Serum Lysophosphatidic Acid Is Produced through Diverse Phospholipase Pathways
2002; Elsevier BV; Volume: 277; Issue: 50 Linguagem: Inglês
10.1074/jbc.m206812200
ISSN1083-351X
AutoresJunken Aoki, Akitsu Taira, Yasukazu Takanezawa, Yasuhiro Kishi, Kotaro Hama, Tatsuya Kishimoto, Koji Mizuno, Keijiro Saku, Ryo Taguchi, Hiroyuki Arai,
Tópico(s)Lipid metabolism and biosynthesis
ResumoLysophosphatidic acid (LPA) is a lipid mediator with multiple biological activities that accounts for many biological properties of serum. LPA is thought to be produced during serum formation based on the fact that the LPA level is much higher in serum than in plasma. In this study, to better understand the pathways of LPA synthesis in serum, we evaluated the roles of platelets, plasma, and phospholipases by measuring LPA using a novel enzyme-linked fluorometric assay. First, examination of platelet-depleted rats showed that half of the LPA in serum is produced via a platelet-dependent pathway. However, the amount of LPA released from isolated platelets after they are activated by thrombin or calcium ionophore accounted for only a small part of serum LPA. Most of the platelet-derived LPA was produced in a two-step process: lysophospholipids such as lysophosphatidylcholine (LPC), lysophosphatidylethanolamine, and lysophosphatidylserine, were released from activated rat platelets by the actions of two phospholipases, group IIA secretory phospholipase A2(sPLA2-IIA) and phosphatidylserine-specific phospholipase A1 (PS-PLA1), which were abundantly expressed in the cells. Then these lysophospholipids were converted to LPA by the action of plasma lysophospholipase D (lysoPLD). Second, accumulation of LPA in incubated plasma was strongly accelerated by the addition of recombinant lysoPLD with a concomitant decrease in LPC accumulation, indicating that the enzyme produces LPA by hydrolyzing LPC produced during the incubation. In addition, incubation of plasma isolated from human subjects who were deficient in lecithin-cholesterol acyltransferase (LCAT) did not result in increases of either LPC or LPA. The present study demonstrates multiple pathways for LPA production in serum and the involvement of several phospholipases, including PS-PLA1, sPLA2-IIA, LCAT, and lysoPLD. Lysophosphatidic acid (LPA) is a lipid mediator with multiple biological activities that accounts for many biological properties of serum. LPA is thought to be produced during serum formation based on the fact that the LPA level is much higher in serum than in plasma. In this study, to better understand the pathways of LPA synthesis in serum, we evaluated the roles of platelets, plasma, and phospholipases by measuring LPA using a novel enzyme-linked fluorometric assay. First, examination of platelet-depleted rats showed that half of the LPA in serum is produced via a platelet-dependent pathway. However, the amount of LPA released from isolated platelets after they are activated by thrombin or calcium ionophore accounted for only a small part of serum LPA. Most of the platelet-derived LPA was produced in a two-step process: lysophospholipids such as lysophosphatidylcholine (LPC), lysophosphatidylethanolamine, and lysophosphatidylserine, were released from activated rat platelets by the actions of two phospholipases, group IIA secretory phospholipase A2(sPLA2-IIA) and phosphatidylserine-specific phospholipase A1 (PS-PLA1), which were abundantly expressed in the cells. Then these lysophospholipids were converted to LPA by the action of plasma lysophospholipase D (lysoPLD). Second, accumulation of LPA in incubated plasma was strongly accelerated by the addition of recombinant lysoPLD with a concomitant decrease in LPC accumulation, indicating that the enzyme produces LPA by hydrolyzing LPC produced during the incubation. In addition, incubation of plasma isolated from human subjects who were deficient in lecithin-cholesterol acyltransferase (LCAT) did not result in increases of either LPC or LPA. The present study demonstrates multiple pathways for LPA production in serum and the involvement of several phospholipases, including PS-PLA1, sPLA2-IIA, LCAT, and lysoPLD. Lysophosphatidic acid (1- or 2-acyl-lysophosphatidic acid, LPA) 1The abbreviations used are: LPA, lysophosphatidic acid; PA, phosphatidic acid; PC, phosphatidylcholine; LPC, lysophosphatidylcholine; LPS, lysophosphatidylserine; LPE, lysophosphatidylethanolamine; LPs, lysophospholipids; PLA1, phospholipase A1; PLA2, phospholipase A2; EDG, endothelial cell differentiated gene; lysoPLD, lysophospholipase D; PS-PLA1, phosphatidylserine-specific PLA1; sPLA2-IIA, secretory PLA2group IIA; LCAT, lecithin-cholesterol acyltransferase; mPA-PLA1, membrane-bound PA-selective PLA1; PAF-AH, platelet-activating factor acetylhydrolase; MGL, monoglyceride lipase; G3PO, glycero-3-phosphate oxidase; GPCP, phosphocholine phosphodiesterase; ACD, acid citrate dextrose; FLD, familial LCAT deficiency; cPLA2, calcium-dependent cytosolic PLA2; iPLA2, calcium-independent cytosolic PLA2; LPI, lysophosphatidylinositol. is a lipid that mediates multiple cellular processes (1Tokumura A. Prog. Lipid Res. 1995; 34: 151-184Google Scholar, 2Moolenaar W.H. Exp. Cell Res. 1999; 253: 230-238Google Scholar), including platelet aggregation, smooth muscle contraction, cell proliferation, and cytoskeletal reorganization (e.g. generation of actin stress fibers and inhibition of neurite outgrowth). LPA evokes its multiple effects through G-protein-coupled receptors that are specific to LPA. Recent studies have identified a new family of receptor genes for LPA (reviewed in Refs. 3Chun J. Contos J.J. Munroe D. Cell Biochem. Biophys. 1999; 30: 213-242Google Scholar and 4Contos J.J. Ishii I. Chun J. Mol. Pharmacol. 2000; 58: 1188-1196Google Scholar). Members of this family include three G-protein-coupled receptors belonging to the EDG (endothelial cell differentiation gene) family, EDG2/LPA1 (5Hecht J.H. Weiner J.A. Post S.R. Chun J. J. Cell Biol. 1996; 135: 1071-1083Google Scholar), EDG4/LPA2 (6An S. Bleu T. Hallmark O.G. Goetzl E.J. J. Biol. Chem. 1998; 273: 7906-7910Google Scholar), and EDG7/LPA3 (7Bandoh K. Aoki J. Hosono H. Kobayashi S. Kobayashi T. Murakami M.K. Tsujimoto M. Arai H. Inoue K. J. Biol. Chem. 1999; 274: 27776-27785Google Scholar), which are coupled with different G-proteins and may explain various cellular responses to LPA. Serum is known to contain micromolar concentrations of LPA, but the levels in plasma are much lower (8Tigyi G. Miledi R. J. Biol. Chem. 1992; 267: 21360-21367Google Scholar). For this reason it has been proposed that LPA in serum is produced as a result of blood coagulation and that platelets are involved, in part, in the production of LPA in serum. In the literature, two pathways have been postulated for the extracellular production of LPA. In the first pathway, LPA is produced by activated platelets. Mauco et al. (9Mauco G. Chap H. Simon M.F. Douste B.L. Biochimie (Paris). 1978; 60: 653-661Google Scholar) showed that LPA is produced by human platelets after the cells were treated with exogenous phospholipase C from Clostridium welchii. LPA is also produced by platelets when they are activated by thrombin (10Gerrard J.M. Robinson P. Biochim. Biophys. Acta. 1989; 1001: 282-285Google Scholar, 11Eichholtz T. Jalink K. Fahrenfort I. Moolenaar W.H. Biochem. J. 1993; 291: 677-680Google Scholar). However, as discussed by Gaits et al. (12Gaits F. Fourcade O. Le B.F. Gueguen G. Gaige B. Gassama D.A. Fauvel J. Salles J.P. Mauco G. Simon M.F. Chap H. FEBS Lett. 1997; 410: 54-58Google Scholar), the amount of LPA produced in the latter two studies is too low to explain the LPA level in serum. Thus, the full contribution of platelets to serum LPA production is currently unknown. In the second pathway, LPA is converted from lysophosphatidylcholine (LPC) by lysophospholipase D (lysoPLD) activity (1Tokumura A. Prog. Lipid Res. 1995; 34: 151-184Google Scholar), which may occur in aged plasma or incubated plasma. The contribution of this pathway to the production of serum LPA is unclear, because little is known about the enzymatic properties of lysoPLD. LPA in serum and produced in platelets is a mixture of various fatty acids. LPA species with both saturated fatty acids (16:0 and 18:0) and unsaturated fatty acids (16:1, 18:1, 18:2, and 20:4) have been detected in serum, plasma, and activated platelets (10Gerrard J.M. Robinson P. Biochim. Biophys. Acta. 1989; 1001: 282-285Google Scholar, 13Xiao Y. Chen Y. Kennedy A.W. Belinson J. Xu Y. Ann. N. Y. Acad. Sci. 2000; 905: 242-259Google Scholar, 14Baker D.L. Umstot E.S. Desiderio D.M. Tigyi G.J. Ann. N. Y. Acad. Sci. 2000; 905: 267-269Google Scholar). Interestingly, these LPA species exhibit differential biological activities (15Tokumura A. Iimori M. Nishioka Y. Kitahara M. Sakashita M. Tanaka S. Am. J. Physiol. 1994; 267: C204-C210Google Scholar, 16Jalink K. Hengeveld T. Mulder S. Postma F.R. Simon M.F. Chap H. van der Marel G. van Boom J.H. van Blitterswijk W.J. Moolenaar W.H. Biochem. J. 1995; 307: 609-616Google Scholar, 17Hayashi K. Takahashi M. Nishida W. Yoshida K. Ohkawa Y. Kitabatake A. Aoki J. Arai H. Sobue K. Circ. Res. 2001; 89: 251-258Google Scholar) by differentially activating three LPA receptors, EDG2, EDG4, and EDG7 (18Bandoh K. Aoki J. Tsujimoto M. Arai H. Inoue K. FEBS Lett. 2000; 478: 159-165Google Scholar). These observations suggest that LPA species are biologically significant and are produced by diverse synthetic pathways. Serum LPA can be produced from phospholipid precursors either in membranes of blood cells or in plasma by sequential actions of phospholipases present in plasma or expressed by blood cells. In rats, two PLAs, secretory phospholipase A2 group IIA (sPLA2-IIA) (19Komada M. Kudo I. Mizushima H. Kitamura N. Inoue K. J. Biochem. 1989; 106: 545-547Google Scholar, 20Kudo I. Murakami M. Hara S. Inoue K. Biochim. Biophys. Acta. 1993; 1170: 217-231Google Scholar) and phosphatidylserine-specific phospholipase A1 (PS-PLA1) (21Sato T. Aoki J. Nagai Y. Dohmae N. Takio K. Doi T. Arai H. Inoue K. J. Biol. Chem. 1997; 272: 2192-2198Google Scholar, 22Aoki J. Nagai Y. Hosono H. Inoue K. Arai H. Biochim. Biophys. Acta. 2002; 1582: 26-32Google Scholar), are expressed predominantly in platelets (23Horigome K. Hayakawa M. Inoue K. Nojima S. J. Biochem. 1987; 101: 53-61Google Scholar). We previously showed that these two PLAs are involved in agonist-induced production of lysophospholipids such as LPC, lysophosphatidylethanolamine (LPE), and lysophosphatidylserine (LPS) in activated platelets by analyzing the phospholipid composition using specific inhibitors of sPLA2-IIA (24Yokoyama K. Kudo I. Inoue K. J. Biochem. 1995; 117: 1280-1287Google Scholar). In addition, other phospholipases capable of producing lysophospholipids, possibly LPC, have been identified in plasma. Two such phospholipases are lecithin-cholesterol acyltransferase (LCAT) and platelet-activating factor acetylhydrolase (PAF-AH). It has been suggested that part of the LPC present in blood is attributed to the transesterification of phosphatidylcholine (PC) and free cholesterol catalyzed by LCAT (25Sekas G. Patton G.M. Lincoln E.C. Robins S.J. J. Lab. Clin. Med. 1985; 105: 190-194Google Scholar). It is also possible that the plasma PAF-AH (26Tjoelker L.W. Wilder C. Eberhardt C. Stafforini D.M. Dietsch G. Schimpf B. Hooper S. Le T.H. Cousens L.S. Zimmerman G.A. Yamada Y. McIntyre T.M. Prescott S.M. Gray P.W. Nature. 1995; 374: 549-553Google Scholar) contributes to LPC production by hydrolyzing oxidized phosphatidylcholine (27Watson A.D. Navab M. Hama S.Y. Sevanian A. Prescott S.M. Stafforini D.M. McIntyre T.M. Du B.N. Fogelman A.M. Berliner J.A. J. Clin. Invest. 1995; 95: 774-782Google Scholar), which has been implicated in various pathological conditions. We have recently identified the above-mentioned lysoPLD by purifying the enzyme (28Umezu-Goto M. Kishi Y. Taira A. Hama K. Dohmae N. Takio K. Yamori T. Mills G.B. Inoue K. Aoki J. Arai H. J. Cell Biol. 2002; 158: 227-233Google Scholar). The identification and cloning of these phospholipases make it possible to examine their contribution to serum LPA production. In this study, to elucidate the one or more synthetic pathways for LPA in serum, we attempted to clarify the roles of blood cells, plasma, and the phospholipases in serum LPA production. Rabbit anti-rat platelet serum was purchased from Inter-Cell Technologies Inc. Bovine serum albumin (fatty acid free, A-6003) was purchased from Sigma. 1-Oleoyl (18:1)-LPA, 1-oleoyl-LPC, 1-oleoyl-LPE, 1-oleoyl-LPS, porcine liver LPI, sphingosylphosphocholine, and dioleoyl-PC were purchased from Avanti Polar Lipids Inc. (Alabaster, AL), and 1-[3H]oleoyl-LPA (18:1) was from Amersham Biosciences (Uppsala, Sweden). Monoglyceride lipase (MGL), glycero-3-phosphate oxidase (G3PO), and phosphocholine phosphodiesterase (GPCP) were kindly donated by Dr. S. Imamura (Asahi Chemical Industry Co. Ltd.). Horseradish peroxidase was purchased from Toyobo (Tokyo, Japan). Other chemicals were purchased from Wako Pure Chemical Industries (Osaka, Japan). Male Wistar rats (15 weeks old, 300–350 g) were purchased from Japan SLC. The rats were fed a standard rat chow until the time of the study. Blood was collected by cardiac puncture. For preparation of serum, the drawn blood samples were incubated at 37 °C for 60 min to allow blood coagulation, and the supernatant after centrifugation at 2300 × g for 20 min at 4 °C was used as “serum.” For plasma, blood was drawn in the presence of one-sixth volume of acid citrate dextrose (ACD, 3% citrate, 2.2% d-glucose, pH 6.0) and was immediately centrifuged at 2300 × g for 20 min at 4 °C. The supernatant was used as “plasma.” For preparation of platelet-depleted rats, anti-rat platelet serum (200 μl) was injected into the animals intravenously, and, after 6 h, serum was prepared as described above. Plasma samples from patients with familial LCAT deficiency (FLD) who were admitted to the Fukuoka Medical University Hospital were drawn as describe above. Written informed consent was obtained from each patient. Control plasma samples were drawn from healthy volunteers (22–45 years old). All blood samples were immediately stored at −80 °C to prevent additional formation of LPA. Blood was drawn from rats or human volunteers in the presence of one-sixth volume of ACD and was immediately centrifuged at 800 × g for 20 min at 4 °C. For platelet preparation, the supernatant (platelet-rich plasma) was further centrifuged at 2300 × g for 20 min at 4 °C, and the resulting cell pellet was washed twice with buffer A (12 mmcitric acid, 15 mm sodium citrate, 113 mm NaCl, 0.4 mm NaH2PO4, 12 mmNaHCO3, pH 7.4) and finally suspended in buffer A. For erythrocyte preparation, the cells in the pellet fraction of the ACD blood were washed with phosphate-buffered saline twice after the buffy coat was removed. White blood cells were isolated using density gradient (Nyco Prep 1.077 Animal (Daiichikagaku, Tokyo, Japan)) according to the manufacturer's protocol. The major cell populations were lymphocytes and monocytes. For experiments to examine LPA production by these cells, the cell numbers of erythrocytes, platelets, and white blood cells were adjusted to 650, 11, and 2.0 × 107 cells/ml, respectively, to match their normal concentration in blood. To evaluate LPA production in these cells, the cells were suspended in phosphate-buffered saline containing 0.1% fatty acid-free bovine serum albumin. Concentrations of LPA and LPC were determined by an enzyme-linked fluorometric method established in the present study. LPA concentration was determined by fluorometry of H2O2 using 3-(4-hydroxyphenyl)propionic acid (7.5 mm, Dojin, Tokyo, Japan) as a peroxidase donor (29Tamaoku K. Ueno K. Akiura K. Ohkura Y. Chem. Pharmacol. Bull. 1982; 30: 2492-2497Google Scholar) generated by the reaction of LPA samples with 10 units/ml monoglyceride lipase (MGL, Asahi Chemical Industry Co. Ltd., Shizuoka, Japan) and 10 units/ml glycero-3-phosphate oxidase (G3PO, Asahi Chemical Industry Co. Ltd.) in a buffer containing 50 mm Tris, 2 mmCaCl2, 0.2% Triton X-100, 0.07 unit/ml peroxidase (Toyobo, Tokyo, Japan), pH 7.4, in a total volume of 1500 μl. The fluorescence intensity of excitation at 320 nm/emission at 404 nm was measured with a fluorometer (Hitachi, Ibaraki), 5 min after mixing the samples. We detected LPA as low as 0.1 nmol and obtained linearity up to 10 nmol of LPA in this system. For determination of the LPA concentration in blood samples, LPA in samples (1 ml) was extracted by the method of Bligh and Dyer (30Bligh E.C. Dyer W.F. Can. J. Biochem. Physiol. 1959; 37: 911-917Google Scholar) under acidic conditions (by lowering the pH to 3.0 with 1n HCl) prior to the LPA quantification. Lipids in the aqueous phase were re-extracted and pooled with the previous organic phase. The extracted lipids were dried and dissolved in phosphate buffered saline containing 0.1% bovine serum albumin. The LPA content of this solution was quantified as described above. The recovery of lipids was monitored by the addition of trace amounts of 1-[3H]oleoyl-LPA to the samples. Based on recovery of 1-[3H]oleoyl-LPA, lipid recovery was always >95% under the above-described conditions. LPC concentration was determined by a similar method except that 10 units/ml phosphocholine phosphodiesterase (GPCP, Asahi Chemical Industry Co. Ltd.) was applied in the assay system. The concentrations of LPA were determined from a standard curve, after subtracting the tentative values obtained from the fluorescence intensities of the MGL (+)/G3PO (+) reaction from those of the MGL (−)/G3PO (+) reaction. Similarly, the concentration of LPC was determined from a standard curve, after subtracting the tentative values obtained from the MGL (+)/G3PO (+)/GPCP (+) reaction from those of the MGL (−)/G3PO (+)/GPCP (+) reaction. LysoPLD activity was assayed as described previously (28Umezu-Goto M. Kishi Y. Taira A. Hama K. Dohmae N. Takio K. Yamori T. Mills G.B. Inoue K. Aoki J. Arai H. J. Cell Biol. 2002; 158: 227-233Google Scholar). Briefly, samples (1–50 μl) were incubated with 1 mm LPC (from egg) in the presence of 100 mmTris-HCl (pH 9.0), 500 mm NaCl, 5 mmMgCl2, and 0.05% Triton X-100 for 1 h at 37 °C. The liberated choline was detected by an enzymatic photometric method using choline oxidase (Asahi Chemical, Tokyo, Japan), horseradish peroxidase (Toyobo, Osaka, Japan), and TOOS reagent (N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline, Dojin, Tokyo, Japan) as a hydrogen donor. To examine lysoPLD activity against LPE, LPS, and LPI (each at 1 mm), the formation of LPA in the reaction mixture was determined as described above. The recombinant enzymes used in this study (PS-PLA1 and lysoPLD) were expressed by a baculovirus system as described previously (28Umezu-Goto M. Kishi Y. Taira A. Hama K. Dohmae N. Takio K. Yamori T. Mills G.B. Inoue K. Aoki J. Arai H. J. Cell Biol. 2002; 158: 227-233Google Scholar, 31Hosono H. Aoki J. Nagai Y. Bandoh K. Ishida M. Taguchi R. Arai H. Inoue K. J. Biol. Chem. 2001; 276: 29664-29670Google Scholar). The recombinant proteins were partially purified from the culture supernatant of Sf9 cells infected with each baculovirus using heparin and Mono Q column chromatography (21Sato T. Aoki J. Nagai Y. Dohmae N. Takio K. Doi T. Arai H. Inoue K. J. Biol. Chem. 1997; 272: 2192-2198Google Scholar, 28Umezu-Goto M. Kishi Y. Taira A. Hama K. Dohmae N. Takio K. Yamori T. Mills G.B. Inoue K. Aoki J. Arai H. J. Cell Biol. 2002; 158: 227-233Google Scholar) and were dialyzed in phosphate-buffered saline (−/−) before use. sPLA2-IIA was purified from cell supernatant of thrombin-activated rat platelets as described previously (28Umezu-Goto M. Kishi Y. Taira A. Hama K. Dohmae N. Takio K. Yamori T. Mills G.B. Inoue K. Aoki J. Arai H. J. Cell Biol. 2002; 158: 227-233Google Scholar, 57). To evaluate the involvement of platelets in the production of serum LPA, we prepared platelet-depleted animals and determined their serum LPA level by a fluorometric method established in this study (see “Experimental Procedures”). The number of platelets in rats treated with rabbit anti-platelet serum was about 1% of that in control animals treated with control rabbit serum (Fig. 1B). The treatment with rabbit anti-platelet serum did not affect the numbers of other blood cells such as erythrocytes and white blood cells (data not shown). Under these conditions, serum LPA levels were 0.94 ± 0.11 μm in anti-platelet-treated animals and 1.95 ± 0.14 μm in control antibody-treated animals (Fig. 1A). Plasma LPA levels in these animals were 0.17 ± 0.02 μm (platelet-depleted) and 0.16 ± 0.02 μm (control). These results confirmed a previous report that the LPA level is high in serum but low in plasma (8Tigyi G. Miledi R. J. Biol. Chem. 1992; 267: 21360-21367Google Scholar). In addition, the present result shows that half of LPA in serum is produced by a platelet-dependent pathway. We next examined the contribution of platelets themselves to the production of LPA in serum by measuring the LPA produced by isolated platelets upon their activation. As shown in Fig. 1C, rat platelets produced and released LPA after they were activated by thrombin or by a calcium ionophore, A23187. However, the levels of LPA produced by these activators (0.10 ± 0.02 and 0.16 ± 0.01 μm, respectively) were too low to account for the LPA produced by the platelet-dependent pathway of LPA production in serum. The human platelets were also found to have a low ability to produce LPA upon activation (Fig. 2B). In view of the findings that much of the lysophospholipids, but not LPA, is produced in activated platelets (24Yokoyama K. Kudo I. Inoue K. J. Biochem. 1995; 117: 1280-1287Google Scholar) and that lysoPLD activity is detected in plasma of several mammalian species, including rat and human (28Umezu-Goto M. Kishi Y. Taira A. Hama K. Dohmae N. Takio K. Yamori T. Mills G.B. Inoue K. Aoki J. Arai H. J. Cell Biol. 2002; 158: 227-233Google Scholar, 32Tokumura A. Harada K. Fukuzawa K. Tsukatani H. Biochim. Biophys. Acta. 1986; 875: 31-38Google Scholar), we hypothesized that part of the LPA in serum is produced in two steps: generation of lysophospholipids in activated platelets and their subsequent conversion to LPA by lysoPLD. To test this possibility, we examined whether the full amount of LPA produced in the platelet-dependent pathway is detected when isolated platelets are activated in the presence of lysoPLD. As was observed previously (24Yokoyama K. Kudo I. Inoue K. J. Biochem. 1995; 117: 1280-1287Google Scholar), a high concentration of LPC was detected in the supernatant of activated platelets stimulated with thrombin or A23187(Fig. 2C). The addition of a physiological concentration of recombinant lysoPLD dramatically increased the amount of LPA in the supernatant of the activated rat or human platelets and slightly decreased the LPC level (Fig. 2B). In the presence of recombinant lysoPLD, the concentration of LPA rose to 0.63 ± 0.09 μm and 0.73 ± 0.12 μm when isolated rat platelets were activated by thrombin and A23187, respectively (Fig. 2B). These concentrations are comparable to the concentration produced in the platelet-dependent pathway (Fig. 1). Among various lysophospholipids detected in the activated rat platelets, only LPC has been shown to be a substrate of lysoPLD. However, as shown in Fig. 2A, lysoPLD hydrolyzed other lysophospholipids, such as LPE, LPS, and LPI, to produce LPA. These results clearly indicate that, in the platelet-dependent pathway for LPA synthesis, lysophospholipids produced by activated platelets are converted to LPA by the action of lysoPLD present in blood plasma. We further examined the roles of sPLA2-IIA and PS-PLA1 in lysoPLD-enhanced LPA production in activated rat platelets, because the two PLAs are predominantly expressed in the cells and have been implicated in lysophospholipid production in activated platelets (24Yokoyama K. Kudo I. Inoue K. J. Biochem. 1995; 117: 1280-1287Google Scholar). As shown in Fig. 3, addition of recombinant sPLA2-IIA or PS-PLA1 dramatically increased the LPA production evoked by addition of recombinant lysoPLD from activated platelets stimulated with either thrombin or A23187. This suggests that the two PLAs are involved in serum LPA production through their roles in supplying lysophospholipids to lysoPLD. We further determined whether erythrocytes or white blood cells are involved in serum LPA production. To examine LPA production in blood cells, we incubated erythrocytes, white blood cells, and platelets prepared from rat blood with a physiological concentration of recombinant lysoPLD. Of the different cell types examined, platelets were by far the most potent in producing LPA and LPC per cell (Fig. 4, A and C). However, a low but significant concentration of LPA (0.17 ± 0.02 μm) was detected in the culture supernatant of erythrocytes, when physiological cell numbers (6.5 × 109 cells/ml) were applied (Fig. 4B). LPC was also detected in the cell supernatant (Fig. 4C). Thus, lysophospholipids associated with erythrocytes, possibly LPC, can be a substrate for lysoPLD, which can explain part of the serum LPA production in the platelet-independent pathway. Both LPA and LPC are produced in plasma during prolonged incubation at 37 °C in rat (32Tokumura A. Harada K. Fukuzawa K. Tsukatani H. Biochim. Biophys. Acta. 1986; 875: 31-38Google Scholar). As shown in Fig. 5, we confirmed that incubation of plasma or serum both from rat and human at 37 °C increased the accumulation of both LPA and LPC in a time course-dependent manner. The formation of LPA in serum is much faster than that in plasma, especially in the initial period (1–6 h). LPC concentration in serum was slightly higher than that in plasma (Fig. 5), indicating that lysophospholipids accumulated in incubated serum can be derived both from plasma phospholipids and, as a result of blood coagulation, from platelet phospholipids. It is likely that lysoPLD converts these lysophospholipids to LPA. Consistent with this idea, the addition of recombinant lysoPLD to the plasma dramatically increased the formation of LPA and resulted in a smaller increase in LPC (Fig. 6). A similar result was obtained in incubated serum (data not shown). Addition of a divalent cation chelator such as EDTA or EGTA to the incubation medium almost completely inhibited the accumulation of LPA but did not affect the accumulation of LPC (Fig. 6, EGTA data not shown). This observation strongly indicates that LPC present or generated during the incubation in plasma is converted to LPA by lysoPLD activity, and it is compatible with the fact that the enzyme requires a divalent cation for its activity (33Tokumura A. Miyake M. Yoshimoto O. Shimizu M. Fukuzawa K. Lipids. 1998; 33: 1009-1015Google Scholar).Figure 6LysoPLD accelerates LPA formation in incubated rat plasma and is sensitive to a divalent cation chelator. Plasma was incubated at 37 °C for the indicated period either in the presence (closed circles) or absence (open circles) of lysoPLD, and the amounts of LPA and LPC formed were determined. The same reaction was performed in the presence of 10 mm EDTA and lysoPLD (closed squares) and in the presence of 10 mm EDTA and in the absence of lysoPLD (open squares). Recombinant lysoPLD was added to a final concentration that was ten times as much as that detected in rat plasma. The data are representative of three different experiments.View Large Image Figure ViewerDownload (PPT) We further examined the mechanism of LPC formation in incubated plasma. It has been suggested that part of the LPC present in blood is due to the activities of lecithin-cholesterol acyltransferase (LCAT) (25Sekas G. Patton G.M. Lincoln E.C. Robins S.J. J. Lab. Clin. Med. 1985; 105: 190-194Google Scholar) or platelet-activating factor acetylhydrolase (PAF-AH) activities (27Watson A.D. Navab M. Hama S.Y. Sevanian A. Prescott S.M. Stafforini D.M. McIntyre T.M. Du B.N. Fogelman A.M. Berliner J.A. J. Clin. Invest. 1995; 95: 774-782Google Scholar). The formation of LPC during incubation was insensitive to EDTA (see Fig. 8), which is compatible with the properties of the two plasma enzymes (34Dobiasova M. Schutzova M. Physiol. Bohemoslov. 1986; 35: 464-472Google Scholar, 35Satoh K. Imaizumi T. Kawamura Y. Yoshida H. Hiramoto M. Takamatsu S. Takamatsu M. J. Clin. Invest. 1991; 87: 476-481Google Scholar). To evaluate the contribution of these enzymes to the accumulation of LPC in plasma during incubation, we examined the plasma LPC level of human subjects deficient in LCAT (FLD) or PAF-AH. As was observed in plasma from normal subjects (Fig. 5), the LPC level increased during incubation at 37 °C in the PAF-AH-deficient plasma. On the other hand, LPC did not form at all in the plasma from LCAT-deficient patients (Fig. 7), which confirmed that LCAT is responsible for the accumulation of LPC in the incubated plasma. In addition, it was revealed that LPA accumulation was significantly suppressed in LCAT-deficient plasma but not in the control or PAF-AH-deficient plasma (Fig. 7). The LCAT-deficient plasma had a comparable level of LPC (∼100 μm) (Fig. 7), indicating that a part of the plasma LPC can also be produced by an LCAT-independent pathway.Figure 7LPA and LPC formation in plasma from LCAT - or PAF-AH-deficient subjects. Plasma samples from an LCAT-deficient patient (FDL, closed circles), a PAF-AH-deficient donor (open squares), and a healthy volunteer (open circles) were incubated at 37 °C, and the time course-dependent accumulations of LPA and LPC were determined. The data are representative of two different experiments.View Large Image Figure ViewerDownload (PPT) The present study was undertaken to clarify how LPA is produced in serum. There are at least four synthetic pathways for serum LPA (Fig. 8). The major two pathways identified in the present study are 1) secretion of lysophospholipids such as LPC, LPE, and LPS from platelets, followed by conversion of the lysophospholipids to LPA (Fig. 2) and 2) g
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