanolic sodium methoxide. The resultant fatty acid methyl esters were extracted and analyzed using a gas chromatograph (GC8A, Shimadzu, Kyoto, Japan) equipped with a fused silica column (SP2330, Supelco, Bellefonte, PA). The structures of the LPAs were confirmed by fast atom bombardment mass spectrometry (FAB MS) using a JEOL JMS-SX102A mass spectrometer. A 2:1 mixture of thioglycerol and dithiothreitol/dithioerythreitol (3:1) was used as the matrix, as previously described (22Sugiura T. Nakane S. Kishimoto S. Waku K. Yoshioka Y. Tokumura A. Hanahan D.J. Occurrence of lysophosphatidic acid and its alkyl ether- linked analog in rat brain and comparison of their biological activities toward cultured neural cells.Biochim. Biophys. Acta. 1999; 1440: 194-204Google Scholar, 34Nakane S. Tokumura T. Waku K. Sugiura T. Hen egg yolk and white contain high amounts of lysophosphatidic acids, growth factor-like lipids: Distinct molecular species compositions.Lipids. 2001; 36: 413-419Google Scholar). The LPAs were treated with 50 μl of N,O-bis(trimethylsilyl)trifluoroacetamide containing 1% trimethylchlorosilane and 50 μl of pyridine at 60°C for 30 min and converted to the tri-trimethylsilyl (TMS) derivative essentially according to the method of Tokumura et al. (44Tokumura A. Harada K. Yoshioka Y. Tsukatani H. Handa Y. Analyses of lysophosphatidic acids by gas chromatography mass spectrometry without hydrolytic pretreatment.Biomed. Mass Spectrom. 1984; 11: 167-171Google Scholar). The electron impact ionization (70 eV) mass spectra of the triTMS derivatives of the LPAs were obtained using a JEOL JMS-SX102A MS (accelerating voltage, 10 kV; ionizing current, 300 μA) coupled with a GC equipped with a fused silica column (DB-1, 30 m × 0.25 mm I.D., 0.25 μm thickness, J&W Scientific, Folsom, CA). The column temperature was increased from 200°C to 320°C at the rate of 10°C/min, and the temperature of the injection port was 300°C (34Nakane S. Tokumura T. Waku K. Sugiura T. Hen egg yolk and white contain high amounts of lysophosphatidic acids, growth factor-like lipids: Distinct molecular species compositions.Lipids. 2001; 36: 413-419Google Scholar). EC-GI-10 esophagus carcinoma cells were cultured in Ham’s F12 medium supplemented with 10% fetal bovine serum (FBS). Detroit 562 pharynx carcinoma cells were incubated in Eagle’s minimun essential medium supplemented with 10% FBS. SCC-9 tongue carcinoma cells were cultured in a 1:1 mixture of Ham’s F12 medium and Dulbecco’s modified Eagle’s medium containing 0.4 μg/ml of hydrocortisone supplemented with 10% FBS. These cells were plated in 24-well plates at the density of 3 × 104 cells/well and incubated for 24 h. The cells were then washed twice with phosphate-buffered saline and further incubated in the medium without FBS for 48 h in the presence of various concentrations of acyl LPA (18:1n-9). After the removal of the medium, the cells were washed and treated with 0.25% trypsin for 2 min. Then, the cells were suspended in phosphate-buffered saline (final 0.5 ml). The suspended cells were counted using a hemocytometer. The viability was checked by the Trypane blue dye exclusion test. The viability was above 99% in each experiment. Cells were cultured on a cover glass (diameter 12 mm) in 24-well plates. The cells were further incubated in the medium without FBS for 24 h. The cells were then incubated in 25 mM Hepes-Tyrode’s solution (−Ca2+) (pH 7.4) containing 5 μM Fura-2/AM and 0.02% Cremophor EL (Sigma, St. Louis, MO) at room temperature for 90 min. After rapid washing with 25 mM Hepes-Tyrode’s solution, the cover glass was placed in a cuvette filled with 3 ml of 25 mM Hepes-Tyrode’s solution. [Ca2+]i was estimated using a CAF-100 Ca2+ analyzer (JASCO, Tokyo, Japan) (22Sugiura T. Nakane S. Kishimoto S. Waku K. Yoshioka Y. Tokumura A. Hanahan D.J. Occurrence of lysophosphatidic acid and its alkyl ether- linked analog in rat brain and comparison of their biological activities toward cultured neural cells.Biochim. Biophys. Acta. 1999; 1440: 194-204Google Scholar). CaCl2 was added 4–5 min before the measurement (the final concentration of Ca2+ in the cuvette, 1 mM). Acyl LPA (18:1n-9) was dissolved in 6 μl of dimethyl sulfoxide and added to the cuvette. Total RNA was isolated from various cell lines using ISOGEN (Nippon Gene Co., Tokyo, Japan). One microgram of the total RNA was treated with DNase I (Invitrogen, 1 U) for 15 min at room temperature in a 10 μl reaction mixture containing 20 mM Tris-HCl (pH 8.4), 2 mM MgCl2 and 50 mM KCl. After the mixture was heated at 70°C for 10 min, the cDNA was synthesized by incubating at 42°C for 60 min in a 20 μl reaction mixture containing 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 10 mM dithiothreitol, 3 mM MgCl2, 0.75 μM dNTPs, 10 μg/ml oligo(dT)18 primer, and 200 U Superscript II reverse-transcriptase (Invitrogen Corp, Carlsbad, CA). After inactivation of the enzyme by heating at 98°C for 5 min, 0.4 μl of the reaction mixture was used for PCR. The sequences of the PCR primer sets were: 5′-CAATCGAGAGGCACATTACGGT-3′ (sense) and 5′-GATGTGAGCATAGAGAACCACC-3′ (antisense) for Edg2 (257 bp); 5′-AGACTGTTGTCATCATCCTGGG-3′ (sense) and 5′-AAGGGTGGAGTCATCAGTGGGT-3′ (antisense) for Edg4 (331 bp); 5′-CTGCTCATTTTGCTTGTCTGGG-3′ (sense) and 5′-CCACAACCATGATGAGGAAGGC-5′ (antisense) for Edg7 (175 bp); and 5′-CAGAGCAAGAGAGGCATCCT-3′ (sense) and 5′-AGGATCTTCATGAGGTAGTC-3′ (antisense) for β-actin (404 bp). Amplification of each gene was conducted with 30 cycles, except for that of the β-actin gene with 25 cycles, consisting of 1 min denaturation at 94°C, 1 min annealing (54°C, 58°C, 57°C, and 55°C for Edg2, Edg4, Edg7, and β-actin, respectively), and 1 min elongation at 72°C in a 20 μl reaction mixture containing 66.7 mM Tris-HCl (pH 8.8), 16.7 mM (NH4)2SO4, 6.67 mM MgCl2, 10 μM 2-mercaptoethanol, 6.67 μM EDTA, 167 μg/ml BSA, 1 μM sense and antisense primers, 15 mM dNTPs, 1 U Taq polymerase (Takara Shuzo, Kyoto, Japan), and 0.4 μl of reverse transcription (RT) reaction mixture. After amplification, 10 μl aliquots of the reaction mixture were electrophoresed on a 2% agarose gel. The gel was then stained with ethidium bromide and photographed under an UV lamp. First, we examined whether human saliva contains LPAs. We found that significant amounts of LPAs are included in human saliva (total 0.889 nmol/ml) (Table 1). A large part of the LPAs was accounted for by acyl LPA (88.3%) and a small part was accounted for by alkyl LPA (11.7%). The fatty acid composition of acyl LPA is shown in Table 2. The major fatty acyl constituent of acyl LPA was 18:1n-9 + n-7 (40.1%) followed by 18:0 (28.5%) and 16:0 (17.9%). We also found that a small amount of 18:2n-6 (8.0%) was present as a fatty acyl constituent. On the other hand, the levels of the C20 and C22 polyunsaturated fatty acid-containing species such as 20:4n-6-containing species were very low.TABLE 1Subclass composition of LPAs obtained from human salivaSubclass%nmol/mlAcyl LPA88.3aSubclass composition of LPAs was determined by mild alkaline hydrolysis as described in Materials and Methods. The data are the means of two separate expe" @default.
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