Original Paper JOL~lof
Biomedical Science
Received: September 16, 1999 Accepted: November 19, 1999
J Biomed Sci 2000;7:311-316
Phosphatidylethanol Stimulates Calcium-Dependent Cytosolic Phospholipase Az Activity of a Macrophage Cell Line (RAW 264.7) Chia-Yu Chang Kathryn R. Farreli Rodney C. Baker Department of Pharmacology and Toxicology, Universityof MississippiMedical Center,Jackson, Miss.,USA
Key Words
Phosphatidylethanol • Phosphatidic acid • Ethanol. Phospholipase A2, cytosolic • Phospholipase D. RAW 264.7 cells
Abstract
The synthesis of inflammation mediators produced from arachidonic acid is regulated primarily by the cellular concentration of free arachidonic acid. Since intracellular arachidonic acid is almost totally present as phospholipid esters, the concentration of intracellular arachidonic acid is primarily dependent on the balance between the release of arachidonic acid from membrane phospholipids and the uptake of arachidonic acid into membrane phospholipids. Cytosolic phospholipase A2 is a calciumdependent enzyme that catalyzes the stimulus-coupled hydrolysis of arachidonic acid from membrane phospholipids. Following exposure of macrophages to various foreign or endogenous stimulants, cytosolic phospholipase A2 is activated. Treatment with these compounds may also stimulate phospholipase D activity, and, in the presence of ethanol, phospholipase D catalyzes the synthesis of phosphatidylethanol. A cell-free system was used to evaluate the effect of phosphatidylethanol on cytosolic phospholipase A2 activity. Phosphatidylethanol (0.5 gM) added to 1-stearoyl-2-[3H]-arachidonoyl-sn-gly -
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cero-3-phosphocholine vesicles stimulated cytosolic phospholipase A2 activity. However, high concentrations (20-100 pM) of phosphatidylethanol inhibited cytosolic phospholipase A2 activity. Phosphatidic acid, the normal phospholipase D product, also stimulated cytosolic phospholipase A2 activity at 0.5 gM, but had an inhibitory effect on cytosolic phospholipase A2 activity at concentrations of 50 and 100 gM. Ethanol (20-200 mM), the precursor of phosphatidylethanol, added directly to the assay did not alter cytosolic phospholipase A2 activity. These results suggest that phosphatidylethanol alters the physical properties of the substrate, and at lower concentrations of anionic phospholipids the substrate is more susceptible to hydrolysis. However, at high concentrations, phosphatidylethanol either reverses the alterations in physical properties of the substrate or phosphatidylethanol may be competing as the substrate. Both interactions may result in lower cytosolic phospholipase A2 activity. Copyright © 2000 National Science Council, ROC and S. Karger AG, Basel
Introduction
Chronic ethanol treatment has been linked with a number of pathological conditions that are associated with disturbances in the synthesis or degradation ofbioac-
Rodney C. Baker Department of Pharmacology and Toxicology University of Mississippi Medical Center 2500 North State Street, Jackson, MS 39216 (USA) Tel. +1 601 984 1620, Fax +1 601 984 1637, E-Mail
[email protected]
tive compounds derived from arachidonic acid [22, 27, 28]. Arachidonic acid that serves as the precursor of bioactive lipids is stored primarily at the sn-2 position of membrane phospholipids, and the release of arachidonic acid from these pools is dependent on the activity ofphospholipase A2. There are at least three classes of phospholipase A2 isozymes: calcium-dependent cytosolic phospholipase A2, calcium-dependent secretory phospholipase A2 and calcium-independent phospholipase A2. Cytosolic phospholipase A2 has been identified as the key enzyme responsible for stimulus-induced hydrolysis of arachidonic acid from membrane phospholipid [24]. This enzyme is an 85-kD protein that is presented in the cytosol of a variety of cells, including macrophages, platelets, renal mesangial cells, and kidney. Stimulants that have been shown to stimulate cytosolic phospholipase A2 activity include calcium ionophores, phorbol myristate acetate, zymosan, lipopolysaccharide, interleukin-1 a, tumor necrosis factor-a, and 5-1ipoxygenase products [ 17, 21, 30, 31]. The regulation of cytosolic phospholipase A2 is dependent, in part, on phosphorylation of the enzyme, and translocation from the cytosol to cellular membranes [ 15, 20]. Both activities are supported by submicromolar concentrations of intracellular calcium. Higher calcium concentrations are required for enzyme activity [12, 29]. In addition to phosphorylation and cellular location, cytosolic phospholipase A2 activity is affected by the chemical/physical state of the membrane. The concentration of arachidonic acid and the specific phospholipid class in which it is located would obviously be expected to influence the release of arachidonic acid. The activity of cytosolic phospholipase A2 is generally highest with choline phospholipids as the substrate. Other phospholipid classes that have been shown to be cytosolic phospholipase A2 substrates include phosphatidylinositol, phosphatidylethanolamine, phosphatidic acid and phosphatidylserine. Using cell-free systems and lipid vesicles prepared from specific phospholipids, phospholipid mixtures, or phospholipid/detergent mixtures, the contribution of the physical state of substrates to modify cytosolic phospholipase A2 activity has been studied extensively [26]. The anionic phospholipids, phosphatidylserine, phosphatidic acid, phosphatidylinositol, phosphatidylinositol-4,5-bisphosphate, phosphatidylethanolamine, and a neutral lipid, diacylglycerol, were found to increase activity of a cytosolic phospholipase A2 isolated from a macrophage cell line (RAW 274.7) [14]. The anionic phospholipid, phosphatidic acid, has also been reported to stimulate secretory phospholipase A2 activity [10], but 2-acylami-
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no-alkylphospholipids, another anionic phospholipids, inhibits the activity of secretory phospholipase A2 [ 11]. A number of studies suggest that ethanol treatment increases the mobilization of [3H]-arachidonic acid [1, 6], and the increase in [3H]-arachidonic acid metabolism is due to increased phospholipase A2 activity [2, 7, 8, 32]. Ethanol treatment has also been shown to alter phospholipase A2 activity in subcellular fractions isolated from the treated animals [2]. Ethanol treatment may influence phospholipase A2 activity through a number of different mechanisms, including altering the physical/chemical properties of the membrane or availability of substrate. This study addresses the possibility that the anionic phospholipid, phosphatidylethanol, may stimulate cytosolic phospholipase A2 activity. When ethanol is present, phosphatidylethanol is produced by phospholipase D rather than the normal product, phosphatidic acid. Phosphatidylethanol is more metabolically stable than phosphatidic acid, and its presence or synthesis has been used extensively as a marker of phospholipase D activity. Phosphatidylethanol has been shown to have unique physical/chemical properties that increase the fluidity of natural and artificial membranes [18]. This phospholipid has been reported to be more effective than other ionic lipids in causing a curvature stress on the lipid bilayer [13]. The transfer of phosphatidylethanol between lipid bilayer is also much faster than that of other phospholipids and this transfer can occur in the absence of membrane proteins [25]. Only a limited number of studies have addressed the effects of phosphatidylethanol directly. In this study we reported that cytosolic phospholipase A2 was stimulated by low concentrations of phosphatidylethanol, but inhibited by concentrations of phosphatidylethanol above 20 gM. The calcium-independent phospholipase A2 was not affected by phosphatidylethanol.
Materials and M e t h o d s Materials RAW 264.7 cells were purchased from the American Type Culture collection, Rockville, Md. Fetal bovine serum was purchased from Summit Biotechnology, Fort Collins, Colo. 1-Stearoyl-2-[3H] arachidonoyl-sn-glycero-3-phosphocholine was purchased from NEN, Boston, Mass. All other chemicals were supplied by Sigma Chemical Co., St. Louis, Mo.
Cell Cultures The RAW 264.7 cells were purchased from American Type Culture collection. This cell line was established from an Abelson leukemia virus-induced tumor. The cells have a number of functions characteristic of mature macrophages including antibody-independent
Chang/Farrell/Baker
and antibody-dependent phagocytosis and the ability to produce a number of lipid mediators of inflammation. The cells were maintained in RPMI 1640 medium containing 10% fetal bovine serum (heat-inactivated), and in a humidified atmosphere of 5 % CO2/95 % air. The cells were subcultured every 4-5 days and cells used in this study were of passages 2-10.
PhospholipaseA2 Assay RAW 264.7 cells were seeded at 0.25 x 106 cells/cm 2 and maintained as described above. After 24 h the cells were collected and washed 3 times with cold HBSS buffer. The cells were suspended in homogenization buffer containing 10 mM Tris buffer (pH 8.0), 1 mM phenylmethylsulfonylfluoride, 50 gg/ml leupeptin, 100 gg/ml aprotonin, 1 mM EGTA, 10 mM NaF, 0.2 mM Na3VO4, and were broken by N2 cavitation after holding at 800 psi for 20 min. The cells were centrifuged at 400 g for 8 min to remove unbroken cells, and the supernatants were centrifuged at 100,000 g for 1 h to separate cytosol and membrane fraction. The supernatant (cytosol) was collected, and the protein content was determined following the method of Bradford [4]. Bovine serum albumin was used as the standard. Macrophage cytosolic phospholipase A2 activity was assayed by measuring the hydrolysis of 1-stearoyl-2-[3Hl-arachidonoyl-sn-glyc ero-3-phosphocholine. All phospholipids were added simultaneously, and the solvent was removed by a stream of N2 gas. The assay mixture, in addition to various concentrations of phosphatidylethanol or phosphatidic acid, contained 50 ttg protein, 10 mM 1-stearoyl2-[3H]-archidonoyl-sn-glycero-3-phosphocholine (50,000 dpm), 10 mM CaCI2, 5 naNrdithiothreitol (DTT), and 10 mM Tris buffer (pH 8.0). 5 mM DTT was added to inhibit activity of secretory phospholipase A2. 10 mM CaC12 was replaced by 5 mM EGTA when calciumindependent phospholipase A2 was determined. Samples were incubated at 37 ° C for 30 min. The reaction was terminated by extracting the lipid fraction [3] and the free fatty acid fraction and phospholipids were separated on silica gel G plates developed in a mobile phase of hexane, diethyl ether and acetic acid (70:30:1). [3H]-arachidonic acid radioactivity was measured by liquid scintillation spectroscopy.
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F i g . 1 . Relationship between incubation time and the hydrolysis of [3H]-arachidonic acid from 1-stearoyl-2-[3H]-arachidonoyl-sn-glyc ero-3-phosphocholine in the cell-free system and using artifical vesicles as substrate. Free [3H]-arachidonic acid was measured after 15, 30, 45 or 60 min incubation at 37 ° C. The incubation mixture contained 10 mM calcium, 5 mM DTT, 10 ~tM 1-stearoyl-2-[3H]-arachi donoyl-sn-glycero-3-phosphocholine, and 10, 50 or 100 gg cytosolic protein. The results are presented as mean + SE (n = 4 experiments). PLA2 = Phospholipase A2.
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Statistical Data Analysis The data are presented as means -+ SEM. The number of individual experiments is given in each figure or table. Statistical significance was determined using one-way ANOVA, followed by the Bonferroni t tests.
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The activity of cytosolic phospholipase A2 was measured by following the release of [3H]-arachidonic acid from 1-stearoyl-2-[3H]-arachidonoyl-sn-glycero-3-phosphocholine in the cell-free system. A constant or linear increase in phospholipase A2 activity was observed over 60 rain using 10 or 50 ~tg of cytosolic protein (fig. 1). In contrast, when using 100 ~tg cytosolic protein per assay, enzyme activity was not linear over the 60 rain (fig. 1). Specific cytosolic phospholipase A2 activity was significantly decreased as the concentration of cytosolic protein per assay was increased (fig. 2). Enzymatic activity at a
Effect of Phosphatidylethanol on Cytosolic Phospholipase A2
-
50 0
10
25
I 50
, E 75
100
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Fig. 2. Effect of cytosolic protein concentration on cytosolic phospholipase A2 (PEA2) activity. Cytosolic phospholipase A2 activity was measured after 30 min incubation at 37 ° C. The incubation mixture contained 10 mM calcium, 5 mM DTT, 10 gM 1-stearoyl-2[3H]-arachidonoyl-sn-glycero-3-phosphocholine, and 10, 25, 50, 75 or 100 ~tg cytosolic protein. The results are presented as mean + SE (n = 4 experiments).
J BiomedSci 2000;7:311-316
313
160 -
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140 Fig. 3. Effect of phosphatidic acid and phosphatidylethanol on cytosolic phospholipase A2 (PLA2) activity. Cytosolic phospholipase A2 activity was measured after 30 rain incubation at 37°C. The incubation mixture contained 10 mac calcium, 5 mM DTT, 10 btM 1-stearoyl-2-[3H]-arachidonoyl-snglycero-3-phosphocholine, 50 gg cytosolic protein, and phosphatidic acid or phosphatidylethanol 0, 0.1, 0.5, 1, 10, 20, 50 and 100 gM. The results are presented as mean -+ SE (n = 3-7 experiments). * p < 0.05, cytosolic phospholipase A2 activity of phosphatidic acid- and phosphatidylethanol-treated groups compared to control.
Table 1. Effect of phosphatidylethanol, phosphatidic acid and ethanol on the activity of calcium-independent phospholipase A2 Treatment
Phosph0iipase A2 activity pmol/mg/min
Control
13.5 -+3.2
Phosphatidylethanol, nmol 1 14.9-+3.5 l0 17.8-+2.5 20 14.4-+3.5 50 13.7-+2.8 100 8.2-+2.6 Phosphatidic acid, nmol 1 27.7+6.7 6 24.2 + 3.9 20 18.8+3.1 50 10.1+1.8 100 8.8-+0.7 Ethanol, mM 20 100
11.3+3.0 10.8+3.2
Calcium-independent phospholipase A2 activity was measured after 30 min incubation at 37 ° C. The incubation mixture contained 5 mM EGTA, 5 mM DTT, 10 laM 1-stearoyl-2-[3H]-arachidonoyl-sn-glycero-3phosphocholine and 50 gg cytosolic protein. The results are presented as mean + SE (n = 2-10 experiments).
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J BiomedSci 2000;7:311-316
Phosphatidic acid Phosphatidylethanol
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concentration of 10 gg cytosolic protein per 100 gl assay volume was 291.35 + 23.89 pmol/mg/min, which decreased to 182.11 + 46.02, 124.28 + 17.35 and 81.08 + 13.45 pmol/mg/min at 25, 50 or 100 ~tg cytosolic protein, respectively (fig. 2). The effects of adding phosphatidylethanol, phosphatidic acid or ethanol to 1-stearoyl-2-[3H]-arachidonyl-snglycero-3-phosphocholine vesicles were measured using 50 gg o f cytosolic protein and an incubation period o f 30 min at 37°C. The responses to the addition of phosphatidylethanol and phosphatidic acid were essentially identical. Cytosolic phospholipase A2 activity was increased from 90.64 + 5.16 pmol/mg/min of control to 141.16 + 6.02 and 127.04 + 13.27 pmol/mg/min in the presence of 0.5 g M phosphatidic acid and phosphatidylethanol, respectively (fig. 3). Both phospholipids inhibited cytosolic phospholipase A2 activity when concentrations of each phospholipid were increased (fig. 3). Cytosolic phospholipase A2 activities were 55.62 + 6.94 and 28.28 + 6.85 pmol/mg/min in the presence of 50 and 100 ~tM of phosphatidic acid, and were 43.02 + 6.90, 46.56 + 5.51 and 9.70 + 1.67 pmol/mg/min when 20, 50 and 100 g M of phosphatidylethanol were added, respectively. Ethanol, over a concentration range of 20-200 m M added directly to the assay mixture, had no effect on cytosolic phospholipase A2 activity. Cytosolic phospholipase A2 activity was 90.64 + 5.16 pmol/mg/min in control and 86.73 + 8.39 pmol/mg/min in the presence of 200 rnM ethanol.
Chang/Farrell/Baker
The addition of phosphatidylethanol had no effect on the activity of calcium-independent phospholipase A2. The activity of this phospholipase A~ enzyme was measured by adding a calcium chelator (EGTA) to inhibit the calcium-dependent phospholipase A2 and by adding DTT to inhibit the low molecular weight secretory phospholipase A2. Under these conditions phosphatidylethanol, phosphatidic acid, or ethanol did not significantly effect the hydrolysis of 1-stearoyl-2-[3H]-arachidonyl-sn-glycero-3-phosphocholine (table 1).
Discussion
Specific cytosolic phospholipase A2 activity decreased significantly as the concentration of protein used in the assay was increased. The lack of linearity with purified or partially purified phospholipase A2 enzymes from P338D1 and RAW 264.7 cells has been reported previously [14, 16]. It has been suggested that the decrease in specific activity may be simply due to product inhibition [16]. However, using the purified or partially purified preparations, products of the reaction, either lysophospholipids or free arachidonic acid, added directly to the assay system did not inhibit phospholipase A2 activity [141. A number of studies addressing cytosolic phospholipase A2 activity, measured in cell-free systems, and using crude cellular fractions or isolated enzymes, have demonstrated that cytosolic phospholipase A2 activity is sensitive to the organization or physical characteristics of the phospholipid substrates [9, 2 3]. Biological membranes are highly asymmetrical and zwitterionic phospholipids, phosphatidylchotine and sphingomyelin are located primarily on the exterior leaflet of the lipid bilayer. Anionic phospholipids, which include phosphatidic acid and the abnormal phospholipid phosphatidylethanol, are predominately incorporated into the inner portion of the membrane [5, 19]. The distribution in biological membranes is controlled or facilitated by enzyme transport mechanisms. The unequal distribution of anionic phospholipids is also seen in artificial membranes, although the basis of differential distribution between exterior and interior portions of artificial membranes is not well characterized [5, 19]. Most investigators have attributed the unequal distribution between bilayers to packing constraints and electrostatic repulsion. Phosphatidylethanol has a small and relatively lipophilic head group compared to the head group of other phospholipids. This physical characteristic causes phos-
Effect of Phosphatidylethanol on Cytosolic Phospholipase A2
phatidylethanol to rapidly accumulate in the inner leaflet of artificial vesicles or in the inner leaflet of isolated membrane fragments or lipid vesicles. The lipophiticity of the head group allows this portion of the phospholipid to partition into the interior of the lipid bilayer to some extent. The unusual distribution of phosphatidylethanol has the potential to change the curvature of the liposomes and may increase the area of the aqueous/lipid interface which should increase phospholipase A2 activity [26].The presence of phosphatidylethanol would also be expected to disrupt the packing of the phospholipid acyl chains, which also has the potential of making the substrate more susceptible to hydrolysis by cytosolic phospholipase A2. At very high concentrations of phosphatidylethanol or phosphatidic acid an inhibition of cytosolic phospholipase A2 activity was seen. The inhibition seen at the high concentrations of phosphatidylethanol may be due to more extensive alterations in the physical structure of the liposomes. The most likely explanation of inhibitory actions of high phosphatidylethanol or phosphatidic acid is, however, dilution of the radioactive substrate. Significant inhibition ofcytosolic phospholipase A2 activity was measured at concentrations of phosphatidylethanol or phosphatidic acid that were at least 2 times the concentration of the substrate, 1-stearoyl-2-arachidonyl-sn-glycero-3phosphocholine. Although cytosolic phosphotipase A2 has been reported to be most active with 1-acyl (or alkyl)2-arachidonyl-sn-glycero-3-phosphocholine as the substrate, other phospholipids can be metabolized by this enzyme [ 14]. This interaction would be expected to inhibit the hydrolysis of the radioactive substrate. Phosphatidylethanol and phosphatidic acid affect cytosolic phospholipase A2 activity measured in a cell-fiee system in a similar manner. Thus, cytosolic phospholipase A2 would not be expected to be immediately influenced by the activation of phospholipase D. However, phosphatidylethanol is more metabolically stable than phosphatidic acid, and may accumulate with extensive or repeated stimulation of phospholipase D. Chronic ethanol treatment has been shown to increase phospholipase A2 activity [7, 8, 32]. Thus, after long-term treatment of ethanol, phosphatidylethanol may contribute to increased cytosolic phospholipase A2 activity.
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Chang/Farrell/Baker
