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Inhibitory effect of docosahexaenoic acid-containing phospholipids on 5-lipoxygenase in rat basophilic leukemia cells
Inhibitory Effect of Docosahexaenoic Acid-containing SLipoxygenase in Rat Basophilic Leukemia Cells
Phospholipids
on
.ABSTRACT.
5-Lipoxygenase has been recognized to be an important enzyme that catalyzes the first step in leukotriene production. In this study we examine whether or not phospholipids containing docosahexaenoic acid (DHA) affect S-lipoxygenase activity of a rat basophilic leukemia cell line (RBL-1). Among the synthesized phospholipids examined, l-oleoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (I-oleoyl-2-DHA-PC) was found to be the most potent inhibitor of S-lipoxygenase. The inhibition was dose-dependent and the ID,, value was 4.0 yM. When the fatty acid at the sn-tposition was replaced by other unsaturated fatty acids, the inhibitory activity decreased with decreasing numbers of both carbon atoms and double bonds in the fatty acids. Substitution at the l-position of the DHA-containing PC also affected the inhibitory potency. If oleic acid was substituted with palmitic acid, the inhibition activity was completely abolished. Lineweaver-Burk plot analysis showed that the inhibition of S-lipoxygenase by I-oleoyl-2-DHA-PC was non-competitive. The inhibition by this synthesized phospholipid was very specific to Uipoxygenase; that is, it did not extend to fatty acid cyclooxygenase, 124ipoxygenase or 154ipoxygenase. These results suggest that endogenously existing DHA-containing phospholipids may affect 54ipoxygenase activity and thus control leukotriene biosynthesis in vivo.
INTRODUCTION
been carried out to elucidate the biophysical function of‘ DHA as a free fatty acid, but it has been commonly~ accepted that DHA incorporated into cellular phospholipids is released from membrane to a much lesser extent than AA and EPA. Therefore, it is necessary to study the biological function of DHA-containing phospholipids. It has been reported that S-lipoxygenase is activated by some phospholipids (8). and recent studies have demonstrated that S-lipoxygenase is translocated from the cytosol to membrane (9). These reports indicate that the quality of membrane phospholipids affects 5lipoxygenase activity. In the present study. we examine the effects of several kinds of synthesized DHA-containing phospholipids on S-lipoxygenase in a rat basophilic leukemia cell line (RBL-1) and show that I-oleoyl-2docosahexaenoyl-sn-glycero-3-phosphocholine ( 1-oleoyl?-DHA-PC) is the most potent inhibitor of 5lipoxygenase.
It is commonly recognized that 4. 7. 10, 13. 16. 19docosahexaenoic acid (DHA) is the longest and most highly unsaturated fatty acid in the n-3 class. 5, 8, 11, I-t. 17-Eicosa-pentaenoic acid (EPA) and DHA have been reported to be substances contributing to a low incidence of myocardial infarction and ischemic heart disease ( 1. 2). Regarding the mechanism underlying this activity. it is believed that both DHA and EPA are incorporated into phospholipids in platelets in place of arachidonic acid (AA), a substrate of fatty acid cyclooxygenase. which leads to a decrease in thromboxane A2 production (3. 4). It is also believed that these unsaturated fatty acids also inhibit cyclooxygenase activity in platelets (5). Regarding other functions of DHA, it is thought that DHA has something to do with the development of the cerebral cortex and retinal membrane. because the ‘k of DHA to total phospholipids is high in both tissues (6, 7). A number of in vivo studies have
MATERIALS
AND METHODS
Chemicals Dale received 76 April 1993’ Dare accepted I7 May 1993
[I-“CJAA Xhl
(2.15 kBq/mol)
was
purchased
from
the
862 Prostaglandins Leukotrienes and Essential Fatty Acids
Radiochemical Centre. Amersham, UK. Unlabeled AA was purchased from Sigma, St Louis, MO. 1,2-Dipalmitoyl-sn-glycero-3-phosphocholines was purchased from Wako Pure Chemical Industries, Osaka. Other phosphatidylcholine (PC) used here were synthesized from 1-acyl-sn-glycero-3-phosphocholine and fatty acid anhydrides. 1-Acyl-sn-glycero-3-phosphocholine ( 1.49 mM). fatty acid anhydride ( 1.64 mM) and N,N,-dimethyl-4amino-pyridine ( 1.66 mM) were stirred in anhydrous chloroform at room temperature for 24 h. The solution was eluted with chloroform through a glass column, 3.0 x 50 cm, packed with an acid-positive-ion-exchange resin, Amberlite 2OOC, purchased from Rohm and Hass Company (25 ml), and basic negative-ion-exchangeresin, Amberlite IRC-50 and IRA-93 (25 ml each), in order to eliminate the N,N.-dimetyl-4-amino-pyridine. After evaporation the residue was eluted with choloform (500 ml) and then chlorofom/methanol (10/l) (1500 ml) through a silica gel packed column, 1.5 x 50 cm, and then the solvent was evaporated off. Identification of the synthesized phospholipids was performed by fast atom bombardment-mass spectrometry (FAB-MS). Firstly, the synthesized phospholipids were directly subjected to FAB-MS. Secondly, the synthesized phospholipids were hydrolyzed with phospholipase A2 (Crotalus adamanteus, Sigma). After extraction with cold acetone, the extract was directly subjected to FAB-MS, or the extract was esterified with diazomethane and then subjected to FAB-MS. For example. in the case of 1-oleoyl-2-DHA3-PC the molecular ion (832, [M+H]+) was detected by FAB-MS, the molecular ions of I-oleoyl IysoPC (522, [M+H]+) and oleic acid methyl ester (297. [M+H]+) were also detected, respectively. Thin layer chromatographic plates of Silica Gel 60F,,, of 0.25 mm thickness were purchased from E. Merck, Darmstandt, Germany.
Enzyme preparation
and assay
5-Lipos_vgenase. RBL-1 cells were grown in Gibco’s Modified Eagle’s Minimum Essential Medium supplemented with 10% fetal bovine serum. The cells were cultured under 5% CO2 at 37’C and then harvested by centrifugation at 400 r.p.m for 5 min. RBL-1 cells (3 x lO’/ml) washed with 50 mM potassium phosphate buffer, pH 7.4. containing 1 mM EDTA were sonicated, followed by centrifugation at 10 000 x g for 30 min. The supematant solution was used to assay 5-lipoxygenase activity. 3.7 kBq [ l-14C]AA (2.15 kBq/mol) was routinely incubated to assay the 5-lipoxygenase activity in a reaction mixture (500 pl) comprising 440 pl of the buffer, 50 pl of the enzyme solution, 10 1.11of a 100 mM calcium chloride solution and 4 mM adenosine triphosphate (ATP) (final concentration). The reaction was carried out at 37°C for 15 min and stopped by acidification with HCl to pH 3. The mixture was extracted with ethyl acetate, and then the extract was subjected to thin-layer chromatography with a solvent system of diethyl ether/ petroleum ether/acetic acid (50/50/l).
12-Lipoxygenase and cyclooqqenuse. Venous blood was collected from male Wister rats and mixed with 3.8% citrate, the volume being about 10% of the blood. Platelet-rich plasma was prepared by centrifugation at 1200 r.p.m for 10 min. The platelet-rich plasma was gently mixed with 0.1 volumes of a 77 mM EDTA solution containing 120 mM NaCl and then recentrifuged at 2500 r.p.m for 10 min. The platelet pellets were then rinsed quickly with 15 mM Tris-HCl buffer, pH 7.4, containing 134 mM NaCl, 1 mM EDTA and 5 mM Dglucose, and centrifuged at 2000 r.p.m for 10 min. The pellets were then resuspended in 2 ml of 67 mM phosphate buffer, pH 8.0. containing 77 mM NaCl. The washed rat platelets were frozen at -80°C and thawed at 37°C using hot water. This procedure was repeated three times to obtain the enzyme source. [ l-‘C]AA (7.4 kBq/ 500 ul) was incubated at 37°C for 3 min to assay the 12lipoxygenase and cyclooxygenase activities, with 500 ul of the enzyme solution. The reaction was stopped by acidification to pH 3. The mixture was extracted with ethyl acetate, and the extract was subjected to thin layer chromatography with a solvent system of chloroform/ methanol/acetic acid/water (90/8/1/0.X). I5-Li@ygenuse. Isolated polymorphonuclear leukocytes (PMNLs) were sonicated at 4°C (15 s, 1OOW: sonicator model 5202, PZT: Ohtake Works, Tokyo, Japan). The sonicated PMNLs were incubated with [ I-‘“C]AA (7.4 kBq/SOO ul) at 37°C for 20 min after the addition of 1 mM CaCl, (final concentration). The reaction was stopped by acidification with HCl to pH 3. The mixture was extracted with ethyl acetate. and the extract was subjected to thin-layer chromatography with a solvent system of ethyl acetate/acetic acid/2,2,4_trimethylpentane/ water (1 l/2/5/10). Rudiouctility meuswemerzt. The radioactivity of each ‘-‘C-AA metabolite was detected and quantified with Berthold TLC-multi Tracemasters LB284 and LB285. respectively.
RESULTS Effects of DHA and DHA-containing lipoxygenase activity in RBL-1 cells
PC on 5-
Firstly. we examined whether or not DHA and DHAcontaining PC affected 5lipoxygenase by using a 10 000 x g supematant of sonicated RBL- 1. DHA did not inhibit the conversion of AA to 5-hydroxyeicosatetraenoic acid (5-HETE), as has been reported (lo), but the synthesized DHA-containing PC inhibited it very strongly (Fig. 1).
Effects of various fatty acids at the sn-2 position in 1-oleoyl-t-acyl-PC on 5-lipoxygenase activity in RBL-1 cells We next tried to compare the inhibitory effects of several kinds of 1-oleoyl-2-acyl-PC on 5-lipoxygenase activity
Inhibitory
di-HETE 0
Effect of Docosahexaenoic
5-HETE 0
15-HETE I.?.-HETE 0
Acid-containing
AA 0
!I
Phospholipids
on S-Lipoxygenase
in Rat Basophilic
Leukemia Cells
863
tor, the ID50 being about 4 pM. At 50 PM, I-oleoyl2-DHA-PC inhibited 5-lipoxygenase by 90% and 1,2diDHA-PC by 40%. while I-palmitoyl-2-DHA-PC hardly inhibited the enzyme.
Kinetic study on the inhibitory activity of I-oleoyl-2DHA-PC on Mipoxygenase activity in RBL-1 To investigate the inhibitory effect of I-oleoyl-2-DHAPC. the conversion of AA to 5HETE by a supernatant of sonicated RBL-I cells was examined, changing the concentration of AA as a substrate. The results of Lineweaver-Burk analysis are shown in Figure 4. In the absence of I-oleoyl-2-DHA-PC in the incubation mixture, V,,, was 50.5 nmol/min and Km 61 FM. However. in the presence of I-oleoyl-2-DHA-PC (7.2 PM), V,,, changed to 2.86 x 10-s mol/min, but Km was almost the same as that in the absence of the inhibitor (67 FM). Thus. the mechanism by which I-oleoyl-2-DHA-PC inhibited 5-lipoxygenase was non-competitive.
Effects of I-oleoyl3-DHA-PC on 12- and 15lipoxygenase, and cyclooxygenase activity
Fig. 1 Radiochromatogram scan showing the conversion of [‘%]AA by the supematant of RBL- 1 cells in the presence of DHA or I oieoyl-2-DHA-PC. RBL-1 cells were sonicated and the 10 000 xg supematant was incubated with 0. I pCi of [‘%Z]AA at 37°C for I5 min Ia), in the presence of DHA, (b) or I-oleoyl-2-DHA-PC (c). The reaction mixture was extracted to be subjected lo thin-layer chromatography. The thin-layer chromatography plates were developed with a solvent system of chloroform/methanol/acetic acid/ water 190/X/1/0.8. v/v,.
in RBL-1. The sn-2 fatty acids used were oleic acid, linoleic acid, AA. EPA and DHA. With 25 pM of each synthesized 1-oleoyl-2-acyl-PC, the inhibitory activity was dependent on the numbers of double bonds and carbon atoms in the molecule of the sn-2 fatty acid; that is, DHA (22:6) > EPA (205) > AA (20:4) > linoleic acid ( 18:2) > oleic acid (18: 1). The maximum inhibition of both 1-oleoyl-2-docosahexaenoyland 1-oleoyl2-eicosapentaenoyl-PC was about 90%, but that of Ioleoyl-2-arachidonyl-PC was only 40% (Fig. 2).
Effects of various fatty acids at the m-1 position in 1-acyl-2-DHA-PC on S-lipoxygenase activity in RBL1 cells In addition, we compared the inhibitory activities of fatty acids at &he sn-1 position in 1-acyl-2-DHA-PC on 5-lipoxygenase activity in RBL- 1 (Fig. 3). The sn- 1 fatty acids tested were palmitic acid, oleic acid and DHA. Of these, I-oleoyl-2-DHA-PC was the most potent inhibi-
We next examined the effects of 1-oleoyl-2-DHA-PC on 12-lipoxygenase and cyclooxygenase activity in rat platelets, and 15-lipoxygenase activity in human neutrophils to investigate its specificity. The data are shown in Figure 5. 1-Oleoyl-2-DHA-PC up to 25 pM showed substantially no effect on 12- or 15-lipoxygenase. or cyclooxygenase, but did cause as much as 90% inhibition of 5-lipoxygenase. At 50 pM, 15-lipoxygenase and cyclooxygenase were not inhibited at ail and 12-lipoxygenase was inhibited by only 30%. These results confinned that the inhibitory effect of 1-oleoyl-2-DHA-PC was specific to Slipoxygenase.
DISCUSSION DHA has been reported to be a poor substrate for 5 lipoxygenase, and it is a weak inhibitor of AA conversion to 5-HETE in RBL-1 ( 10). In this paper, we confirmed this, but a series of DHA-containing PC were found to be very strong inhibitors of 5-lipoxygenase. Among the DHA-containing PC examined, 1-oleoyl-2DHA-PC was the most potent inhibitor of 5-lipoxygenase, the ID,, value being 4.0 pM (Fig. 2). This value was almost equal to that of caffeic acid, which was previously reported by us to be a potent inhibitor of 5lipoxygenase (11). In several 1-acyl-2-DHA-PC, the fatty acid at the first position was found to be responsible for the inhibitory activity toward 5-lipoxygenase to a great extent (Fig. 3). On the other hand, the fatty acid occupying the second position in I-oleoyl-2-acyl-PC was more important for the inhibition of 5-lipoxygenase. The inhibitory activities of such PC toward 5lipoxygenase were correlated well with the number of double bonds
864
Prostaglandins
Leukotrienes
and Essential Fatty Acids
lOOk
2 ..+
n .s
s .-
Concentration Fig. 2 reaction position or DHA mean of
of lipids
Effect of the \n-2-fatty acid of I-oleoyl-2-acyl-PC on 5-lipoxygenase in RBL-I cells. The conditions were identical with those given in the legend to Figure I The fatty acid at the sn-2. in I-oleoyl-2-acyl-PC was oleic acid (A--A). linoleic acid (( .)--<~)). AA (n--n), EPA (B-4) (@--0). respectively. Values are expressed as o/ of the control. Each point represents the duplicate determinations for two different experiments.
Concentration
of lipids
(PM)
Fig. 3 Effect of the l-fatty acid of I-acyl-2-DHA-PC on S-lipoxygenase activity in RBL-I cells. The reaction conditions were identical with those given in the legend to Figure I, The fatty acid at the l-position in I-acyl-2-DHA-PC was palmitoic acid ((_~)-J~~),oleic acid (e--e) or DHA (O--O). Values are expressed as % of the control. Each point represents the mean of duplicate determinations for two different experiments.
and the chain-length of the fatty acid of the second tion, that is, DHA > EPA > AA > linoleic acid > acid (Fig. 2). If the PC was replaced by phophatidic the inhibitory activity decreased by 70% at 10 pM not shown). The mechanism by which the DHA-containing
posioleic acid, (data PC
inhibit S-lipoxygenase was non-competitive. The reason why I-oleoyl-2-DHA-PC is a potent inhibitor of 5 lipoxygenase remains unclear. It has been reported that Slipoxygenase is activated by Ca*+ and ATP (12, 13), and some phospholipids (8). Recently, Miller et al (9) and Dixon et al (14) reported that some proteins are
Inhibitory Et’i’ectof DocosahexaenoicAcid-containing Phospholipidson 5-Lipoxygenasein Rat
l/Substrate
(FM)-’
Fig. 4 Kinclic s~utiy on the inhibition by I-oleoyl-2-DHA-PC. The reaction conditions of the reaction were identical with those given in the legend IO Figure I except for the AA concentration. The concentration of AA used as a substrate was 0.5, I, 2. 5 or 10 pg. Control (0) and presence of I-oleoyl-2-DHA-PC (A).
Basophillc Leukemia Cell\
X65
rated into PC, phosphatidylethanolamine (PE). phosphatidylinositol (PI) and phosphatidylserine (PS) in human platelets ( IS), neutrophils ( 16) and brain membranes (6). According to the reports. DHA was incorporated into PE by 45% and PC by 17%.. The DHA incorporated was not easily released by stimulants which could release AA and EPA ( 16). It was also reported that DHA inhibited AA metabolism to prostaglandins in both human endothelial cells ( 17) and platelets (I 8, 19). In these cases the inhibitory mechanism of DHA is competitive. However. in the present study the inhibition by DHA-containing PC was found to be non-competitive. Therefore, it is unlikely that DHA-containing PC were decomposed during the incubation period. The present data may explain the fact that the inpestion of DHA causes a low incidence of cardiovascular diseases, and may also indicate that DHA-containing PC could be expected to attenuate leukotriene B1- mediated leukocyte chemotaxis and neutrophil adhesion on endothelial cells. Thus, DHA-containing PC may bring about an anti-inflammatory effect.
References required for the activation of 5-lipoxygenase. Therefore, it is thought that S-lipoxygenase is translocated from cytosol to membrane. However, these studies were not able to demonstrate the role of phospholipids in Slipoxygenase activation. If the 5lipoxygenase activating protein (FLAP) has different affinity to phospholipids. the data obtained in this study will be more interesting. However. to obtain a more conclusive picture of the inhibitory mechanism. it may be necessary to use purified S-lipoxygenase. DHA as well as EPA has been reported to be incorpo-
Bang H 0. Dyerbeg J. Sinclar J M. The composition oi food comsumed by Greenland Eskimos. Acta Med Scand 1916: ‘00: 69-75. Dyerbcrg J. Bang H 0. Fatty acid composition of the plasma lipids in Greenland Eskimos. Am J Clin Nut! 1975: 18: YSU-Y66. Nagakawa Y. Orimo H. Harasawa M, Morlta I. Ya\hnv K, Murota S. Effect of eicosapentaenoic acid on platelet aggregation and composition of fatty acid in man. Atheroaclerosih IYX.3: 47: 71-75. Needleman P. Minkes M F, Ferrendelli F A. Sprecher H. Triene prostaglandins: prostacyclin and thromhoxane biosynthejih and unique biological propertie\. f’roc Nat1 Acad Sci (ISA lY7Y: 76: Y44-Y4X.
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Fig. 5 Effech of‘ I-oleoyl-2.DHA-PC on S-lipoxypenase. I?-lipoxygenase. 1%lipoxygenasc XXI cyclooxygcnase activities. Each enzyme solution, S-lipoxygenase in RBL- I (0). 17.lipoxygenase (m) and cyclooxygenase ( 0 ) 111 washed rut platelets. and IS-lipoxygenase ( q ) in human neutrophils was incubated with 0. I pCi of 1I’CjAA m the pre\encc or absence ot I -oleoyl-2-DHA-PC. Values are expressed as p/r of the control. Each bar reprchenL\ the menns ? SE for al Icast duplicate determinations for two different expcrimcnts.
866
Prostaglandins
Leukotrienes
and Essential Fatty Acids
5. Siess W, Roth P. Scherer B, Kurzmann 1. Bohlig B. Weber PC. Plateiet-membrane fatty acids, platelet aggregation, and thromboxane formation during a mackerel diet, Lancet 1980; 441-444 6. Onuma Y. Masuzawa Y, Ishima Y. Waku K. Selective incorporation of docosahexaenoic acid in rat brain. Biochim Biophys Acta 1984: 793: 80-85. 7. Anderson R E, Benolken R M, Duley P A. Landis D J. Wheler T G. Polyunsaturated fatty acids of photoreceptoer protein. Exp Eye Res 1974: 18: 205-213. 8. Goetze A M, Fayer L. Carter G W. Purification of mammalian 5-lipoxygenase from rat basophilic leukemia cells. Prostaglandins 1985: 29: 689-701. 9. Miller D K, Gillard J W. Vickers P J. Identification and isolation of a membrane protein necessary for leukotriene production. Nature 1990: 343( 18): 278-28 I IO. Comey E J, Shih C, Cashman J R. Docosahexaenoic acid is a strong inhibitor of prostaglandins but not leukotriene biosynthesis. Proc Nat1 Acad Sci USA 1983; 80: 3581-3584. 11. Koshihara Y, Neichi T, Murota S, Lao A. Fujimoto Y. Tatsuno T. Selective inhibition of 5-lipoxygenase of rat basophilic leukemia cells. FEBS Lett 1983: 158: 41-44. 12. Furukawa M. Yoshimoto T, Ochi K. Yoshimoto S. Studies on arachidonate 5-lipoxygenase of rat basophilic
13.
14.
15. 16.
17.
18.
19.
leukemia cells. Biochim Biophys Acta 1984: 795: 458-465. Ochi K. Yoshimoto T. Taniguchi K. Miyamoto T. Arachidonate 5-lipoxygenase of guinea pig peritoneal polymotphonuclear leukocytes. J Biol Chem 1983: 258: 5754-5758. Dixon R A. Diehl R E. Opas E. Requirement of a 5 lipoxygenase activating protein for leukotriene synthesis. Nature 1990: 343( 18): 282-284. Weiner T W, Sprecher H. 22.Carbon polyenoic acids. J Biol Chem 1985; 260: 60326038. Fisher S, Schacky C V. Siess E, Strasser T H. Weber PC. Uptake. release and metabolism of docosahexaenoic acid (DHA. C22:6w3) in human platelets and neutrophils. Biochem Biophys Res Commun 1984: 120: 907-9 18. Nordoy A, Lyngmo V, Vartun A, Svenson 9. Docosahexaenoic fatty acids and human endothelial cells. Biochim Biophys Acta 1986: 877: 31-36. Rae G H, Radha E. White J G. Effect of docosahexaenoic acid (DHA) on arachidonic acid metabolism and platelet function. Biochem Biophys Res Comm 1983: 117: 549-555. Srivastava K C. Docosahexaenic acid (C22:6w3) and linoleic acid are anti-aggregatory, and alter arachidonic acid metabolism in human platelets. Prostaglandins Leuko Med 1985; 17: 319-327.
Phospholipids
on
.ABSTRACT.
5-Lipoxygenase has been recognized to be an important enzyme that catalyzes the first step in leukotriene production. In this study we examine whether or not phospholipids containing docosahexaenoic acid (DHA) affect S-lipoxygenase activity of a rat basophilic leukemia cell line (RBL-1). Among the synthesized phospholipids examined, l-oleoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (I-oleoyl-2-DHA-PC) was found to be the most potent inhibitor of S-lipoxygenase. The inhibition was dose-dependent and the ID,, value was 4.0 yM. When the fatty acid at the sn-tposition was replaced by other unsaturated fatty acids, the inhibitory activity decreased with decreasing numbers of both carbon atoms and double bonds in the fatty acids. Substitution at the l-position of the DHA-containing PC also affected the inhibitory potency. If oleic acid was substituted with palmitic acid, the inhibition activity was completely abolished. Lineweaver-Burk plot analysis showed that the inhibition of S-lipoxygenase by I-oleoyl-2-DHA-PC was non-competitive. The inhibition by this synthesized phospholipid was very specific to Uipoxygenase; that is, it did not extend to fatty acid cyclooxygenase, 124ipoxygenase or 154ipoxygenase. These results suggest that endogenously existing DHA-containing phospholipids may affect 54ipoxygenase activity and thus control leukotriene biosynthesis in vivo.
INTRODUCTION
been carried out to elucidate the biophysical function of‘ DHA as a free fatty acid, but it has been commonly~ accepted that DHA incorporated into cellular phospholipids is released from membrane to a much lesser extent than AA and EPA. Therefore, it is necessary to study the biological function of DHA-containing phospholipids. It has been reported that S-lipoxygenase is activated by some phospholipids (8). and recent studies have demonstrated that S-lipoxygenase is translocated from the cytosol to membrane (9). These reports indicate that the quality of membrane phospholipids affects 5lipoxygenase activity. In the present study. we examine the effects of several kinds of synthesized DHA-containing phospholipids on S-lipoxygenase in a rat basophilic leukemia cell line (RBL-1) and show that I-oleoyl-2docosahexaenoyl-sn-glycero-3-phosphocholine ( 1-oleoyl?-DHA-PC) is the most potent inhibitor of 5lipoxygenase.
It is commonly recognized that 4. 7. 10, 13. 16. 19docosahexaenoic acid (DHA) is the longest and most highly unsaturated fatty acid in the n-3 class. 5, 8, 11, I-t. 17-Eicosa-pentaenoic acid (EPA) and DHA have been reported to be substances contributing to a low incidence of myocardial infarction and ischemic heart disease ( 1. 2). Regarding the mechanism underlying this activity. it is believed that both DHA and EPA are incorporated into phospholipids in platelets in place of arachidonic acid (AA), a substrate of fatty acid cyclooxygenase. which leads to a decrease in thromboxane A2 production (3. 4). It is also believed that these unsaturated fatty acids also inhibit cyclooxygenase activity in platelets (5). Regarding other functions of DHA, it is thought that DHA has something to do with the development of the cerebral cortex and retinal membrane. because the ‘k of DHA to total phospholipids is high in both tissues (6, 7). A number of in vivo studies have
MATERIALS
AND METHODS
Chemicals Dale received 76 April 1993’ Dare accepted I7 May 1993
[I-“CJAA Xhl
(2.15 kBq/mol)
was
purchased
from
the
862 Prostaglandins Leukotrienes and Essential Fatty Acids
Radiochemical Centre. Amersham, UK. Unlabeled AA was purchased from Sigma, St Louis, MO. 1,2-Dipalmitoyl-sn-glycero-3-phosphocholines was purchased from Wako Pure Chemical Industries, Osaka. Other phosphatidylcholine (PC) used here were synthesized from 1-acyl-sn-glycero-3-phosphocholine and fatty acid anhydrides. 1-Acyl-sn-glycero-3-phosphocholine ( 1.49 mM). fatty acid anhydride ( 1.64 mM) and N,N,-dimethyl-4amino-pyridine ( 1.66 mM) were stirred in anhydrous chloroform at room temperature for 24 h. The solution was eluted with chloroform through a glass column, 3.0 x 50 cm, packed with an acid-positive-ion-exchange resin, Amberlite 2OOC, purchased from Rohm and Hass Company (25 ml), and basic negative-ion-exchangeresin, Amberlite IRC-50 and IRA-93 (25 ml each), in order to eliminate the N,N.-dimetyl-4-amino-pyridine. After evaporation the residue was eluted with choloform (500 ml) and then chlorofom/methanol (10/l) (1500 ml) through a silica gel packed column, 1.5 x 50 cm, and then the solvent was evaporated off. Identification of the synthesized phospholipids was performed by fast atom bombardment-mass spectrometry (FAB-MS). Firstly, the synthesized phospholipids were directly subjected to FAB-MS. Secondly, the synthesized phospholipids were hydrolyzed with phospholipase A2 (Crotalus adamanteus, Sigma). After extraction with cold acetone, the extract was directly subjected to FAB-MS, or the extract was esterified with diazomethane and then subjected to FAB-MS. For example. in the case of 1-oleoyl-2-DHA3-PC the molecular ion (832, [M+H]+) was detected by FAB-MS, the molecular ions of I-oleoyl IysoPC (522, [M+H]+) and oleic acid methyl ester (297. [M+H]+) were also detected, respectively. Thin layer chromatographic plates of Silica Gel 60F,,, of 0.25 mm thickness were purchased from E. Merck, Darmstandt, Germany.
Enzyme preparation
and assay
5-Lipos_vgenase. RBL-1 cells were grown in Gibco’s Modified Eagle’s Minimum Essential Medium supplemented with 10% fetal bovine serum. The cells were cultured under 5% CO2 at 37’C and then harvested by centrifugation at 400 r.p.m for 5 min. RBL-1 cells (3 x lO’/ml) washed with 50 mM potassium phosphate buffer, pH 7.4. containing 1 mM EDTA were sonicated, followed by centrifugation at 10 000 x g for 30 min. The supematant solution was used to assay 5-lipoxygenase activity. 3.7 kBq [ l-14C]AA (2.15 kBq/mol) was routinely incubated to assay the 5-lipoxygenase activity in a reaction mixture (500 pl) comprising 440 pl of the buffer, 50 pl of the enzyme solution, 10 1.11of a 100 mM calcium chloride solution and 4 mM adenosine triphosphate (ATP) (final concentration). The reaction was carried out at 37°C for 15 min and stopped by acidification with HCl to pH 3. The mixture was extracted with ethyl acetate, and then the extract was subjected to thin-layer chromatography with a solvent system of diethyl ether/ petroleum ether/acetic acid (50/50/l).
12-Lipoxygenase and cyclooqqenuse. Venous blood was collected from male Wister rats and mixed with 3.8% citrate, the volume being about 10% of the blood. Platelet-rich plasma was prepared by centrifugation at 1200 r.p.m for 10 min. The platelet-rich plasma was gently mixed with 0.1 volumes of a 77 mM EDTA solution containing 120 mM NaCl and then recentrifuged at 2500 r.p.m for 10 min. The platelet pellets were then rinsed quickly with 15 mM Tris-HCl buffer, pH 7.4, containing 134 mM NaCl, 1 mM EDTA and 5 mM Dglucose, and centrifuged at 2000 r.p.m for 10 min. The pellets were then resuspended in 2 ml of 67 mM phosphate buffer, pH 8.0. containing 77 mM NaCl. The washed rat platelets were frozen at -80°C and thawed at 37°C using hot water. This procedure was repeated three times to obtain the enzyme source. [ l-‘C]AA (7.4 kBq/ 500 ul) was incubated at 37°C for 3 min to assay the 12lipoxygenase and cyclooxygenase activities, with 500 ul of the enzyme solution. The reaction was stopped by acidification to pH 3. The mixture was extracted with ethyl acetate, and the extract was subjected to thin layer chromatography with a solvent system of chloroform/ methanol/acetic acid/water (90/8/1/0.X). I5-Li@ygenuse. Isolated polymorphonuclear leukocytes (PMNLs) were sonicated at 4°C (15 s, 1OOW: sonicator model 5202, PZT: Ohtake Works, Tokyo, Japan). The sonicated PMNLs were incubated with [ I-‘“C]AA (7.4 kBq/SOO ul) at 37°C for 20 min after the addition of 1 mM CaCl, (final concentration). The reaction was stopped by acidification with HCl to pH 3. The mixture was extracted with ethyl acetate. and the extract was subjected to thin-layer chromatography with a solvent system of ethyl acetate/acetic acid/2,2,4_trimethylpentane/ water (1 l/2/5/10). Rudiouctility meuswemerzt. The radioactivity of each ‘-‘C-AA metabolite was detected and quantified with Berthold TLC-multi Tracemasters LB284 and LB285. respectively.
RESULTS Effects of DHA and DHA-containing lipoxygenase activity in RBL-1 cells
PC on 5-
Firstly. we examined whether or not DHA and DHAcontaining PC affected 5lipoxygenase by using a 10 000 x g supematant of sonicated RBL- 1. DHA did not inhibit the conversion of AA to 5-hydroxyeicosatetraenoic acid (5-HETE), as has been reported (lo), but the synthesized DHA-containing PC inhibited it very strongly (Fig. 1).
Effects of various fatty acids at the sn-2 position in 1-oleoyl-t-acyl-PC on 5-lipoxygenase activity in RBL-1 cells We next tried to compare the inhibitory effects of several kinds of 1-oleoyl-2-acyl-PC on 5-lipoxygenase activity
Inhibitory
di-HETE 0
Effect of Docosahexaenoic
5-HETE 0
15-HETE I.?.-HETE 0
Acid-containing
AA 0
!I
Phospholipids
on S-Lipoxygenase
in Rat Basophilic
Leukemia Cells
863
tor, the ID50 being about 4 pM. At 50 PM, I-oleoyl2-DHA-PC inhibited 5-lipoxygenase by 90% and 1,2diDHA-PC by 40%. while I-palmitoyl-2-DHA-PC hardly inhibited the enzyme.
Kinetic study on the inhibitory activity of I-oleoyl-2DHA-PC on Mipoxygenase activity in RBL-1 To investigate the inhibitory effect of I-oleoyl-2-DHAPC. the conversion of AA to 5HETE by a supernatant of sonicated RBL-I cells was examined, changing the concentration of AA as a substrate. The results of Lineweaver-Burk analysis are shown in Figure 4. In the absence of I-oleoyl-2-DHA-PC in the incubation mixture, V,,, was 50.5 nmol/min and Km 61 FM. However. in the presence of I-oleoyl-2-DHA-PC (7.2 PM), V,,, changed to 2.86 x 10-s mol/min, but Km was almost the same as that in the absence of the inhibitor (67 FM). Thus. the mechanism by which I-oleoyl-2-DHA-PC inhibited 5-lipoxygenase was non-competitive.
Effects of I-oleoyl3-DHA-PC on 12- and 15lipoxygenase, and cyclooxygenase activity
Fig. 1 Radiochromatogram scan showing the conversion of [‘%]AA by the supematant of RBL- 1 cells in the presence of DHA or I oieoyl-2-DHA-PC. RBL-1 cells were sonicated and the 10 000 xg supematant was incubated with 0. I pCi of [‘%Z]AA at 37°C for I5 min Ia), in the presence of DHA, (b) or I-oleoyl-2-DHA-PC (c). The reaction mixture was extracted to be subjected lo thin-layer chromatography. The thin-layer chromatography plates were developed with a solvent system of chloroform/methanol/acetic acid/ water 190/X/1/0.8. v/v,.
in RBL-1. The sn-2 fatty acids used were oleic acid, linoleic acid, AA. EPA and DHA. With 25 pM of each synthesized 1-oleoyl-2-acyl-PC, the inhibitory activity was dependent on the numbers of double bonds and carbon atoms in the molecule of the sn-2 fatty acid; that is, DHA (22:6) > EPA (205) > AA (20:4) > linoleic acid ( 18:2) > oleic acid (18: 1). The maximum inhibition of both 1-oleoyl-2-docosahexaenoyland 1-oleoyl2-eicosapentaenoyl-PC was about 90%, but that of Ioleoyl-2-arachidonyl-PC was only 40% (Fig. 2).
Effects of various fatty acids at the m-1 position in 1-acyl-2-DHA-PC on S-lipoxygenase activity in RBL1 cells In addition, we compared the inhibitory activities of fatty acids at &he sn-1 position in 1-acyl-2-DHA-PC on 5-lipoxygenase activity in RBL- 1 (Fig. 3). The sn- 1 fatty acids tested were palmitic acid, oleic acid and DHA. Of these, I-oleoyl-2-DHA-PC was the most potent inhibi-
We next examined the effects of 1-oleoyl-2-DHA-PC on 12-lipoxygenase and cyclooxygenase activity in rat platelets, and 15-lipoxygenase activity in human neutrophils to investigate its specificity. The data are shown in Figure 5. 1-Oleoyl-2-DHA-PC up to 25 pM showed substantially no effect on 12- or 15-lipoxygenase. or cyclooxygenase, but did cause as much as 90% inhibition of 5-lipoxygenase. At 50 pM, 15-lipoxygenase and cyclooxygenase were not inhibited at ail and 12-lipoxygenase was inhibited by only 30%. These results confinned that the inhibitory effect of 1-oleoyl-2-DHA-PC was specific to Slipoxygenase.
DISCUSSION DHA has been reported to be a poor substrate for 5 lipoxygenase, and it is a weak inhibitor of AA conversion to 5-HETE in RBL-1 ( 10). In this paper, we confirmed this, but a series of DHA-containing PC were found to be very strong inhibitors of 5-lipoxygenase. Among the DHA-containing PC examined, 1-oleoyl-2DHA-PC was the most potent inhibitor of 5-lipoxygenase, the ID,, value being 4.0 pM (Fig. 2). This value was almost equal to that of caffeic acid, which was previously reported by us to be a potent inhibitor of 5lipoxygenase (11). In several 1-acyl-2-DHA-PC, the fatty acid at the first position was found to be responsible for the inhibitory activity toward 5-lipoxygenase to a great extent (Fig. 3). On the other hand, the fatty acid occupying the second position in I-oleoyl-2-acyl-PC was more important for the inhibition of 5-lipoxygenase. The inhibitory activities of such PC toward 5lipoxygenase were correlated well with the number of double bonds
864
Prostaglandins
Leukotrienes
and Essential Fatty Acids
lOOk
2 ..+
n .s
s .-
Concentration Fig. 2 reaction position or DHA mean of
of lipids
Effect of the \n-2-fatty acid of I-oleoyl-2-acyl-PC on 5-lipoxygenase in RBL-I cells. The conditions were identical with those given in the legend to Figure I The fatty acid at the sn-2. in I-oleoyl-2-acyl-PC was oleic acid (A--A). linoleic acid (( .)--<~)). AA (n--n), EPA (B-4) (@--0). respectively. Values are expressed as o/ of the control. Each point represents the duplicate determinations for two different experiments.
Concentration
of lipids
(PM)
Fig. 3 Effect of the l-fatty acid of I-acyl-2-DHA-PC on S-lipoxygenase activity in RBL-I cells. The reaction conditions were identical with those given in the legend to Figure I, The fatty acid at the l-position in I-acyl-2-DHA-PC was palmitoic acid ((_~)-J~~),oleic acid (e--e) or DHA (O--O). Values are expressed as % of the control. Each point represents the mean of duplicate determinations for two different experiments.
and the chain-length of the fatty acid of the second tion, that is, DHA > EPA > AA > linoleic acid > acid (Fig. 2). If the PC was replaced by phophatidic the inhibitory activity decreased by 70% at 10 pM not shown). The mechanism by which the DHA-containing
posioleic acid, (data PC
inhibit S-lipoxygenase was non-competitive. The reason why I-oleoyl-2-DHA-PC is a potent inhibitor of 5 lipoxygenase remains unclear. It has been reported that Slipoxygenase is activated by Ca*+ and ATP (12, 13), and some phospholipids (8). Recently, Miller et al (9) and Dixon et al (14) reported that some proteins are
Inhibitory Et’i’ectof DocosahexaenoicAcid-containing Phospholipidson 5-Lipoxygenasein Rat
l/Substrate
(FM)-’
Fig. 4 Kinclic s~utiy on the inhibition by I-oleoyl-2-DHA-PC. The reaction conditions of the reaction were identical with those given in the legend IO Figure I except for the AA concentration. The concentration of AA used as a substrate was 0.5, I, 2. 5 or 10 pg. Control (0) and presence of I-oleoyl-2-DHA-PC (A).
Basophillc Leukemia Cell\
X65
rated into PC, phosphatidylethanolamine (PE). phosphatidylinositol (PI) and phosphatidylserine (PS) in human platelets ( IS), neutrophils ( 16) and brain membranes (6). According to the reports. DHA was incorporated into PE by 45% and PC by 17%.. The DHA incorporated was not easily released by stimulants which could release AA and EPA ( 16). It was also reported that DHA inhibited AA metabolism to prostaglandins in both human endothelial cells ( 17) and platelets (I 8, 19). In these cases the inhibitory mechanism of DHA is competitive. However. in the present study the inhibition by DHA-containing PC was found to be non-competitive. Therefore, it is unlikely that DHA-containing PC were decomposed during the incubation period. The present data may explain the fact that the inpestion of DHA causes a low incidence of cardiovascular diseases, and may also indicate that DHA-containing PC could be expected to attenuate leukotriene B1- mediated leukocyte chemotaxis and neutrophil adhesion on endothelial cells. Thus, DHA-containing PC may bring about an anti-inflammatory effect.
References required for the activation of 5-lipoxygenase. Therefore, it is thought that S-lipoxygenase is translocated from cytosol to membrane. However, these studies were not able to demonstrate the role of phospholipids in Slipoxygenase activation. If the 5lipoxygenase activating protein (FLAP) has different affinity to phospholipids. the data obtained in this study will be more interesting. However. to obtain a more conclusive picture of the inhibitory mechanism. it may be necessary to use purified S-lipoxygenase. DHA as well as EPA has been reported to be incorpo-
Bang H 0. Dyerbeg J. Sinclar J M. The composition oi food comsumed by Greenland Eskimos. Acta Med Scand 1916: ‘00: 69-75. Dyerbcrg J. Bang H 0. Fatty acid composition of the plasma lipids in Greenland Eskimos. Am J Clin Nut! 1975: 18: YSU-Y66. Nagakawa Y. Orimo H. Harasawa M, Morlta I. Ya\hnv K, Murota S. Effect of eicosapentaenoic acid on platelet aggregation and composition of fatty acid in man. Atheroaclerosih IYX.3: 47: 71-75. Needleman P. Minkes M F, Ferrendelli F A. Sprecher H. Triene prostaglandins: prostacyclin and thromhoxane biosynthejih and unique biological propertie\. f’roc Nat1 Acad Sci (ISA lY7Y: 76: Y44-Y4X.
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Fig. 5 Effech of‘ I-oleoyl-2.DHA-PC on S-lipoxypenase. I?-lipoxygenase. 1%lipoxygenasc XXI cyclooxygcnase activities. Each enzyme solution, S-lipoxygenase in RBL- I (0). 17.lipoxygenase (m) and cyclooxygenase ( 0 ) 111 washed rut platelets. and IS-lipoxygenase ( q ) in human neutrophils was incubated with 0. I pCi of 1I’CjAA m the pre\encc or absence ot I -oleoyl-2-DHA-PC. Values are expressed as p/r of the control. Each bar reprchenL\ the menns ? SE for al Icast duplicate determinations for two different expcrimcnts.
866
Prostaglandins
Leukotrienes
and Essential Fatty Acids
5. Siess W, Roth P. Scherer B, Kurzmann 1. Bohlig B. Weber PC. Plateiet-membrane fatty acids, platelet aggregation, and thromboxane formation during a mackerel diet, Lancet 1980; 441-444 6. Onuma Y. Masuzawa Y, Ishima Y. Waku K. Selective incorporation of docosahexaenoic acid in rat brain. Biochim Biophys Acta 1984: 793: 80-85. 7. Anderson R E, Benolken R M, Duley P A. Landis D J. Wheler T G. Polyunsaturated fatty acids of photoreceptoer protein. Exp Eye Res 1974: 18: 205-213. 8. Goetze A M, Fayer L. Carter G W. Purification of mammalian 5-lipoxygenase from rat basophilic leukemia cells. Prostaglandins 1985: 29: 689-701. 9. Miller D K, Gillard J W. Vickers P J. Identification and isolation of a membrane protein necessary for leukotriene production. Nature 1990: 343( 18): 278-28 I IO. Comey E J, Shih C, Cashman J R. Docosahexaenoic acid is a strong inhibitor of prostaglandins but not leukotriene biosynthesis. Proc Nat1 Acad Sci USA 1983; 80: 3581-3584. 11. Koshihara Y, Neichi T, Murota S, Lao A. Fujimoto Y. Tatsuno T. Selective inhibition of 5-lipoxygenase of rat basophilic leukemia cells. FEBS Lett 1983: 158: 41-44. 12. Furukawa M. Yoshimoto T, Ochi K. Yoshimoto S. Studies on arachidonate 5-lipoxygenase of rat basophilic
13.
14.
15. 16.
17.
18.
19.
leukemia cells. Biochim Biophys Acta 1984: 795: 458-465. Ochi K. Yoshimoto T. Taniguchi K. Miyamoto T. Arachidonate 5-lipoxygenase of guinea pig peritoneal polymotphonuclear leukocytes. J Biol Chem 1983: 258: 5754-5758. Dixon R A. Diehl R E. Opas E. Requirement of a 5 lipoxygenase activating protein for leukotriene synthesis. Nature 1990: 343( 18): 282-284. Weiner T W, Sprecher H. 22.Carbon polyenoic acids. J Biol Chem 1985; 260: 60326038. Fisher S, Schacky C V. Siess E, Strasser T H. Weber PC. Uptake. release and metabolism of docosahexaenoic acid (DHA. C22:6w3) in human platelets and neutrophils. Biochem Biophys Res Commun 1984: 120: 907-9 18. Nordoy A, Lyngmo V, Vartun A, Svenson 9. Docosahexaenoic fatty acids and human endothelial cells. Biochim Biophys Acta 1986: 877: 31-36. Rae G H, Radha E. White J G. Effect of docosahexaenoic acid (DHA) on arachidonic acid metabolism and platelet function. Biochem Biophys Res Comm 1983: 117: 549-555. Srivastava K C. Docosahexaenic acid (C22:6w3) and linoleic acid are anti-aggregatory, and alter arachidonic acid metabolism in human platelets. Prostaglandins Leuko Med 1985; 17: 319-327.