- Email: [email protected]
Eicosapentaenoic acid: Its effects on arachidonic acid metabolism by cells in culture
Eico~m& arachi&mic in culture* Lawrence
acid: Its effwzts on acid methoilism by cells
Levine, SC.%**
and Nancy Worth Waitham,
Mass.
Some eukatytic, ceils in culture synthesize u varieq qf 1ipoxygvgcna.w und!or cylooxygenasr products when stirnuluted by appropriate agonist.c Under normal nutritional conditions, these fatty ucid (PUFA) 5, 8, 11, lo-eic,osatetraenoi( product.s are derived from the polvunsuturated a(id (ET.4). urachidonic acid. which before metubo(isn1 ~nu,st he liberawd.from cellular lipids b> &ester@cation. Jf’ the <,elluiar lipids are prelouded with 3,X, 1 I , I J. / 7-eicosclpent(lPnoic ucid I EPA4i alEd cells are then stimulated to metaboiize the PUFA P, JCIYJIJof c~ycloox~~enase and lipoxygcnnse products synthesized are altered. C~c~I~~oqv~~~n(~sc productr\ dec,rease, while lipoxygenuse product.5 are not .signifcantl~ ajyected and ~nq even increase. The dec,rease in the production of cylooxygenase products results ,fiom redu(,ed utiiizution of the substrute (ETA). Decreased prostaglandin production by rat basophil teuketnia-I wit preloaded with EPA and rctdiotaheled with pHH]E;TA and [“CJEP.4 (an u1.w be denwnstr.crted tg high-pe$ortnance liquid ~,hrorrrrcto~ruphi~~ w~~!\.ws ctf [“HI- and [’ ‘C] radiolabeled nwtabolites in c~utturc ,juids qf cells ~riniutated lo metaho~i~c PIIFA bv the CO“ - iorzophr~rc~ ,42.1 I&‘. i .I ilI.LErRGY Cl.l,V ~MMlJhVL ‘i
.I.?i,,
!#J
j
The metabolism of PUFAs through cyclooxygenase and lipoxygenase pathways by cells in which the content of two main classes of PLJFAs (w-3 and w-6) varies qualitatively and/or quantitatively appears to have biologicalIy important consequences. i -” The effects of EPA on cyciooxygenase activity have been studied extensively because of the findings of decreased platelet function and cardiovascular diseases in Greenland Eskimos.’ Such decreases have been associated with increased levels of the w-3 class of PUFAs in the diet.’ It has been suggested that these biologic effects reflect metabolism of PUFA.” We have found that the levels of some cyclooxygenase products in peripheral sera of the NZB X NZWF,
Supported by National Institutes ol Health Grant Nos. GM 17256 md CA l-7 309. Rrp~int requests: Lawrence Levine, Sc.D., Department of Riochemistr). Brandeis University. Waltham, MA 02253, Publication No. 1486. Department of Biochemistq. Brandeiq, ilnivers~. Wattham, Mass. ’ .Amencan Cancer Society Research Professor of Biochcmistr) !AwarJ PRP-21)
Pli FA:
EPA. MEM: HE’lx a i: KlA, HPLC, SRS: PG. ‘i’k EGF~ ADH, ‘r PA KHI.: bl-A. I I-r-L.1
Polyunsaturated fatty acid 5,X, 1I, 14,17-Eicosapentaenoic acid Minimal essential medium Hydroxy-eicosatetraenoic acid Leukotriene Radioimmunoassay High-performance liquid chromatography &Sulfidopeptide-containing leukotriene Prostaglandin Thromboxane Epidermal growth factor Vasopressin : 2-O-Tetradecanoyl-phorbo- 1Sacetate Rat basophil leukemia 5.8, I 1. id-Eicosatetraenoic acid j t-l W/IS isomer of a peptidoleukotriene
Grain* and in peripheral plasma and some tissues of the Swiss albino strains? of mice that are fed menhaden oil diets decrease significantly, while levels of lipoxygenase products do not. Such analyses of ‘Robinson DR. Levine 1,: Unpublished observations. TTashjian AH Jr. Voelkel EF. Robinson DR, Levine L: linpubiichetl ohscrvatlons.
VOLUME NUMBER
74 3, PART 2
Effects of eicosapentaenoic
2
6
B
7
6-KETO-pGF#
PGE2
acid
431
mF2a
O.l!j
t
MEM
Serum
TPA
EGJ-ADI
MEM
TW
EGF-AtNi
MEM
TF’A
EGF-ADH
FIG. 1. Effect of EPA on PG production by rat liver cells (the C-9 cell line) after stimulation (8 x 10” cells/60 mm dish) of PUFA metabolism by 10% fetal calf serum (MEM), TPA (0.01 pglml), and EGF (0.1 pglml) plus ADH (0.06pM) for 20 hours. The C-9 liver cells were preloaded with EPA, 15 cLgl2 x IO5 (El), or 5 cLg/2 x IO5 cells ( ), or were not preloaded (a). EPA treatment is nontoxic as’ measured by trypan blue exclusion. Each bar is the mean value for groups of four dishes and brackets represent standard deviations. Where there are no brackets, a pool was used and the RIA was performed in duplicate.
arachidonic acid metabolites do not specify the source of the substrate; i.e., is the PUFA derived from plasma lipoproteins or cells, and if cells, which cells? We have now analyzed culture fluids of several types of cells preloaded with EPA for metabolic products of both cyclooxygenase and lipoxygenase pathways. Preloading of cellular lipids with EPA results in decreased production of cyclooxygenase products and, in cells that synthesize lipoxygenase as well as cyclooxygenase products, the decrease of cyclooxygenase products is accompanied by unaltered or even increased levels of lipoxygenase products.
MATERIAL AND METHODS Tissue culture. Sourcesand growth condifions for the cell lines used in this study have been described.6-10 Exponentially growing cells were treated with 1 ml trypsin (0.5 mg)/ethylenediaminetetra-acetate (0.2 mg) solution and cells were seeded into tissue culture dishes with Eagle MEM containing 10% fetal calf serum, 2mM L-glutamine, penicillin (250 U/ml), and streptomycin (250 $J/ml).
After 24 hours of incubation, cells were washedtwice with the MEM. Cells then were incubated with MEM containing penicillin and streptomycin in the presence or absence of the agonist. For radiolabeling of the cells, r3H] arachidonic acid (.5,6,8,9,11,12,14,15-[3H(N)], 87.4 Ciimmol) and
[Y]EPA
(5,8,11,14,17-[‘4C(U)], 212 mCi/mmol; New
England Nuclear, Boston, Mass.) were added to cells at the time of seeding. After incubation for 24 hours, the unincorporated [3H]arachidonic acid and [Y]EPA were removed by the washing procedures. For preloading cells with EPA, EPA (Sigma Chemical Co., St. Louis, MO.) was also added (from a 50 mg/ml ethanol stock) to cells at the time of seeding, and 24 hours later the unincorporated EPA was removed during the washing procedure. Ethanol alone had
40
45
50
40
45
50
Fti!!TUN
FIG. 2. lmmunochromatography of tissue culture extracts of the C-9 liver cells before (A) and after (A) preloading with EPA (5 pg12 x lo5 cells). Cells (8 x 105/60 mm dish) were stimulated to metabolize PUFA by incubation with TPA (0.01 pglml) for 20 hours.
no effect on the prostaglandins, HETEs, or LTs synthesized by control cells. The quantity of EPA used for preloading the cells varied from cell to cell and was recorded in each experiment. After stimulation, cell culture media were collected and centrifuged to remove floating cells and cellular debris and were assayed by RIA for cyclooxygenase and lipoxygenase products. HPLC. HPLC was performed by reverse phase on a p-Bondapak phenyl column (7.8 X 300 mm, Waters Associates). The Waters Model 6000A pumps, Model 660 solvent programmer, and Model U6K injector were used for
HPLC. Each sample was run on a linear gradient program from 100% solvent A to 100% solvent B for 110 minutes at a flow rate of 1 ml/min, and 1 ml fractions were collected. Solvent A consisted of 93.4% O.OlM phosphate buffer (pH 7.4), 6% methanol (HPLC grade, Fisher), and 0.6% tert-
J ALLERGY
432 L.evine and Worth TABLE I. Stimulation of PUFA metabolism by cells in culture on PUFA metabolism ._ --_ .-.. _.~.~. ~._.~-.--_--- -___I____. “. lmmunoreactive Cell
Stimulus
Rat glomerular epithelium Methylcholanthrcne-transformed rar tibrobtaht ~MC-5 i Mouse fibro sarcoma tHSDM,j
MEM ‘E4.
0. I p#lll
A23187. 2&M MEM Serum 15% fetal calf ) A231X7. If*%4
and effects of EPA preloading -.
PGE, hglml) -...__l_--
EPA preloaded
CLIN. !MMlJNOL. SEPTEMBER 1984
-.
--
Control
lmmunoreactive
PGF,, (nglml) Control
EPA preloaded
Cl.IO
o.th
(1 04
0.0’)
0. I:!
0.24
0.06
0.24
0. so 1,18 r 0.259 (4) x.53 2 0.1314(4) ‘.I0 + 0,822 t31
1.90 0.86 0.7?. 2.19 ” 0.336 (4) 0.14 s 0.030 (4) 0.23 i 0.015 (4) I I.04 c 0.717 (4) 1.64 i- 0.152 (4) 2. IO +- 0. IS2 (4) 7 63 + 0.65 14) 0.28 + 0.060 (4) 0.90 t 0.063 (4)
MEM 0.61 L 0.124 (Ij 1.55 I!Z0.30 (4) 0.09 Serum (5% fctai IO.85 i- 2.490 (4, 13 19 t 0.819 (4) 2.08 calf: iZ.hY I+ 0.925 (4) 0.34 A’3187. 2).&f 2.89 i- 0.968 (41 MEM 0.76 T 0.091 (3) 0.99 z!z 0.066 (4) Rat mesangial ADH. ! FM I .Yh .+ 0. I 13 (4) .! 08 rt 0.30.5 (4) MEIM *: 41.i 0 65 z 0.1 15 141 Dog kidnq TP4. 0.001 ~giml I.71 .T 0.170 (3) -1.74 ? 0.408 (4) 1.17 epithelium Norepinephrinc 1 !).17 .I 0.061 C4) 0.81 -+ 0.100i4) ~MDCK I 5OJdf 0,x 0.3.: Mouse tibroblast ,MEM j 3’1‘31 Melittin. &g/m! ! .30 7 0.084 (3) 2. IO i 0.1’3 (3) 0.65 AL3 187. 2)LM OYY + 0.13‘J (31 I .6X + 0.1 17 (3‘1 0.32 I{ovmc aort;k MEM m00e-l Tl’A. 0.01 kg:ml muwli: EsiF. 0 OS tJ-piml. dnd rerotonin i .O p,M Monocytic leLJ MEM 0.06 --i 0.010 (4~ 0. I4 ” 0.043 (4) 0.02 i;cmi;i ( 1 / I i i TPA. 1.O w&ml 11.2I 1 0.039 (4 I ii,63 3 0.035 (4) 0.03 A23 I 87, 2pM 0 so -i- 11.039(3) 13.75 .+ 0.04Y (4) 0.18 -- .---.. -- -...-.-. ..--.-. _-.----__ “---------.---__-1__--_-._....---- --.---I..-I_...
t 0.020 (4) 0.24 t 0.038 (4) -c 0.356 (4) 2.90 ?Y 0.173 (4) 1 0.055 (4) 1.49 + 0.065 (4)
0.21 f 0.027 (4) 0.1 ” 0.023 (4) 5.0s t 0.618 (4) 0.33 rt O.OXI (4) i 0, 1 0.036 t 0.105 (3) t 0.057 (3)
0.092 1.96 k 0.170 (3) 0.84 + 0.038 (3)
rr 0.002 (4) 0.03 + 0.004 (4) i 0.003 (4) 0. 14 t 0.014 (4) r?- 0,017 (4J 0.44 t 0.034 (4) ____-
!):rta are % i- SD. The number ot dishes is rndicated m parentheses. Where onI\ _ ;: single value IS given. duplicate dishes were assayed and the average is presented. The EPA concentration used to preload the cells ranged from 10 to 50 pg/ 2 X 10’ cells during the seeding procedure, in which 5% fetal calf serum wiis present. With most bur not a11 cella. controi and EPA-loaded cells were counted and +xamincd t’or trypan hluc exclusion. Under the conditions of prelnadirq. nr, cell toxicity was recorded
amyi-alcohol (Aldrich) (percentage by volume). Solvent B :&it> 99.4%
tuethzmol
with
0.6%
trrf. amyl-.alcohol.
Stan-
dsrd cyclooxygenase products were obtained from The UpJohn Co. (Kalamazoo, Mich.), HETE WG obtained from Dr. Walter Hubbard. Vanderbilt University. and the l-T\ ,tnd SRSs were obtained from Dr. Roberl A. Lewis. Harmd Medical School Sc;ro/o~ic~ ~nnl~.~r,,\. Culture Ruids or the appropriate HPLC fmctions were assayed by RlAs with antibodies directed toward PC&, PGF?,a, 6-keto-PGF,,,: TxB,, PC&. METES. and SRSx. Dr. E. C. Hayes of Merck Institute for Therapeutic Research supplied the anti-SRS used in this h~udy. Serologic specificity of this antiserum has been described.” The radiolabeled haptens were purchased from New England Nuclear. RIA procedures and serologic spec:kities of Ihe other RIA systems have been published ‘:
When the unsaturated fatty acid metabolites were to be meaby RIA after their resolution by HPLC. solvents were removed either under a nitrogen stream or by lyophilization, dnd residues were dissolved in Tris (hydroxymethylaminomethane) buffer. xtred
RESULTS Rat liver cells (the C-9 cell line) synthesize
PGI,,
PGE2, and PGF,,; more than 90% of these synthesized cyclooxygenase
products
are PGIZ.’ The C-9
liver cells can be stimulated to metabolize arachidonic acid by incubation with several agonists including Wmor promoters, growth factors such as EGF, platelet-derived growth factor, and serum, and synergisticafy by incubation with EGF and ADH, EGF
VOLUME NUMBER
74 3, PART 2
lmmunoreactive
Effects of eicosapentaenoic
6-keto-PGF,,
EPA preloaded
0.32 k 0.070(4) 0.36 it 0.061 (4) 6.65 +- 0.26 (4)
0.04 1.55 0.11 < 0.03 0.06 < 0.03 0.03 IL 0.008 (4) 0.06 IL 0.017 (4) 0.05 + 0.008 (4)
433
(nglml) Control
0.63 IL 0.178 (4) 2.80 k 0.10 (4) 19.5 k 2.26 (4)
FIG. 3. Inhibition of [JH]PGEZ anti-PGE, (100 ~1 of a rabbit antiserum, diluted 1125) binding by increasing quantities of PGE, (c), PGE;, (01, and PGE, (A); 16,500 cpm of [3H]PGE, were added; 1976 cpm were bound specifically and 384 cpm were bound nonspecifically.
0.20 10.6 0.30 -c 0.03 0.12 0.09 0.05 -+ 0.019 (4) 0.21 +- 0.018 (4) 0.24 -+ 0.046 (4)
and angiotensin II, or EGF and thrombin.* Such stimulation by serum, tumor promoter TPA, and synergistic stimulation by EGF and ADH of the C-9 cells and the effect of preloading cells with EPA are shown in Fig. 1. Stimulations by these agonists are reduced in a dose-dependent manner when cells are preloaded with EPA. Synthesis of all three cyclooxygenase products is reduced, suggesting that inhibition is taking place at or before the generation of the endoperoxide PGG,(H,). Inhibition of the TPA-stimulated synthesis of PG12, measured as its stable hydrolytic product 6-keto-PGF,,, by the EPA-preloaded cells also can be demonstrated by immunochromatographic analyses’” (Fig. 2). It is possible that cyclooxygenase activity is not being affected and EPA is being metabolized to a product not recognized by the RIA, such as PGEa, a cyclooxygenase product of EPA.r4 This was ruled out *Levine L: Unpublished observations.
acid
by use of an antiserum prepared with x3 bovine albumin-PGE, immunogen. This anti-PGE, does not distinguish among PGEl, PGE2, and PGE, (Fig. 3). Thus the reduction in PGEz serologic activity by the EPA-preloaded cells reflects a decrease in PGE2 synthesis. The effect of EPA on stimulated amchidonic acid metabolism by a variety of cells is shown in Table I. In all of the cells preloaded with EPA, stimulated levels of cyclooxygenase products PGE2, PGF,,, or cketo-PGF,, are decreased. Again, since the levels of PGEZ were measured by RIA with the antiserum whose serologic specificity is such that the cis double bond of PGE, at the C- 17 terminal is not recognized, it is likely that PGE, synthesis is being inhibited. When stimulated by melittin, a polypeptide from bee venom, a steroid-secreting cell line isolated from a Leydig cell testicular tumor of the rat (the LC-540 cell line) synthesizes PGF,,, PGE2, TxAz (measured as TxB2), 6keto-PGF,,, and also serologically active HETEs and SRSs. Again, the levels of cyclooxygenase products PGE2, PGF2,, 6-keto-PGF,,, and TxB2 are reduced in culture media of cells preloaded with EPA. The levels of lipoxygenase products HETE and SRS, however, are not reduced in these culture media; they are increased (Fig. 4), suggesting that EPA is affecting generation of the PGG,(H,) endoperoxide and not that of the lipoxygenase-generated epoxide . Similar reductions of cyclooxygenase-generated
434
J ALLERGY
Levine and Worth
CLIN IMMUNOL. SEPTEMBER 1994
FIG. 4, Effect of EPA on PUFA metabolism by rat teydig ceils (LC-540 cell line), Cells (5 x IO5160 mm dish) were preloaded with EPA (5 Fg/Z x 1O”cells) and, after washing to remove unincorporated EPA, they were stimulated with melittin (5 pgiml) for 20 hours. Stippled &KS are EPApreloaded cells and striped bars are control cells. Each bar is the xii- SD for groups (n in parentheses). Where there are no brackets, an appropriate pool was made and the RIA was oerformed in duplicate. f : immunoreactive.
-.
i A-23187
A-23187
FIG. 5. Effect of EPA on PUFA metabolism by RBL-1 cell line. For preloading cells with EPA, ceils were seeded in the prasence of EPA (5 pgi1.25 x IO5 cells) as described in Material and Methods. After washing to remove unincorporated EPA, cells (5 x IO?60 mm dish) were stimulated to metabolize PUFA by incubation with the Ca2- ionophore A23187 (0.7 yM) for 60 minutes. Stippled bars are EPA-pwioaded cells and striped bars are control cells. Each bar is thex? SD for groups (n in parentheses). Where there are no brackets, an appropriate pool was made and the RJA was performed in duplicate. i = Immunoreactive.
PC&, PGF,,, and TxB, are found in culture media oi KBL-1 ceils preloaded with EPA that are stimulated tay the Ca-‘- ionophore A23187. Again, levels of lipoxygenase products SRS and HETE are not atfccted significantly or are even increased by ceils preloaded with EPA (Fig. 5). RBL- 1 cells have also been Iabeled with E”H]ETA and [‘*C]EPA. The doublelabeled cells, with and without preloading with EPA, were then stimulated to metabolize arachidnnic acid by 60 minutes of incubation with the Ca2+ ionophore A.23187; after extraction with ethanol, culture media were analyzed by HPLC and HPLC fractions were counted for tritium and Y. Radioactivity in the fraciions of the entire chromatogram is shown in Fig. 6 and radioactivity in the fractions in which authentic
cyclooxygenase and lipoxygenase products as well as the free fatty acid chromatograph’” are shown in Fig. 7” Levels of tritium in the fraction where authentic PGEL, is found (fraction No. 53) are decreased as a result of preloading with EPA (Fig. 6). There is little “C in this fraction. Tritium is also found in fractions that cochromatograph with authentic LTB,. HETE. and ETA, but unlike the reduction of tritium in the fraction that cochromatographs with authentic PGE,. levels of the radiolabel in the latter fractions are not decreased in culture fluids of cells preloaded with EPA. The level of the radiolabel that cochromatographs with authentic HETE and ETA is increased. Since products of EPA metabolism are not characterized. the j14C/-containing compounds in the
VOLUME NUMBER
d 8 M
74 3, PART 2
Effects of eicosapentaenoic
.acid
435
B 16-
12-
FIG. 6. Reverse-phase HPLC of extracted culture fluids of [3H]ETA- and [Y]EPA-labeled RBL-1 cells after stimulation of PUFA metabolism by the Ca2+ ionophore A23187 (1 PM) for 60 minutes. The percentage of recovered tritium (dashedlines) and 14C(so/id lines) in culture fluids of controt cells (A/ and cells preloaded with EPA/B) are shown. Recovery was about 60% for both control and EPApreloaded cells. For radiolabeling of cells, [3H]ETA (0.02 &i/1.25 x IO5 cells) and [Y]EPA (0.002 #S/1.25 x ‘IO5 cells) were used during the seeding procedure. Cells were preloaded with EPA as described in the legend of Fig. 5. Culture fluids from seven identically treated dishes were pooled and extracted for the HPLC analyses. The counts per minute that were incorporated into 5 x lo5 cells were 15,013 (tritium) and 4972 (“C) by the EPA-preloaded cells and 18,808 (tritium) and 8,571 (“C) by control cells. The counts per minute released by 5 x IO5 cells after treatment with A23187 were 4300 (tritium) and 1060 (‘*C) by the EPA-preloaded cells and 3540 (tritium) and 1120 (‘“C) by control cells.
chromatogram cannot be unequivocally identified. Both tritium and 14C are found in fractions in which authentic phospholipids and cholesterol esters chromatograph (fraction Nos. 85 to 110). DISCUSSION Increases in levels of EPA relative to those of arachidonic acid have been associated with altered
FIG. 7. Reverse-phase HPLC of the extracted culture fluids described in Fig. 6. The radioactivity in fractions in which authentic cyclooxygenase and lipoxygenase products of ETA metabolism as well as authentic ETA and EPA chromatography are shown.
platelet function,’ decreased episodes of thrombosis as determined epidemiologically,’ decreased focal cerebral infarctions,* and decreased renal disease in NZB X NZWFi mice.” These biologic properties may be causally related to cyclooxygenase or lipoxygenase utilization of EPA or ETA.‘, 3, 5 Preloading cells with EPA and analyses of the PUFA metabolic products of these cells after appropriate stimulation of PUFA metabolism should distinguish between syntheses of the class of products having double bonds at the C-5, C-13, and C-17 positions (e.g., PGEQ)” and a decrease in syntheses of the class of products having double bonds at the C-5 and C-13 positions (e.g., PGE,). Thus if the analyses for PGE2 are made with antiserum that does not distinguish between PGE, and PGE3, a decrease in serologic activity demonstrates inhibition of cyclooxygenase-mediated oxygenation of ETA by the incorporated EPA, as opposed to utilization of the incorporated EPA for synthesis of PGE,. Such decreases in the immunologic activity of PGE, are found in cells preloaded with EPA. In contrast to
436
J ALLERGY CLIN. IMMUNOL SEPTEMBER 1984
Levine and Worth
decreases in PGE2, PGFzu, 6-keto-PGF,,, and TX& levels that result from preloading cells with EPA. there is no significant decrease in the lipoxygenase products SRS and HETE. These findings in cells are in agreement with the known specificities of these substrates for cyclooxygenase’“. ” and 12-lipoxygrnase lx as determined
in vitro
REFERENCES Dyerberg J, Bang HO: Dietary fat and thmmbosls. Lancer !:152, 1978 Black KL, Culp B, Madison I), Randall OS, Laads WEM: The protective effects of dietary fish oil on focal cerebral i&arc fion. Prostaglandins Med 3:257, 1979 Prickett JD, Kohinson DR, Steinberg AD: Dietary enrichment with the polyunsaturated fatty acid eicosapentaenoic acid pre l’ents proteinuria and prolongs survival in NZB X NZW F mice. J Clin Invest 68:556, 1981 Dyerberg J, Bang HO, Hjome N: Fatty acid composition of the plasma lipids in Greenland Eskimos. Am J Clin Nutr 283958 1975 Dyerberg J, Bang HO, Stoffersen E, Moncada S. Vane JR Eicosapentaenoic acid and prevention of thrombosis and atherosclerosis. Lancet 2: 117, 1978 Hong SC, Polsky-Cynkin R, Levine L: Stimulation of prostaglandin biosynthesis by vasoactive substances in methylcholanthrene-transformed mouse BAIBI3T3. J Biol Chem 251 776. 1976 Levine L: Chemical carcinogens stimulate kidney (MDCK! cells to produce prostaglandins. Nature 26&447. 1977 Snoek FT, Levine L: Requirements for protein synthesis and calcium for stimulation of prostaglandin synthes+s in cultured rat liver cells by tumor promoters. Cancer Res 43:4743, 1983 Levine L, Morgan KA, Lewis RA, Austen KF, Clark 1). Marfat A, Corey EJ: Radioimmunoassay of the leukotrienes of slow reacting substance of anaphylaxis. Proc Nat1 Acad SCI USA 78:7692, 1981 Coughbn SK, Moskowitz MA, Antoniades HN, Levine 1. Serotonin receptor-mediated stimulation of bovine smooth muscle cells: prostacyclin synthesis and its modulation b) platelet-derived growth factor. Proc Nat1 Acad Sci USA 78:7134, 1981 Hayes EC, Lombard0 DL, Girard Y, Maycock AL, Rokach J Rosenthal S. Young RN, Egan KW, Zweerink HJ: Measuring leukotrienes of slow reacting substance of anaphylaxis: Development of a specific radioimmunoassay. 3 Immunol 131:429. 1; Levine L: Arachidonic acid transformation and tumor production. Adv Cancer Res 35:49, 1981 13 Alam 1. Ohuchi K, Levine L: Determination of cycloox>genase products and prostaglandin metabolites using highpressure liquid chromatography and radioimmunoassay 4nal Biochem 93:339, 1979 14. Bergstrom S, Carlson LA, Weeks JR: The prustaglandins: A family of biologically active lipids. Pharmacol Rev 20: 1, 1968
i’i. Kobayashi ‘I‘, Levine L: Arachidonic acid metabolism by erythrocytes. J Biol Chem 258:9116, 1983 16. Gulp BR, Titus BG, Lands WEM: Inhibition of prostaglandin biosynthesis by eieosapentaenoic acid. Prostaglandins Med 3269, 1979 17. Lands WEM, LeTeIlier PK. Rome LH, Vanderhoek JY: Inhibition of prostaglandin biosynthesis. In Bergstrom S, editor: Advances in the biosciences, vol 9. Oxford, 1973, Pergamon Press, pp 15-28 IX. Nugteren DH: Arachidonate lipoxygenase in blood platelets. Biochim Biophys Acta 380:299. 1975
DlSCtJSSlON Priscilla Piper: First, did you further identify the LTC, Immunoreactive material? Have you any evidence that it was LTC,? Second, have you tried experiments with EPA on RBL- I cells grown in spinner culture? When we used RBL-1 cells to produce SRS with A23187 stimulation, the cells grown in spinner culture produced more than cells srown on monolayers. Lawrence Levine: We have measured the constituents of SRS produced by A23 187-stimulated RBL- 1 in the absence of EPA. The immunoreactive constituents eluted from HPLC with 1l-{runs-LTD, and I I-trans-LTE,. We have to repeat these studies with RBL- 1 cells preloaded with EPA. I have never looked at SRS production by RBL-1 cells in suspension, but with several other cell lines the production is greater with monolayers than in suspension cultures. Marc Goldyne: Do you know whether the elevations in immunoreactive SRS and LTB in EPA-treated cells reflect elevations in arachidonic acid-derived hydroxy acids, EPA-derived hydroxy acids, or a combination of both? L. Levine: We have not completed these analyses. Edward Goetzl: I am interested in the specificity of the anti-LTB, you use. Our rabbit anti-LTB, reacts with LTB, with an affinity two and one-half to three times higher than that characteristic of LTB,, so that we need to resolve LTB, and LTB, befoFe RIA when specimens contain the latter. L. Levine: I used an anti- IZHETE to assay LTB,/ LTB, , but binding affinity of the individual constituents has not been established. For the SRS LTs of both series, I used the antibody of Dr. Hayes at Merck Laboratories. This antibody reacts best with LTC4, but cross-reacts 40% to 50% with LTD, and 5% to 6% with LTE,. We do not know its affinity for EPA LTs. Robert Lewis: We have utilized EPA as a substrate for pentane LT synthesis in the immune complex-activated rat peritoneal cavity and it is about as good a substrate for 5lipoxygenase as arachidonic acid.
acid: Its effwzts on acid methoilism by cells
Levine, SC.%**
and Nancy Worth Waitham,
Mass.
Some eukatytic, ceils in culture synthesize u varieq qf 1ipoxygvgcna.w und!or cylooxygenasr products when stirnuluted by appropriate agonist.c Under normal nutritional conditions, these fatty ucid (PUFA) 5, 8, 11, lo-eic,osatetraenoi( product.s are derived from the polvunsuturated a(id (ET.4). urachidonic acid. which before metubo(isn1 ~nu,st he liberawd.from cellular lipids b> &ester@cation. Jf’ the <,elluiar lipids are prelouded with 3,X, 1 I , I J. / 7-eicosclpent(lPnoic ucid I EPA4i alEd cells are then stimulated to metaboiize the PUFA P, JCIYJIJof c~ycloox~~enase and lipoxygcnnse products synthesized are altered. C~c~I~~oqv~~~n(~sc productr\ dec,rease, while lipoxygenuse product.5 are not .signifcantl~ ajyected and ~nq even increase. The dec,rease in the production of cylooxygenase products results ,fiom redu(,ed utiiizution of the substrute (ETA). Decreased prostaglandin production by rat basophil teuketnia-I wit preloaded with EPA and rctdiotaheled with pHH]E;TA and [“CJEP.4 (an u1.w be denwnstr.crted tg high-pe$ortnance liquid ~,hrorrrrcto~ruphi~~ w~~!\.ws ctf [“HI- and [’ ‘C] radiolabeled nwtabolites in c~utturc ,juids qf cells ~riniutated lo metaho~i~c PIIFA bv the CO“ - iorzophr~rc~ ,42.1 I&‘. i .I ilI.LErRGY Cl.l,V ~MMlJhVL ‘i
.I.?i,,
!#J
j
The metabolism of PUFAs through cyclooxygenase and lipoxygenase pathways by cells in which the content of two main classes of PLJFAs (w-3 and w-6) varies qualitatively and/or quantitatively appears to have biologicalIy important consequences. i -” The effects of EPA on cyciooxygenase activity have been studied extensively because of the findings of decreased platelet function and cardiovascular diseases in Greenland Eskimos.’ Such decreases have been associated with increased levels of the w-3 class of PUFAs in the diet.’ It has been suggested that these biologic effects reflect metabolism of PUFA.” We have found that the levels of some cyclooxygenase products in peripheral sera of the NZB X NZWF,
Supported by National Institutes ol Health Grant Nos. GM 17256 md CA l-7 309. Rrp~int requests: Lawrence Levine, Sc.D., Department of Riochemistr). Brandeis University. Waltham, MA 02253, Publication No. 1486. Department of Biochemistq. Brandeiq, ilnivers~. Wattham, Mass. ’ .Amencan Cancer Society Research Professor of Biochcmistr) !AwarJ PRP-21)
Pli FA:
EPA. MEM: HE’lx a i: KlA, HPLC, SRS: PG. ‘i’k EGF~ ADH, ‘r PA KHI.: bl-A. I I-r-L.1
Polyunsaturated fatty acid 5,X, 1I, 14,17-Eicosapentaenoic acid Minimal essential medium Hydroxy-eicosatetraenoic acid Leukotriene Radioimmunoassay High-performance liquid chromatography &Sulfidopeptide-containing leukotriene Prostaglandin Thromboxane Epidermal growth factor Vasopressin : 2-O-Tetradecanoyl-phorbo- 1Sacetate Rat basophil leukemia 5.8, I 1. id-Eicosatetraenoic acid j t-l W/IS isomer of a peptidoleukotriene
Grain* and in peripheral plasma and some tissues of the Swiss albino strains? of mice that are fed menhaden oil diets decrease significantly, while levels of lipoxygenase products do not. Such analyses of ‘Robinson DR. Levine 1,: Unpublished observations. TTashjian AH Jr. Voelkel EF. Robinson DR, Levine L: linpubiichetl ohscrvatlons.
VOLUME NUMBER
74 3, PART 2
Effects of eicosapentaenoic
2
6
B
7
6-KETO-pGF#
PGE2
acid
431
mF2a
O.l!j
t
MEM
Serum
TPA
EGJ-ADI
MEM
TW
EGF-AtNi
MEM
TF’A
EGF-ADH
FIG. 1. Effect of EPA on PG production by rat liver cells (the C-9 cell line) after stimulation (8 x 10” cells/60 mm dish) of PUFA metabolism by 10% fetal calf serum (MEM), TPA (0.01 pglml), and EGF (0.1 pglml) plus ADH (0.06pM) for 20 hours. The C-9 liver cells were preloaded with EPA, 15 cLgl2 x IO5 (El), or 5 cLg/2 x IO5 cells ( ), or were not preloaded (a). EPA treatment is nontoxic as’ measured by trypan blue exclusion. Each bar is the mean value for groups of four dishes and brackets represent standard deviations. Where there are no brackets, a pool was used and the RIA was performed in duplicate.
arachidonic acid metabolites do not specify the source of the substrate; i.e., is the PUFA derived from plasma lipoproteins or cells, and if cells, which cells? We have now analyzed culture fluids of several types of cells preloaded with EPA for metabolic products of both cyclooxygenase and lipoxygenase pathways. Preloading of cellular lipids with EPA results in decreased production of cyclooxygenase products and, in cells that synthesize lipoxygenase as well as cyclooxygenase products, the decrease of cyclooxygenase products is accompanied by unaltered or even increased levels of lipoxygenase products.
MATERIAL AND METHODS Tissue culture. Sourcesand growth condifions for the cell lines used in this study have been described.6-10 Exponentially growing cells were treated with 1 ml trypsin (0.5 mg)/ethylenediaminetetra-acetate (0.2 mg) solution and cells were seeded into tissue culture dishes with Eagle MEM containing 10% fetal calf serum, 2mM L-glutamine, penicillin (250 U/ml), and streptomycin (250 $J/ml).
After 24 hours of incubation, cells were washedtwice with the MEM. Cells then were incubated with MEM containing penicillin and streptomycin in the presence or absence of the agonist. For radiolabeling of the cells, r3H] arachidonic acid (.5,6,8,9,11,12,14,15-[3H(N)], 87.4 Ciimmol) and
[Y]EPA
(5,8,11,14,17-[‘4C(U)], 212 mCi/mmol; New
England Nuclear, Boston, Mass.) were added to cells at the time of seeding. After incubation for 24 hours, the unincorporated [3H]arachidonic acid and [Y]EPA were removed by the washing procedures. For preloading cells with EPA, EPA (Sigma Chemical Co., St. Louis, MO.) was also added (from a 50 mg/ml ethanol stock) to cells at the time of seeding, and 24 hours later the unincorporated EPA was removed during the washing procedure. Ethanol alone had
40
45
50
40
45
50
Fti!!TUN
FIG. 2. lmmunochromatography of tissue culture extracts of the C-9 liver cells before (A) and after (A) preloading with EPA (5 pg12 x lo5 cells). Cells (8 x 105/60 mm dish) were stimulated to metabolize PUFA by incubation with TPA (0.01 pglml) for 20 hours.
no effect on the prostaglandins, HETEs, or LTs synthesized by control cells. The quantity of EPA used for preloading the cells varied from cell to cell and was recorded in each experiment. After stimulation, cell culture media were collected and centrifuged to remove floating cells and cellular debris and were assayed by RIA for cyclooxygenase and lipoxygenase products. HPLC. HPLC was performed by reverse phase on a p-Bondapak phenyl column (7.8 X 300 mm, Waters Associates). The Waters Model 6000A pumps, Model 660 solvent programmer, and Model U6K injector were used for
HPLC. Each sample was run on a linear gradient program from 100% solvent A to 100% solvent B for 110 minutes at a flow rate of 1 ml/min, and 1 ml fractions were collected. Solvent A consisted of 93.4% O.OlM phosphate buffer (pH 7.4), 6% methanol (HPLC grade, Fisher), and 0.6% tert-
J ALLERGY
432 L.evine and Worth TABLE I. Stimulation of PUFA metabolism by cells in culture on PUFA metabolism ._ --_ .-.. _.~.~. ~._.~-.--_--- -___I____. “. lmmunoreactive Cell
Stimulus
Rat glomerular epithelium Methylcholanthrcne-transformed rar tibrobtaht ~MC-5 i Mouse fibro sarcoma tHSDM,j
MEM ‘E4.
0. I p#lll
A23187. 2&M MEM Serum 15% fetal calf ) A231X7. If*%4
and effects of EPA preloading -.
PGE, hglml) -...__l_--
EPA preloaded
CLIN. !MMlJNOL. SEPTEMBER 1984
-.
--
Control
lmmunoreactive
PGF,, (nglml) Control
EPA preloaded
Cl.IO
o.th
(1 04
0.0’)
0. I:!
0.24
0.06
0.24
0. so 1,18 r 0.259 (4) x.53 2 0.1314(4) ‘.I0 + 0,822 t31
1.90 0.86 0.7?. 2.19 ” 0.336 (4) 0.14 s 0.030 (4) 0.23 i 0.015 (4) I I.04 c 0.717 (4) 1.64 i- 0.152 (4) 2. IO +- 0. IS2 (4) 7 63 + 0.65 14) 0.28 + 0.060 (4) 0.90 t 0.063 (4)
MEM 0.61 L 0.124 (Ij 1.55 I!Z0.30 (4) 0.09 Serum (5% fctai IO.85 i- 2.490 (4, 13 19 t 0.819 (4) 2.08 calf: iZ.hY I+ 0.925 (4) 0.34 A’3187. 2).&f 2.89 i- 0.968 (41 MEM 0.76 T 0.091 (3) 0.99 z!z 0.066 (4) Rat mesangial ADH. ! FM I .Yh .+ 0. I 13 (4) .! 08 rt 0.30.5 (4) MEIM *: 41.i 0 65 z 0.1 15 141 Dog kidnq TP4. 0.001 ~giml I.71 .T 0.170 (3) -1.74 ? 0.408 (4) 1.17 epithelium Norepinephrinc 1 !).17 .I 0.061 C4) 0.81 -+ 0.100i4) ~MDCK I 5OJdf 0,x 0.3.: Mouse tibroblast ,MEM j 3’1‘31 Melittin. &g/m! ! .30 7 0.084 (3) 2. IO i 0.1’3 (3) 0.65 AL3 187. 2)LM OYY + 0.13‘J (31 I .6X + 0.1 17 (3‘1 0.32 I{ovmc aort;k MEM m00e-l Tl’A. 0.01 kg:ml muwli: EsiF. 0 OS tJ-piml. dnd rerotonin i .O p,M Monocytic leLJ MEM 0.06 --i 0.010 (4~ 0. I4 ” 0.043 (4) 0.02 i;cmi;i ( 1 / I i i TPA. 1.O w&ml 11.2I 1 0.039 (4 I ii,63 3 0.035 (4) 0.03 A23 I 87, 2pM 0 so -i- 11.039(3) 13.75 .+ 0.04Y (4) 0.18 -- .---.. -- -...-.-. ..--.-. _-.----__ “---------.---__-1__--_-._....---- --.---I..-I_...
t 0.020 (4) 0.24 t 0.038 (4) -c 0.356 (4) 2.90 ?Y 0.173 (4) 1 0.055 (4) 1.49 + 0.065 (4)
0.21 f 0.027 (4) 0.1 ” 0.023 (4) 5.0s t 0.618 (4) 0.33 rt O.OXI (4) i 0, 1 0.036 t 0.105 (3) t 0.057 (3)
0.092 1.96 k 0.170 (3) 0.84 + 0.038 (3)
rr 0.002 (4) 0.03 + 0.004 (4) i 0.003 (4) 0. 14 t 0.014 (4) r?- 0,017 (4J 0.44 t 0.034 (4) ____-
!):rta are % i- SD. The number ot dishes is rndicated m parentheses. Where onI\ _ ;: single value IS given. duplicate dishes were assayed and the average is presented. The EPA concentration used to preload the cells ranged from 10 to 50 pg/ 2 X 10’ cells during the seeding procedure, in which 5% fetal calf serum wiis present. With most bur not a11 cella. controi and EPA-loaded cells were counted and +xamincd t’or trypan hluc exclusion. Under the conditions of prelnadirq. nr, cell toxicity was recorded
amyi-alcohol (Aldrich) (percentage by volume). Solvent B :&it> 99.4%
tuethzmol
with
0.6%
trrf. amyl-.alcohol.
Stan-
dsrd cyclooxygenase products were obtained from The UpJohn Co. (Kalamazoo, Mich.), HETE WG obtained from Dr. Walter Hubbard. Vanderbilt University. and the l-T\ ,tnd SRSs were obtained from Dr. Roberl A. Lewis. Harmd Medical School Sc;ro/o~ic~ ~nnl~.~r,,\. Culture Ruids or the appropriate HPLC fmctions were assayed by RlAs with antibodies directed toward PC&, PGF?,a, 6-keto-PGF,,,: TxB,, PC&. METES. and SRSx. Dr. E. C. Hayes of Merck Institute for Therapeutic Research supplied the anti-SRS used in this h~udy. Serologic specificity of this antiserum has been described.” The radiolabeled haptens were purchased from New England Nuclear. RIA procedures and serologic spec:kities of Ihe other RIA systems have been published ‘:
When the unsaturated fatty acid metabolites were to be meaby RIA after their resolution by HPLC. solvents were removed either under a nitrogen stream or by lyophilization, dnd residues were dissolved in Tris (hydroxymethylaminomethane) buffer. xtred
RESULTS Rat liver cells (the C-9 cell line) synthesize
PGI,,
PGE2, and PGF,,; more than 90% of these synthesized cyclooxygenase
products
are PGIZ.’ The C-9
liver cells can be stimulated to metabolize arachidonic acid by incubation with several agonists including Wmor promoters, growth factors such as EGF, platelet-derived growth factor, and serum, and synergisticafy by incubation with EGF and ADH, EGF
VOLUME NUMBER
74 3, PART 2
lmmunoreactive
Effects of eicosapentaenoic
6-keto-PGF,,
EPA preloaded
0.32 k 0.070(4) 0.36 it 0.061 (4) 6.65 +- 0.26 (4)
0.04 1.55 0.11 < 0.03 0.06 < 0.03 0.03 IL 0.008 (4) 0.06 IL 0.017 (4) 0.05 + 0.008 (4)
433
(nglml) Control
0.63 IL 0.178 (4) 2.80 k 0.10 (4) 19.5 k 2.26 (4)
FIG. 3. Inhibition of [JH]PGEZ anti-PGE, (100 ~1 of a rabbit antiserum, diluted 1125) binding by increasing quantities of PGE, (c), PGE;, (01, and PGE, (A); 16,500 cpm of [3H]PGE, were added; 1976 cpm were bound specifically and 384 cpm were bound nonspecifically.
0.20 10.6 0.30 -c 0.03 0.12 0.09 0.05 -+ 0.019 (4) 0.21 +- 0.018 (4) 0.24 -+ 0.046 (4)
and angiotensin II, or EGF and thrombin.* Such stimulation by serum, tumor promoter TPA, and synergistic stimulation by EGF and ADH of the C-9 cells and the effect of preloading cells with EPA are shown in Fig. 1. Stimulations by these agonists are reduced in a dose-dependent manner when cells are preloaded with EPA. Synthesis of all three cyclooxygenase products is reduced, suggesting that inhibition is taking place at or before the generation of the endoperoxide PGG,(H,). Inhibition of the TPA-stimulated synthesis of PG12, measured as its stable hydrolytic product 6-keto-PGF,,, by the EPA-preloaded cells also can be demonstrated by immunochromatographic analyses’” (Fig. 2). It is possible that cyclooxygenase activity is not being affected and EPA is being metabolized to a product not recognized by the RIA, such as PGEa, a cyclooxygenase product of EPA.r4 This was ruled out *Levine L: Unpublished observations.
acid
by use of an antiserum prepared with x3 bovine albumin-PGE, immunogen. This anti-PGE, does not distinguish among PGEl, PGE2, and PGE, (Fig. 3). Thus the reduction in PGEz serologic activity by the EPA-preloaded cells reflects a decrease in PGE2 synthesis. The effect of EPA on stimulated amchidonic acid metabolism by a variety of cells is shown in Table I. In all of the cells preloaded with EPA, stimulated levels of cyclooxygenase products PGE2, PGF,,, or cketo-PGF,, are decreased. Again, since the levels of PGEZ were measured by RIA with the antiserum whose serologic specificity is such that the cis double bond of PGE, at the C- 17 terminal is not recognized, it is likely that PGE, synthesis is being inhibited. When stimulated by melittin, a polypeptide from bee venom, a steroid-secreting cell line isolated from a Leydig cell testicular tumor of the rat (the LC-540 cell line) synthesizes PGF,,, PGE2, TxAz (measured as TxB2), 6keto-PGF,,, and also serologically active HETEs and SRSs. Again, the levels of cyclooxygenase products PGE2, PGF2,, 6-keto-PGF,,, and TxB2 are reduced in culture media of cells preloaded with EPA. The levels of lipoxygenase products HETE and SRS, however, are not reduced in these culture media; they are increased (Fig. 4), suggesting that EPA is affecting generation of the PGG,(H,) endoperoxide and not that of the lipoxygenase-generated epoxide . Similar reductions of cyclooxygenase-generated
434
J ALLERGY
Levine and Worth
CLIN IMMUNOL. SEPTEMBER 1994
FIG. 4, Effect of EPA on PUFA metabolism by rat teydig ceils (LC-540 cell line), Cells (5 x IO5160 mm dish) were preloaded with EPA (5 Fg/Z x 1O”cells) and, after washing to remove unincorporated EPA, they were stimulated with melittin (5 pgiml) for 20 hours. Stippled &KS are EPApreloaded cells and striped bars are control cells. Each bar is the xii- SD for groups (n in parentheses). Where there are no brackets, an appropriate pool was made and the RIA was oerformed in duplicate. f : immunoreactive.
-.
i A-23187
A-23187
FIG. 5. Effect of EPA on PUFA metabolism by RBL-1 cell line. For preloading cells with EPA, ceils were seeded in the prasence of EPA (5 pgi1.25 x IO5 cells) as described in Material and Methods. After washing to remove unincorporated EPA, cells (5 x IO?60 mm dish) were stimulated to metabolize PUFA by incubation with the Ca2- ionophore A23187 (0.7 yM) for 60 minutes. Stippled bars are EPA-pwioaded cells and striped bars are control cells. Each bar is thex? SD for groups (n in parentheses). Where there are no brackets, an appropriate pool was made and the RJA was performed in duplicate. i = Immunoreactive.
PC&, PGF,,, and TxB, are found in culture media oi KBL-1 ceils preloaded with EPA that are stimulated tay the Ca-‘- ionophore A23187. Again, levels of lipoxygenase products SRS and HETE are not atfccted significantly or are even increased by ceils preloaded with EPA (Fig. 5). RBL- 1 cells have also been Iabeled with E”H]ETA and [‘*C]EPA. The doublelabeled cells, with and without preloading with EPA, were then stimulated to metabolize arachidnnic acid by 60 minutes of incubation with the Ca2+ ionophore A.23187; after extraction with ethanol, culture media were analyzed by HPLC and HPLC fractions were counted for tritium and Y. Radioactivity in the fraciions of the entire chromatogram is shown in Fig. 6 and radioactivity in the fractions in which authentic
cyclooxygenase and lipoxygenase products as well as the free fatty acid chromatograph’” are shown in Fig. 7” Levels of tritium in the fraction where authentic PGEL, is found (fraction No. 53) are decreased as a result of preloading with EPA (Fig. 6). There is little “C in this fraction. Tritium is also found in fractions that cochromatograph with authentic LTB,. HETE. and ETA, but unlike the reduction of tritium in the fraction that cochromatographs with authentic PGE,. levels of the radiolabel in the latter fractions are not decreased in culture fluids of cells preloaded with EPA. The level of the radiolabel that cochromatographs with authentic HETE and ETA is increased. Since products of EPA metabolism are not characterized. the j14C/-containing compounds in the
VOLUME NUMBER
d 8 M
74 3, PART 2
Effects of eicosapentaenoic
.acid
435
B 16-
12-
FIG. 6. Reverse-phase HPLC of extracted culture fluids of [3H]ETA- and [Y]EPA-labeled RBL-1 cells after stimulation of PUFA metabolism by the Ca2+ ionophore A23187 (1 PM) for 60 minutes. The percentage of recovered tritium (dashedlines) and 14C(so/id lines) in culture fluids of controt cells (A/ and cells preloaded with EPA/B) are shown. Recovery was about 60% for both control and EPApreloaded cells. For radiolabeling of cells, [3H]ETA (0.02 &i/1.25 x IO5 cells) and [Y]EPA (0.002 #S/1.25 x ‘IO5 cells) were used during the seeding procedure. Cells were preloaded with EPA as described in the legend of Fig. 5. Culture fluids from seven identically treated dishes were pooled and extracted for the HPLC analyses. The counts per minute that were incorporated into 5 x lo5 cells were 15,013 (tritium) and 4972 (“C) by the EPA-preloaded cells and 18,808 (tritium) and 8,571 (“C) by control cells. The counts per minute released by 5 x IO5 cells after treatment with A23187 were 4300 (tritium) and 1060 (‘*C) by the EPA-preloaded cells and 3540 (tritium) and 1120 (‘“C) by control cells.
chromatogram cannot be unequivocally identified. Both tritium and 14C are found in fractions in which authentic phospholipids and cholesterol esters chromatograph (fraction Nos. 85 to 110). DISCUSSION Increases in levels of EPA relative to those of arachidonic acid have been associated with altered
FIG. 7. Reverse-phase HPLC of the extracted culture fluids described in Fig. 6. The radioactivity in fractions in which authentic cyclooxygenase and lipoxygenase products of ETA metabolism as well as authentic ETA and EPA chromatography are shown.
platelet function,’ decreased episodes of thrombosis as determined epidemiologically,’ decreased focal cerebral infarctions,* and decreased renal disease in NZB X NZWFi mice.” These biologic properties may be causally related to cyclooxygenase or lipoxygenase utilization of EPA or ETA.‘, 3, 5 Preloading cells with EPA and analyses of the PUFA metabolic products of these cells after appropriate stimulation of PUFA metabolism should distinguish between syntheses of the class of products having double bonds at the C-5, C-13, and C-17 positions (e.g., PGEQ)” and a decrease in syntheses of the class of products having double bonds at the C-5 and C-13 positions (e.g., PGE,). Thus if the analyses for PGE2 are made with antiserum that does not distinguish between PGE, and PGE3, a decrease in serologic activity demonstrates inhibition of cyclooxygenase-mediated oxygenation of ETA by the incorporated EPA, as opposed to utilization of the incorporated EPA for synthesis of PGE,. Such decreases in the immunologic activity of PGE, are found in cells preloaded with EPA. In contrast to
436
J ALLERGY CLIN. IMMUNOL SEPTEMBER 1984
Levine and Worth
decreases in PGE2, PGFzu, 6-keto-PGF,,, and TX& levels that result from preloading cells with EPA. there is no significant decrease in the lipoxygenase products SRS and HETE. These findings in cells are in agreement with the known specificities of these substrates for cyclooxygenase’“. ” and 12-lipoxygrnase lx as determined
in vitro
REFERENCES Dyerberg J, Bang HO: Dietary fat and thmmbosls. Lancer !:152, 1978 Black KL, Culp B, Madison I), Randall OS, Laads WEM: The protective effects of dietary fish oil on focal cerebral i&arc fion. Prostaglandins Med 3:257, 1979 Prickett JD, Kohinson DR, Steinberg AD: Dietary enrichment with the polyunsaturated fatty acid eicosapentaenoic acid pre l’ents proteinuria and prolongs survival in NZB X NZW F mice. J Clin Invest 68:556, 1981 Dyerberg J, Bang HO, Hjome N: Fatty acid composition of the plasma lipids in Greenland Eskimos. Am J Clin Nutr 283958 1975 Dyerberg J, Bang HO, Stoffersen E, Moncada S. Vane JR Eicosapentaenoic acid and prevention of thrombosis and atherosclerosis. Lancet 2: 117, 1978 Hong SC, Polsky-Cynkin R, Levine L: Stimulation of prostaglandin biosynthesis by vasoactive substances in methylcholanthrene-transformed mouse BAIBI3T3. J Biol Chem 251 776. 1976 Levine L: Chemical carcinogens stimulate kidney (MDCK! cells to produce prostaglandins. Nature 26&447. 1977 Snoek FT, Levine L: Requirements for protein synthesis and calcium for stimulation of prostaglandin synthes+s in cultured rat liver cells by tumor promoters. Cancer Res 43:4743, 1983 Levine L, Morgan KA, Lewis RA, Austen KF, Clark 1). Marfat A, Corey EJ: Radioimmunoassay of the leukotrienes of slow reacting substance of anaphylaxis. Proc Nat1 Acad SCI USA 78:7692, 1981 Coughbn SK, Moskowitz MA, Antoniades HN, Levine 1. Serotonin receptor-mediated stimulation of bovine smooth muscle cells: prostacyclin synthesis and its modulation b) platelet-derived growth factor. Proc Nat1 Acad Sci USA 78:7134, 1981 Hayes EC, Lombard0 DL, Girard Y, Maycock AL, Rokach J Rosenthal S. Young RN, Egan KW, Zweerink HJ: Measuring leukotrienes of slow reacting substance of anaphylaxis: Development of a specific radioimmunoassay. 3 Immunol 131:429. 1; Levine L: Arachidonic acid transformation and tumor production. Adv Cancer Res 35:49, 1981 13 Alam 1. Ohuchi K, Levine L: Determination of cycloox>genase products and prostaglandin metabolites using highpressure liquid chromatography and radioimmunoassay 4nal Biochem 93:339, 1979 14. Bergstrom S, Carlson LA, Weeks JR: The prustaglandins: A family of biologically active lipids. Pharmacol Rev 20: 1, 1968
i’i. Kobayashi ‘I‘, Levine L: Arachidonic acid metabolism by erythrocytes. J Biol Chem 258:9116, 1983 16. Gulp BR, Titus BG, Lands WEM: Inhibition of prostaglandin biosynthesis by eieosapentaenoic acid. Prostaglandins Med 3269, 1979 17. Lands WEM, LeTeIlier PK. Rome LH, Vanderhoek JY: Inhibition of prostaglandin biosynthesis. In Bergstrom S, editor: Advances in the biosciences, vol 9. Oxford, 1973, Pergamon Press, pp 15-28 IX. Nugteren DH: Arachidonate lipoxygenase in blood platelets. Biochim Biophys Acta 380:299. 1975
DlSCtJSSlON Priscilla Piper: First, did you further identify the LTC, Immunoreactive material? Have you any evidence that it was LTC,? Second, have you tried experiments with EPA on RBL- I cells grown in spinner culture? When we used RBL-1 cells to produce SRS with A23187 stimulation, the cells grown in spinner culture produced more than cells srown on monolayers. Lawrence Levine: We have measured the constituents of SRS produced by A23 187-stimulated RBL- 1 in the absence of EPA. The immunoreactive constituents eluted from HPLC with 1l-{runs-LTD, and I I-trans-LTE,. We have to repeat these studies with RBL- 1 cells preloaded with EPA. I have never looked at SRS production by RBL-1 cells in suspension, but with several other cell lines the production is greater with monolayers than in suspension cultures. Marc Goldyne: Do you know whether the elevations in immunoreactive SRS and LTB in EPA-treated cells reflect elevations in arachidonic acid-derived hydroxy acids, EPA-derived hydroxy acids, or a combination of both? L. Levine: We have not completed these analyses. Edward Goetzl: I am interested in the specificity of the anti-LTB, you use. Our rabbit anti-LTB, reacts with LTB, with an affinity two and one-half to three times higher than that characteristic of LTB,, so that we need to resolve LTB, and LTB, befoFe RIA when specimens contain the latter. L. Levine: I used an anti- IZHETE to assay LTB,/ LTB, , but binding affinity of the individual constituents has not been established. For the SRS LTs of both series, I used the antibody of Dr. Hayes at Merck Laboratories. This antibody reacts best with LTC4, but cross-reacts 40% to 50% with LTD, and 5% to 6% with LTE,. We do not know its affinity for EPA LTs. Robert Lewis: We have utilized EPA as a substrate for pentane LT synthesis in the immune complex-activated rat peritoneal cavity and it is about as good a substrate for 5lipoxygenase as arachidonic acid.