Lysophosphatidylcholine Stimulates the Release of Arachidonic Acid in Human Endothelial Cells*

Jason T. WongDagger §, Khai TranDagger §par , Grant N. Pierce**, Alvin C. ChanDagger Dagger , Karmin ODagger , and Patrick C. ChoyDagger §§

From the Departments of Dagger  Biochemistry and Molecular Biology and ** Physiology, University of Manitoba, Winnipeg, Manitoba R3E 0W3, Canada and the Dagger Dagger  Department of Biochemistry, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Lysophosphatidylcholine (lyso-PC) is a product of phosphatidylcholine hydrolysis by phospholipase A2 (PLA2) and is present in cell membranes, oxidized lipoproteins, and atherosclerotic tissues. It has the ability to alter endothelial functions and is regarded as a causal agent in atherogenesis. In this study, the modulation of arachidonate release by lyso-PC in human umbilical vein endothelial cells was examined. Incubation of endothelial cells with lyso-PC resulted in an enhanced release of arachidonate in a time- and concentration-dependent manner. Maximum arachidonate release was observed at 10 min of incubation with 50 µM lyso-PC. Lyso-PC species containing palmitoyl (C16:0) or stearoyl (C18:0) groups elicited the enhancement of arachidonate release, while other lysolipids such as lysophosphatidylethanolamine, lysophosphatidylserine, lysophosphatidylinositol, or lysophosphatidate were relatively ineffective. Lyso-PC-induced arachidonate release was decreased by treatment of cells with PLA2 inhibitors such as para-bromophenacyl bromide and arachidonoyl trifluoromethyl ketone. Furthermore, arachidonate release was attenuated in cells grown in the presence of antisense oligodeoxynucleotides that specifically bind cytosolic PLA2 mRNA. Treatment of cells with lyso-PC resulted in a translocation of PLA2 activity from the cytosolic to the membrane fractions of cells. Lyso-PC induced a rapid influx of Ca2+ from the medium into the cells, with a simultaneous enhancement of protein kinase C (PKC) activity in the membrane fractions. The lyso-PC-induced arachidonate release was attenuated when cells were preincubated with specific inhibitors of PKC (staurosporine and Ro31-8220) or a specific inhibitor of mitogen-activated protein kinase/extracellular regulated kinase kinase (PD098059). Taken together, the results of this study show that lyso-PC caused the elevation of cellular Ca2+ and the activation of PKC, which stimulated cytosolic PLA2 in an indirect manner and resulted in an enhanced release of arachidonate.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

The release of arachidonate from phospholipids is the rate-limiting step in the synthesis of eicosanoids via the arachidonate cascade (1). Arachidonate and its metabolites possess diverse biological properties, many of which are related to vascular homeostasis (1). In endothelial cells, arachidonate is converted to prostacyclin, a potent vasodilator and platelet antiaggregator (2). Although different mechanisms have been proposed for the release of arachidonate in mammalian cells, the hydrolysis of the acyl chain at the sn-2 position of glycerophospholipids by phospholipase A2 (PLA2)1 is regarded as the primary pathway for this reaction (1, 3). In mammalian cells, several forms of PLA2 have been identified. Those that have been purified and well characterized include the "type II" 14-kDa secretory PLA2 (sPLA2) and the "type IV" 85-kDa cytosolic PLA2 (cPLA2) (for reviews, see Refs. 3-5). These two isoforms are products of distinct genes (5) and have different properties. The cPLA2 preferentially hydrolyzes phospholipid substrates containing arachidonate at the sn-2 position (6), while sPLA2 does not exhibit any preference with respect to substrate acyl composition. The sPLA2 requires millimolar concentrations of Ca2+ for maximum activity, while cPLA2 contains a calcium-dependent lipid binding domain and requires submicromolar levels for translocation to cellular membranes (6, 7). In stimulated cells, cPLA2 activity is enhanced by phosphorylation at serine 505 by mitogen-activated protein kinase (MAPK) (3, 8). Protein kinase C (PKC) also appears to play a role in the regulation of PLA2 activity, although PKC is not thought to directly phosphorylate cPLA2 in vivo (3, 9). Both isoforms are found in human endothelial cells and have been implicated in arachidonate release and prostacyclin production (10-13).

Lysophosphatidylcholine (lyso-PC) is a product of phosphatidylcholine hydrolysis by PLA2. This lysophospholipid possesses detergent properties at high concentrations (14) but is quickly metabolized or reacylated within cells (15, 16). Lyso-PC is a normal constituent of blood plasma (17), vascular tissue (18), and lipoproteins (19, 20), but its levels are greatly elevated in hyperlipidemia (21), atherosclerotic tissue (18), oxidized lipoproteins (19, 20), and ischemic hearts (22). A growing body of evidence has implicated lyso-PC in the pathogenesis of cardiovascular diseases. For example, lyso-PC in oxidized low density lipoproteins impairs vascular relaxation (20, 23, 24) and induces mitogenesis of macrophages (25). Lyso-PC is chemotactic for monocytes (26) and T lymphocytes (27). In endothelial cells, lyso-PC can induce the expression of genes for various growth factors (28, 29) and cellular adhesion molecules (30, 31). The perturbation of vascular endothelial function and recruitment of various cell types to sites of lesion have been implicated as early events in atherogenesis (32, 33). Thus, given its many biological properties, lyso-PC has been postulated to be an important causal agent in inflammation and atherosclerosis (34, 35).

The interactions among phosphatidylcholine, fatty acids, lyso-PC, and sPLA2 have been examined in in vitro kinetic studies (36). An abrupt increase in PLA2 activity after an initial lag period was observed in these studies. This pattern of activity was attributed to the accumulation of fatty acid and lysophospholipids, which together altered the organization of substrate vesicles (36). In light of the many biological effects of lyso-PC, we hypothesize that it can modulate PLA2 in intact cells. In the present study, the effects of lyso-PC on the release of arachidonate in endothelial cells was examined. The involvement of Ca2+, PKC, and MAPK in the modulation of PLA2 activity was examined.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- Medium 199 with Hanks' salt and L-glutamine, heat-inactivated fetal calf serum, and other standard culture reagents were obtained from Life Technologies, Inc. Type I collagenase was obtained from Worthington. Endothelial cell growth supplement was obtained from Collaborative Biomedical Products (Bedford, MA). Phorbol 12-myristate 13-acetate, staurosporine, para-bromophenacyl bromide, and all other chemicals were purchased from Sigma. PD098059 was a product of Calbiochem. [5,6,8,11,12,14,15-3H]arachidonate (230.5 Ci/mmol) was obtained from NEN Life Science Products, and 1-stearoyl-2-[1-14C]arachidonoyl-L-3-phosphatidylcholine (55 mCi/mmol) was obtained from Amersham Corp. Arachidonoyl trifluoromethyl ketone (AACOCF3) and H89 were obtained from Biomol Inc. (Plymouth Meeting, PA). Ro31-8220 was a gift from Roche Research Center (Welwyn Garden City, Hertfordshire, United Kingdom). Lysophospholipids and all lipid standards were obtained from Serdary Research Laboratory (London, Ontario, Canada). Thin layer chromatography plates (silica gel G) were products of Fisher. Anti-cPLA2 polyclonal antibody was a generous gift from Drs. J. L. Knopf and L-L. Lin of the Genetics Institute (Boston, MA). Anti-human sPLA2 monoclonal antibody was a product of Upstate Biotechnology Inc. (Lake Placid, NY).

Cell Culture-- Endothelial cells were harvested from human umbilical veins using Type I collagenase as described previously (37, 38). The cells were grown in flasks or culture dishes pretreated with 0.2% gelatin, in medium 199 (pH 7.4) supplemented with 25 mM HEPES, 30 µg/ml endothelial cell growth supplement, 90 µg/ml heparin, 10% fetal calf serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 1.25 µg/ml Fungizone. The cells were subcultured at a 1:3 ratio using 0.05% trypsin to free the cells from the culture ware. Near-confluent cell monolayers from the third passage were used for all experiments.

Radiolabeling and Challenge of Cells-- Cells were radiolabeled as described previously (39). Cell monolayers grown to near-confluence in 35-mm culture dishes were incubated for 20 h with 1 µCi/ml [3H]arachidonate in medium 199 containing 10% fetal calf serum. The cells were washed three times with HEPES-buffered saline (140 mM NaCl, 4 mM KCl, 5.5 mM glucose, 10 mM HEPES, 1.5 mM CaCl2, and 1.0 mM MgCl2, pH 7.4) containing 0.025% (w/v) essentially fatty acid-free bovine serum albumin. Aliquots of lysophospholipids were dissolved in chloroform/methanol (2:1, v/v). The solvent was evaporated under N2, and the lysophospholipid samples were then resuspended in HEPES-buffered saline containing bovine serum albumin.

Measurement of Arachidonate Release-- The arachidonate released from cells was determined as described previously (39). Briefly, the lysophospholipid was added to the cell culture and incubated for the prescribed period. The buffer was then removed and acidified with 50 µl of glacial acetic acid. A 0.8-ml aliquot was used for lipid extraction in a solvent mixture consisting of chloroform/methanol/water (4:3:2, by volume). Oleic acid was added as an internal fatty acid standard. The free fatty acid fraction in the organic phase was resolved by thin layer chromatography in a solvent system consisting of hexane/diethyl ether/acetic acid (70:30:1, v/v). The fatty acid fraction was visualized by iodine vapor, and its radioactivity was determined by liquid scintillation counting.

Binding of Lyso-PC to Endothelial Cells-- Endothelial cells were cultured on 60-mm plates and incubated with medium 199 containing 100 nM [14C]lyso-PC (57 nCi/nmol) for 15 min. The medium was removed, and the cells were incubated for 15 min with medium 199 (control) or medium 199 containing 10 µM lyso-PC (a 100-fold excess of nonradioactive lyso-PC). The media were subsequently removed, and the cells were dislodged from the culture dish in HEPES-buffered saline. Samples were taken for protein determination or scintillation counting.

Immunoblotting Analysis of Phospholipase A2-- Immunoblotting analysis of cPLA2 or sPLA2 was performed as described previously (39). Cell lysates containing approximately 50 µg of protein were subjected to sodium dodecylsulfate, 7.5% polyacrylamide gel electrophoresis. The protein fractions from the gels were transferred to nitrocellulose membranes and then allowed to react with a polyclonal anti-cPLA2 antibody or with an anti-sPLA2 antibody. The nitrocellulose membranes were then exposed to a goat anti-rabbit antibody that was coupled to horseradish peroxidase. The cPLA2 or sPLA2 bands were detected on film using a Western blotting detection reagent kit (from Amersham), which yields a fluorescent compound via a reaction catalyzed by the peroxidase.

Oligonucleotide Treatment-- The antisense oligonucleotides for group II PLA2 (ASsA2, 5'-GAT CCT CTG CCA CCC ACA CC-3') (40) and for cPLA2 (AScA2, 5'-GTA AGG ATC TAT AAA TGA CAT-3') (11) with phosphorothioate linkages were synthesized by the University Core DNA Services, University of Calgary (Alberta, Canada). Complementary sense oligomers were used as controls. Seventy-two hours prior to challenge with lyso-PC, the cells were incubated with medium containing 10 µM oligonucleotides. The cells were supplied with fresh medium containing 10 µM oligonucleotides at 24-h intervals thereafter. The presence of oligonucleotides did not affect cell viability or arachidonate labeling.

Determination of Phospholipase A2 Activity-- 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 60 min. The supernatant was designated as the cytosolic fraction, while the pellet was designated as the membrane fraction and resuspended in the buffer described above. PLA2 activity in the subcellular fractions was determined by the hydrolysis of 1-stearoyl-2-[1-14C]arachidonoyl-sn-glycero-3-phosphocholine to yield free radiolabeled arachidonate. The assay mixture contained 50 mM Tris-HCl (pH 8.0), 1.5 mM CaCl2, 0.9 nmol of 1-stearoyl-2-[1-14C]arachidonoyl-sn-glycero-3-phosphocholine (100,000 dpm/assay), and approximately 10 µg of protein in a final volume of 100 µl. The reaction mixtures were incubated at 37 °C for 30 min, and the reactions were terminated by the addition of 1.5 ml of chloroform/methanol (2:1, by volume). Total lipid was extracted, and the radioactivity of the arachidonate released was determined as described above. The amounts of protein in the samples were determined by the bicinchoninic acid method (41).

Monitoring of Intracellular Ca2+-- Changes in cytosolic free Ca2+ were monitored using the fluorescent Ca2+ indicator fura-2 as described previously (42). Briefly, monolayers grown on microscope coverslips were incubated in medium with 5 µM fura-2/AM for 30 min. Fura-2/AM is permeable to cells, and once inside the cells the compound is hydrolyzed by endogenous esterases to yield the cell-impermeable fura-2. The cells on the coverslip were transferred into a cuvette, rinsed with HEPES-buffered saline containing 0.025% bovine serum albumin, and immersed in the same buffer. Fluorescent signals were monitored on a SPEX fluorescence spectrophotometer at the excitation and emission wavelengths of 340 and 380 nm, respectively. Cells were then challenged with lyso-PC or A23187 for 10 min, and the ratio of the fluorescence at the two wavelengths was monitored as an indicator of changes in cytosolic Ca2+ levels. The isosbestic (cross-over) point of fura-2 remained constant during lyso-PC treatment.

Determination of PKC Activity-- 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% beta -mercaptoethanol, 10 µM benzamidine, 1 mM PMSF, 10 µg/ml leupeptin, and 10 µg/ml aprotinin) and were centrifuged at 1500 × g for 10 min. The supernatants were subjected to ultracentrifugation at 100,000 × g for 60 min to obtain the soluble and membrane fractions. Approximately 15-30 µg of protein from these fractions were used to determine PKC activity using a PKC assay kit (Amersham), which is based on the incorporation of 32P from [gamma -32P]ATP into a PKC-specific substrate peptide.

Statistical Analysis-- The data were analyzed using a two-tailed independent Student's t test. The level of statistical significance was defined as p < 0.05.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Lyso-PC Stimulates Arachidonate Release in Endothelial Cells-- To determine the effect of lyso-PC on arachidonate release, human umbilical vein endothelial cells were labeled with [3H]arachidonate in medium 199 containing 10% fetal calf serum for 20 h. The cells were rinsed, and then incubated with HEPES-buffered saline containing 0.025% bovine serum albumin and 0 or 50 µM lyso-PC for various time periods (Fig. 1A). Lyso-PC elicited a time-dependent arachidonate release, which reached a maximum at 10 min of incubation, after which arachidonate release was slightly diminished. A nominal amount of bovine serum albumin was required to bind the arachidonate that is released into the buffer. The optimal concentration of lyso-PC for the induction of arachidonate release was determined at bovine serum albumin concentrations ranging from 0.025 to 0.1% (w/v) (4-16 µM albumin). The effect of lyso-PC on arachidonate release was affected by the albumin concentration (Fig. 1B). Higher concentrations of lyso-PC was required to elicit a stimulation of arachidonate release at higher albumin concentrations. For example, at 0.025% albumin, the maximal stimulation of arachidonate release was observed at 50 µM lyso-PC. Lyso-PC at this concentration has been found to be nonlethal to endothelial cells (43), and we confirmed cell viability under the incubation conditions by the exclusion of trypan blue dye. Hence, these conditions were routinely used in subsequent experiments.


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Fig. 1.   Lyso-PC stimulates arachidonate release in endothelial cells. Cells were incubated with 1 µCi/ml of [3H]arachidonate in medium containing 10% fetal calf serum for 20 h. Cells were washed three times with HEPES-buffered saline containing 0.025% (w/v) bovine serum albumin prior to challenge. A, cells were challenged with 0 µM (bullet ) or 50 µM (black-square) lyso-PC in HEPES-buffered saline containing 0.025% bovine serum albumin for the indicated times. B, cells were challenged for 10 min with the indicated concentrations of lyso-PC in HEPES-buffered saline containing 0.025% (black-triangle), 0.05% ([black-square), or 0.10% (bullet ) bovine serum albumin (w/v). Arachidonate release was determined as described under "Experimental Procedures." Values represent means ± S.D. of three separate experiments.

Release of Arachidonate by Long Chain Lyso-PC and Other Lysolipids-- Initial experiments on the effect of lyso-PC on arachidonate release were performed using lyso-PC derived from egg lecithin. Since egg lysolecithin contains mainly saturated acyl species, we tested the ability of palmitoyl (C16:0)- and stearoyl (C18:0)-lyso-PC to stimulate arachidonate release. Fig. 2 shows that lyso-PC containing palmitoyl and stearoyl chains induced a high release of arachidonate. To determine if the stimulation of arachidonate release is specific to lyso-PC or if it is a property common to all lysolipids, we tested the effect of other lysophospholipids such as lysophosphatidylethanolamine, lysophosphatidylserine, lysophosphatidylinositol, and lysophosphatidate on arachidonate release. As shown in Fig. 2, lysolipids with head groups other than choline were minimally effective in the stimulation of arachidonate release. Based on these results, lyso-PC containing a palmitoyl (C16:0) chain was used in subsequent experiments.


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Fig. 2.   Stimulation of arachidonate release by lysolipids. Cells prelabeled with [3H]arachidonate were challenged for 10 min with 0 µM (Control) or 50 µM of the indicated lyso-PCs or other lysolipids. Egg, lysolecithin from egg; 16:0, palmitoyl-lyso-PC; 18:0, stearoyl-lyso-PC; LysoPE, lysophosphatidylethanolamine; LysoPS, lysophosphatidylserine; LysoPI, lysophosphatidylinositol; LysoPA, lysophosphatidic acid. Arachidonate release was then determined as in Fig. 1. Values represent means ± S.D. of three separate experiments.

Lyso-PC is an amphiphilic molecule and can incorporate into lipid membranes. Thus, we performed binding studies as described under "Experimental Procedures" to determine the nature of the association of lyso-PC with the endothelial cells. Cells were labeled with [14C]lyso-PC (100 nM, 57 nCi/nmol), followed by incubation with control medium (without lyso-PC) or medium containing 10 µM nonradioactive lyso-PC. The majority of radioactivity remained associated with cells in the presence of excess lyso-PC (results not shown), indicating that the association of lyso-PC with cells is mainly nonspecific.

Involvement of Phospholipase A2 in Lyso-PC-induced Arachidonate Release-- To determine whether the release of arachidonate is mediated by PLA2, we examined the effects of the PLA2 inhibitors para-bromophenacyl bromide (pBPB) and arachidonoyl trifluoromethyl ketone (AACOCF3), the latter of which specifically inhibits the cPLA2 (44). As shown in Table I, arachidonate release was significantly inhibited in those cells that were preincubated with these inhibitors prior to challenge with lyso-PC. The inhibition of arachidonate release by up to 62% by AACOCF3 indicates that the cPLA2 may be involved in the arachidonate release induced by lyso-PC. However, sPLA2 is also present in endothelial cells and may also participate in arachidonate release (10).

                              
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Table I
Inhibition of lyso-PC-induced arachidonate release by PLA2 inhibitors
Cells prelabeled with [3H]arachidonate were incubated with the indicated concentrations of AACOCF3 for 2 min or pBPB for 15 min prior to challenge with 50 µM lyso-PC for 10 min. Arachidonate release was then determined as described under "Experimental Procedures." Results are expressed as mean ± S.D. of three separate experiments.

To further delineate the type of PLA2 that was involved in the lyso-PC-induced arachidonate release, we used antisense oligonucleotides toward cPLA2 and sPLA2. These oligonucleotides were designed to bind specifically to the respective mRNAs and prevent the translation and synthesis of the enzyme protein (11, 40). Complementary sense oligonucleotides were used as negative controls. Cells were grown in the presence of sense or antisense oligonucleotides to either PLA2 isoform for 3 days prior to challenge with lyso-PC. Treatment of the cells with either sense or antisense oligonucleotides did not alter the total incorporation of [3H]arachidonate. However, lyso-PC-induced arachidonate release was significantly attenuated in cells grown in the presence of antisense oligonucleotides for cPLA2, compared with cells grown without oligonucleotides or with sense oligonucleotides (Fig. 3). The level of cPLA2 protein after the treatment with antisense cPLA2 oligonucleotides was determined by immunoblotting analysis with a polyclonal antibody for cPLA2. The level of cPLA2 protein was decreased (40% reduction) by the antisense oligonucleotide treatment (Fig. 4A). In cells treated with antisense sPLA2 oligonucleotides, the lyso-PC-induced arachidonate release was not significantly affected (Fig. 3), despite a 35% decrease in the sPLA2 protein level in those cells (Fig. 4B).


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Fig. 3.   Effect of antisense oligonucleotides for PLA2s on arachidonate release. Cells were cultured in the presence of oligonucleotides for 72 h as described under "Experimental Procedures." Cells were labeled with [3H]arachidonate for 20 h prior to challenge with 50 µM lyso-PC for 10 min. Control, cells cultured without oligonucleotides and not challenged with lyso-PC; No oligo, cells cultured without oligonucleotide treatment; ScA2, cells cultured in the presence of sense oligonucleotide for cPLA2; AScA2, cells cultured in the presence of antisense oligonucleotide for cPLA2; SsA2, cells cultured in the presence of sense oligonucleotide for sPLA2; ASsA2, cells cultured in the presence of antisense oligonucleotide for sPLA2. Values represent means ± S.D. of three separate experiments. *, p < 0.05, compared with cells cultured in the absence of oligonucleotides prior to being challenged with lyso-PC.


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Fig. 4.   Immunoblots of cPLA2 and sPLA2 in cells grown in the presence of sense or antisense oligonucleotides to cPLA2. Cells were cultured in the absence or presence of 10 µM sense or antisense oligonucleotides to cPLA2 or sPLA2 for 72 h. The levels of cPLA2 (A) or sPLA2 (B) in the cell lysates were quantitated by immunoblotting as described under "Experimental Procedures." A, control, cells cultured without oligonucleotides; ScA2, cells cultured in the presence of sense oligonucleotides for cPLA2; AScA2, cells cultured in the presence of antisense oligonucleotides for cPLA2. B, control, cells cultured without oligonucleotides; SsA2, cells cultured in the presence of sense oligonucleotides for sPLA2; ASsA2, cells cultured in the presence of antisense oligonucleotides for sPLA2.

Taken together, the inhibition of arachidonate release by antisense oligonucleotides for cPLA2 and by AACOCF3 indicate that the cPLA2 is involved in the lyso-PC-induced arachidonate release. Therefore, we assayed in vitro the PLA2 activity in subcellular fractions from control cells and from cells treated with lyso-PC. As shown in Table II, specific PLA2 activity was decreased by 52% in the cytosolic fractions of lyso-PC-treated cells, while activity in the membrane fractions was increased by 33%. The increase in the membrane PLA2 activity is consistent with the notion that the enzyme translocated to cell membranes. The membrane-bound enzyme is regarded as the active form, since the cell membrane contains the endogenous phospholipid substrates of cPLA2 (3). This notion was supported by an in vitro study, in which PLA2 activity was determined after the treatment of cells with the antisense cPLA2 oligonucleotides. The total PLA2 activity was reduced in those cells, with a corresponding (30%) decrease in the activity associated with the membrane fractions.

                              
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Table II
Effect of lyso-PC on PLA2 activity in endothelial cells
Cells were treated with 0 µM (-) or 50 µM (+) lyso-PC in HEPES-buffered saline containing 0.025% bovine serum albumin. Cells were lysed, and PLA2 activity was assayed in the cytosolic and membrane fractions as described under "Experimental Procedures." Results are expressed as mean ± S.D. of three separate experiments.

In separate experiments, the direct effect of lyso-PC on PLA2 activity was examined by adding lyso-PC to the PLA2 assays. Lyso-PC had no effect on PLA2 activity when added directly to the enzyme assays (results not shown). Thus, the enhancement of PLA2 activity observed in the membrane fraction of lyso-PC-treated cells was probably due to an indirect mechanism occurring in the intact cells. Therefore, we investigated the potential roles of Ca2+ and protein kinases on the induction of arachidonate release by lyso-PC.

Extracellular Ca2+ Is Required for Arachidonate Release by Lyso-PC-- Since both sPLA2 and cPLA2 are regulated by Ca2+ and since Ca2+ stimulates the translocation of cPLA2 to cell membranes (7), the role of Ca2+ in arachidonate release was investigated. Cells were challenged with lyso-PC in the presence of 0-1.5 mM Ca2+. As shown in Fig. 5, the induction of arachidonate release by lyso-PC was progressively suppressed at the lower Ca2+ concentrations. Arachidonate release was completely abolished when Ca2+ was absent from the buffer (the calcium-free buffer also contained 1 mM EDTA and 1 mM EGTA). Thus, the lyso-PC-induced arachidonate release was dependent on the Ca2+ concentration in the buffer. Lyso-PC has been shown to cause increases in intracellular Ca2+ concentrations (43). In the current study, treatment of the cells with 50 µM lyso-PC in the presence of 1.5 mM Ca2+ in the buffer caused an approximately 3-fold increase in the intracellular Ca2+ level (Fig. 5, inset). In the absence of extracellular Ca2+, neither lyso-PC nor the calcium ionophore bromo-A23187 was able to cause any change in cell Ca2+. It appears that an influx of Ca2+ from an extracellular source was a prerequisite for the induction of arachidonate release by lyso-PC and that the rise of cellular Ca2+ was not derived from an intracellular pool.


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Fig. 5.   Requirement of extracellular Ca2+ for lyso-PC-induced arachidonate release. Cells were labeled with [3H]arachidonate for 20 h and then challenged with 0 µM (bullet ) or 50 µM (black-square) lyso-PC for 10 min in the presence of the indicated Ca2+ concentrations. Arachidonate release was determined as described in Fig. 1. Values represent means ± S.D. of three separate experiments. Inset, cells were challenged with 50 µM lyso-PC in HEPES-buffered saline containing 0.025% bovine serum albumin, and the intracellular Ca2+ was monitored using fura-2 as described under "Experimental Procedures." A typical trace is shown.

Involvement of PKC and MAPK in the Stimulation of Arachidonate Release by Lyso-PC-- In addition to Ca2+, phosphorylation events have also been shown to play a role in the regulation of cPLA2 activity in a number of cell types (3, 45, 46). Thus, we investigated whether PKC or MAPK are involved in the lyso-PC-induced arachidonate release. Cells were pretreated with the PKC inhibitor staurosporine (47) or Ro31-8220 (48) prior to challenge with lyso-PC. For comparison, we also investigated the involvement of the cAMP-dependent protein kinase A by using the protein kinase A inhibitor H89 (49). Staurosporine and Ro31-8220 inhibited the arachidonate release induced by lyso-PC by up to almost 70% (Table III). In contrast, H89 did not cause any significant inhibition, up to a dose (1 µM) far exceeding its Ki (0.05 µM) for protein kinase A (49). Lyso-PC has previously been shown to modulate PKC activity in both cell-free and cell-based assays (50-52). Consistent with these findings, we observed that treatment of the cells with lyso-PC for 5 min caused a 58% increase in PKC activity in the membrane fraction of the cells (Table IV).

                              
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Table III
Effect of PKC and PKA inhibitors on lysoPC-induced arachidonate release
Cells prelabeled with [3H]arachidonate were treated with indicated concentrations of staurosporine, Ro31-8220, or H89 for 10 min prior to challenge with 50 µM lyso-PC for an additional 10 min. Arachidonate release was then determined as described under "Experimental Procedures." Results are expressed as mean ± S.D. of three separate experiments.

                              
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Table IV
Effect of lyso-PC on PKC activity
Cells were challenged with 50 µM lyso-PC or 200 nM phorbol 12-myristate 13-acetate (PMA) for 5 min. PKC activity in the membrane fractions of the cells was determined as described under "Experimental Procedures." Results are expressed as mean ± S.D. of three separate experiments.

The phosphorylation of cPLA2 by MAPK results in enhanced phospholipase activity, and MAPK is thought to be responsible for cPLA2 phosphorylation in vivo (8, 45, 46). Thus, we investigated the involvement of the MAPK pathway by determining the release of arachidonate in the presence of PD098059, an inhibitor of the MAPK/extracellular regulated kinase kinase (53). Cells were preincubated with the indicated concentrations of PD098059 for 30 min prior to challenge with lyso-PC (Table V). Pretreatment of cells with PD098059 resulted in as much as a 38% decrease in the arachidonate release induced by lyso-PC, relative to the arachidonate release caused by lyso-PC alone.

                              
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Table V
Effect of PD098059 on lyso-PC-induced arachidonate release
Cells prelabeled with [3H]arachidonate were treated with the indicated concentrations of PD098059 for 30 min prior to challenge with 50 µM lyso-PC for 10 min. Arachidonate release was then determined as described under "Experimental Procedures." Results are expressed as mean ± S.D. of three separate experiments.

It has been shown that lyso-PC can modulate the activities of adenylyl cyclase and guanylyl cyclase, leading to alterations of cAMP and cGMP levels within cells (54-56). To determine whether these cyclic nucleotides can affect the release of arachidonate induced by lyso-PC, cells were pretreated with forskolin, a direct activator of adenylyl cyclase, or with cell-permeable analogs of cAMP (8-bromo-cAMP) or cGMP (8-bromo-cGMP) prior to lyso-PC exposure. These compounds had little effect on lyso-PC-stimulated arachidonate release (data not shown). This result is consistent with the observation that H89 had no effect on arachidonate release (Table III) and supports the noninvolvement of cAMP- or cGMP-dependent protein kinases in the lyso-PC-induced arachidonate release.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

The present study was conducted to study the effects of lyso-PC on the release of arachidonate in endothelial cells. We found that exposure of the cells to lyso-PC containing long saturated acyl chains induced a dose-dependent increase in the release of arachidonate and that the effect was mediated through PLA2. Our findings support a model in which the induction of arachidonate release by lyso-PC is dependent on Ca2+ influx and the activation of PKC. These processes result in the stimulation of cPLA2 activity to give rise to an enhanced arachidonate release.

A major pathway for arachidonic acid release from agonist-stimulated cells is via hydrolysis of phospholipids by PLA2 (4). Within the PLA2 subtypes, the preference for arachidonate-containing substrates and the low (intracellular concentrations) requirement for Ca2+ of cPLA2 have led many investigators to believe that this isoform is the main enzyme responsible for arachidonate release (3). The present study shows that the arachidonate release induced by lyso-PC was inhibited in a dose-dependent manner by the PLA2 inhibitors pBPB and the cPLA2-specific AACOCF3. Furthermore, the arachidonate release by lyso-PC was also attenuated in cells grown in the presence of antisense oligonucleotides for the cPLA2. The observed enhancement of membrane-associated PLA2 activity is consistent with an activation and translocation of cPLA2 to membranes. The decrease in soluble PLA2 activity was not quantitatively recovered in the membrane fraction of the cell lysates. This result is not entirely surprising, since it was previously documented that the membrane component may interfere with the cPLA2 activity when assayed in vitro (57). This phenomenon was also observed with other enzymes upon their association with membranes and was attributed to a reduced accessibility of exogenous radioactive substrate to the enzyme and/or to a "dilution" effect on the exogenous radioactive substrate; i.e. the presence of endogenous membrane phospholipids lowered the effective specific activity of the radioactive substrate (57-59). The normal Ca2+ concentration in endothelial cells is approximately 70 nM (60, 61), and in the current study lyso-PC was found to cause a 3-fold increase in the intracellular Ca2+ level. The increased Ca2+ level induced by lyso-PC is similar to those Ca2+ concentrations that were shown to cause the association of cPLA2 with membranes (7, 57). We conclude that the cPLA2 is involved in the lyso-PC-stimulated arachidonate release.

The contribution of sPLA2 was also considered. Although antisense oligonucleotides for this isoform caused a reduction in sPLA2 protein, a corresponding attenuation of the lyso-PC-induced arachidonate release was not detected in those cells. This result suggests that the sPLA2 does not contribute significantly to the arachidonate release stimulated by lyso-PC. This finding is in accord with studies in which hormone-stimulated arachidonate release and eicosanoid production was attributed to cPLA2 but not sPLA2 (11-13). It is interesting to note that the involvement of both the cytosolic and secretory PLA2 subtypes in the release of arachidonate for prostacyclin synthesis has also been reported (10).

Lyso-PC, with the participation of diacylglycerol, phosphatidylserine, and Ca2+ (50-52), has been shown to modulate PKC activity both in vitro and in vivo. The increase in intracellular Ca2+ caused by lyso-PC may contribute to the enhancement of membrane-associated PKC activity. Although cPLA2 is an in vitro substrate for PKC, the direct phosphorylation of cPLA2 by PKC does not result in enhanced phospholipase activity (8), nor has PKC been shown to directly phosphorylate cPLA2 in vivo. However, PKC is a known activator of the p42/p44 MAPK signaling cascade via phosphorylation of Raf-1 (62). Our results with the MAPK/extracellular regulated kinase kinase 1 inhibitor PD098059 implicate the involvement of the p42/p44 MAPK cascade in the arachidonate release by lyso-PC. The concentrations of PD098059 used in this study (up to 30 µM) were similar to those used to almost completely inhibit the activation of MAPK/extracellular regulated kinase kinase 1 and to inhibit the activation of p42 MAPK by up to 80% (53, 63). The partial inhibition of arachidonate release by PD098059 suggests that lyso-PC may also act through pathways other than the recruitment of p42/p44 MAPK. For example, the p38 MAPK is thought to participate in the activation of cPLA2 by agonists (64, 65).

Reported plasma concentrations of lyso-PC range from approximately 130-150 µM in healthy subjects (66, 67) to 1.7 mM in hyperlipidemic patients (21), while reported concentrations of human serum albumin ranged from approximately 185 to 850 µM (68, 69). These figures would correspond to theoretical molar lyso-PC:albumin ratios in serum that range from 0.15 to 9.2. In our studies, arachidonate release was stimulated by lyso-PC at concentrations that correspond to lyso-PC:albumin ratios of 6.2-25. We selected a lyso-PC:albumin ratio of 12.5 (50 µM lyso-PC and 0.025% or approximately 4 µM albumin) for the subsequent experiments, because this ratio represented the lowest lyso-PC concentration that elicited the maximum effect on arachidonate release. Although the complexities of other serum components would probably complicate the in vivo situation, the lyso-PC:albumin ratios used in this study may mimic conditions found in physiological or pathophysiological situations.

Lyso-PC is a natural amphiphile and incorporates into lipid membranes and affects membrane fluidity and permeability (14, 70, 71). Indeed, lyso-PC (at concentrations higher than those used in this study) has been used as an agent for permeabilizing cells (72). However, the detergent properties of lyso-PC do not fully account for its myriad biological effects. For example, lyso-PC increases intracellular Ca2+ levels (43, 73, 74) and yet inhibits receptor-mediated Ca2+ mobilization (52, 74). It exhibits both vasorelaxant (75) and vasoconstrictive (23) properties. It perturbs nitric oxide synthase mRNA and protein levels in endothelial cells, and up- or down-regulation is dependent on the concentration and incubation conditions used in each study (76-78). Lyso-PC increases the expression of various growth factors and adhesive molecules in endothelial cells (28-31), and it was shown recently that lysophosphatidylcholine can modulate gene expression independently of PKC and MAPK (31, 79, 80). In our study, lyso-PC was the only lysolipid tested that stimulates arachidonate release, despite the fact that other lysolipids also possess detergent properties (81, 82). Furthermore, the stimulation of arachidonate release by lyso-PC parallels earlier observations that the ability of lyso-PC to stimulate PKC is unique among lysolipids (50, 51). The findings of the current study demonstrate a novel role of lyso-PC in the modulation of endothelial cell functions.

It is clear from this study that lyso-PC may modulate a pathway that is responsible for its generation in vivo. It is therefore tempting to speculate that the activation of phosphatidylcholine hydrolysis by PLA2 could be regulated via a positive feedback mechanism that is mediated by its product lyso-PC. Lyso-PC may thus function as an intracellular messenger molecule. A prolonged activation and overactivation of the system may subsequently create adverse effects to the cells. Hence, the physiological consequences of the stimulation of arachidonate release in endothelial cells by lyso-PC will be an interesting area for further study. Since arachidonate and its metabolites have many biological properties related to vascular homeostasis, the perturbation of arachidonate release by lyso-PC may be a further mechanism whereby this lysolipid could contribute to vascular dysfunction.

    ACKNOWLEDGEMENTS

We thank Monroe Chan and Thane Maddaford for excellent technical assistance. We thank the staff at the Labor Unit of Women's Hospital in Winnipeg for providing umbilical cords for cell isolation. Ro31-8220 was a generous gift from Roche Research Center. The cPLA2 polyclonal antibody was a generous gift from Drs. J. L. Knopf and L-L. Lin of the Genetics Institute (Boston, MA).

    FOOTNOTES

* This work was supported by grants from the Heart and Stroke Foundation of Canada and the Medical Research Council of Canada (for Ca2+ determinations).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ The first two authors contributed equally to this work.

Recipient of a Research Traineeship from the Heart and Stroke Foundation of Canada.

par Recipient of a Medical Research Council Fellowship.

§§ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Faculty of Medicine, University of Manitoba, 770 Bannatyne Ave., Winnipeg, Manitoba R3E 0W3, Canada. Tel.: 204-789-3723; Fax: 204-789-3900.

1 The abbreviations used are: PLA2, phospholipase A2; sPLA2, secretory phospholipase A2; cPLA2, cytosolic phospholipase A2; pBPB, para-bromophenacyl bromide; AACOCF3, arachidonoyl trifluoromethyl ketone; PKC, protein kinase C; MAPK, mitogen-activated protein kinase; lyso-PC, lysophosphatidylcholine.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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