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Docosapolyenoic fatty acids and human endothelial cells
Biochimica et Biophysics Acta 877 (1986) 31-36
31
Elsevier
BBA 52246
Docosapolyenoic fatty acids and human endothelial cells
A. Nordoy *, V. Lyngmo, A. V&m Department
ofMedicine,University Hospital, 9012 (Received
Key words:
and B. Svensson
Lipid metabolism;
December
Tromso (Norway)
2nd, 1985)
Prostacyclin
synthesis;
(Endothelial
cell)
The total fatty acids in human endothelial cells include approximately 5% each of 22: 4(n - 6), 22 : 5(n - 3) and 22: 6(n - 3), whereas 22: 5(n - 6) is present only in trace amounts. This study evaluates the effect of three of these fatty acids bound to albumin on lipid composition and prostacyclin (prostaglandin I*) synthesis in primary cultures of endothelial cell monolayers. 22: 4(n - 6), 22: 5(n - 6) and 22: 6(n - 3) were all incorporated into total phospholipids. 20: 4(n - 6) was reduced in phospholipids in all cells incubated with the three different docosaenoic fatty acids. This reduction was abolished when equimolar concentrations of 20: 4(n - 6) and the separate docosaenoic fatty acid were added to the medium simultaneously. 22: 4(n - 6) incorporation into the free fatty acids was associated with an increase of 20: 4(n - 6) in this fraction. 22 : 4( n - 6), 22 : S(n - 6) and 22 : 5(n - 3) all reduced the synthesis of prostacylin measured as 6-ketoprostaglandin F,,. These effects were reversed by simultaneous incubation with 20: 4(n - 6). This study shows that three of the docosaenoic fatty acids present in human endothelial cells of the (n - 6) and (n - 3) family were all incorporated into endothelial cells with a simultaneous reduction in 20: 4(n - 6). The three fatty acids reduced the synthesis of prostacylin. Introduction
Dietary linoleic (n - 6) and linolenic (n - 3) acids are metabolized by desaturation and elongation to two series of very-long-chain polyunsaturated fatty acids [l]. The docosaenoic fatty acids of the (n - 6) family, 22 : 4 (7,10,13,16) and 22 : 5 (4,7,10,13,16) are present in human platelets and endothelial cells [2,3] and may be further metabolized by platelets into dihomothromboxane B, and 4-dihomothromboxane B,, respectively [4]. Also, hydroxyacids are formed by the lipoxygenase pathway [4,5]. The docosaenoic fatty acids of the (n - 3) family 22 : 5 (7,10,13,16,19) and 22 : 6 (4,7,10,13,16,19) are present in marine oils and fish. Recent studies have indicated that particularly 20 : 5 (5,8,11,14,17) of the (n - 3)
family may reduce platelet aggregation when it is incorporated into platelet phospholipids [6]. Small amounts of (n - 3) docosaenoic acids are also present in human platelets and endothelial cells [2,3]. They may be further metabolized by platelets into hydroxy acid isomers and may depress thromboxane A 2 production [7,8]. In the present study we have examined the content of docosaenoic acids in human endothelial cells and investigated the metabolism of 22: 4 (7,10,13,16), 22 : 5 (4,7,10,13,16) and 22 : 6 (4,7,10,13,16,19) and their effects on prostacyclin production. Methods Cell
human cell monolayers were Jaffe et al. [9] modifications [lo]. The cells were in 35 x 10 umbillical vein
* To whom correspondence
0005-2760/86/$03.50
should be addressed.
1986 Elsevier Science Publishers
B.V. (Biomedical
Division)
32
mm culture dishes in modified medium E 199 (medium E 199) containing 20% of foetal calf serum until confluency (4-5 days). Test procedure. At the time of confluence the medium was pipetted off, and the cells were washed twice with 2 ml medium E 199 without foetal calf serum. The endothelial cells were then incubated with 2 ml medium E 199 containing 2.5% foetal calf serum and supplemented with the following fatty acids (NuCheck Prep. Elysian MN) bound to human serum albumin (essential fatty acid-free, Sigma Chem. Corp.) in mol ratio 2 : 1, 20 : 4(n - 6) 22 : 4(n - 6) 22 : 5(n - 6) 22 : 6(n 3) and 20 : 4( n - 6) combined with the other in equal concentrations. 22 : 5( n - 6) was a generous gift from Dr. Howard Sprecher, University of Ohio, Columbus, OH, U.S.A. Each fatty acid was in a final cont. of 50 PM and was incubated with endothelial cells for 24 h at 37°C. At the end of the incubation the medium was pipetted off, and the cells were washed twice with 2 ml medium E 199 without supplement. Lipid analysis. Endothelial cells were dislodged from the dishes and extracted as described earlier [3]. The extract was used for lipid separation by thin-layer chromatography on 0.5 mm thick silica gel plates (Type D.O. without CaSO, binder) (Camag, mutterz, Switzerland) using light petroleum (40-60”(Z)/ diethyl ether/ acetic acid (82 : 18 : 1, v/v). Reference standards (The Hormel Institute, Austin, MN) were run in parallel to the sample. Spots corresponding to cholesterol ester triacylglycerols, cholesterol, free fatty acids and total phospholipids were visualized, scraped off and methylated by BF,/methanol as described earlier [3] after addition of internal standard, heptadecaenoic acid (17 : 0). Analysis of fatty acid methyl esters were carried out with a Hewlett Packard 5830 A gas chromatograph with single column and flame detector. The glass column (8.0 m x 2 mm i.d.) was packed with 8% SP-2340 on chromosorb W AW (Supelco Inc., Bellefonte, PA). Each run was programmed from 160 to 200°C. The carrier gas was argon at a flow rate of 30 ml/min. Peaks were identified by known fatty acid methyl ester mixtures (Supelco Inc., Bellefonte PA) and purified 22 : 5( n - 6). Peak areas were measured by an H-P 18850 A GC terminal. CKetoprostaglandin F,, measurement. When the
endothelial cells had been incubated with medium supplemented with the various fatty acids for 24 h, the medium (I) was removed for further analysis. The cells were washed twice with 2 ml Tris buffer (140 mM NaCl/6 mM KCl/ 3 mM CaCl,/16 mM Tris-HCl, pH 7.4) and once with 2 ml of this buffer supplemented with 0.125 mM bovine serum albumin (buffer A). The washing solutions were preheated to 37°C. The cells were then incubated with 1.5 ml buffer A for 5 min at 37°C. The incubation medium (II) was collected for further analysis. The cells were then incubated with 1.5 ml Tris-buffered saline with albumin containing human thrombin (Sigma) (1.0 U/ml final cont.) for 5 min at 37°C (III). Finally, the cells were washed once with buffer A before they were frozen and thawed three times (IV). 6-Ketoprostaglandin Fi,, the stable metabolite of prostacylin was measured by radioimmunoassay following the method of Salmon [ll] as described in detail recently [12]. The cross reactivity has been given before [12] and the detection limit was 50 pg. The antibodies against 5-ketoprostaglandin F,, were a generous gift from the Wellcome Research Laboratories, Beckenham, U.K. Statistics. Student’s t-test was used to determine the significance of differences. Results Endothelial cell free fatty acids The distribution of fatty acids in the free fatty acids of endothelial cells is given in Table I. In cells grown for the last 24 h in growth medium containing human serum albumin as the only supplement, the fatty acid composition was, with one exception, similar to that reported earlier [3]. Consistently, we observed an unidentified peak in this fraction, containing more than 10% of the total free fatty acids and with a retention time in our system about 1 min longer than that registered for 22 : 5( n - 6). In endothelial cell monolayers incubated with 20 : 4(n - 6), a significant increase of this fatty acid was observed; however, no increase of the other fatty acids of the (n - 6) family was registered. However, when cells were grown with 22 : 4( n - 6) the relative contents of both 20 : 4( n - 6) and 20 : 3( n - 6) increased in addition to
33
22 : 4( n - 6). When equimolar supplements of 20 : 4(n - 6) and 22 : 4( n - 6) were used, only a moderate increase of 22 : 4( n - 6) was observed (only one experiment). When 22 : 6(n - 3) was added to the medium, a significant, but very modest increase of this acid was seen in the free fatty acids, with no increase of the other (n - 3) fatty acids. When equimolar supplements of 20 : 4( n - 6) and 22 : 6( n - 3) were used, an increase of the n - 6 fatty acids was observed, similar to that registered when 20 : 4( n - 6) was given separately. Endothelial cell total phospholipids The distribution of the fatty acids in total phospholipids is given in Table II. The composition in cells grown in medium supplemented only with human serum albumin was similar to that reported earlier [3]. When cells were incubated with 20 : 4( n - 6) a significant increase of both 20 : 4( n
- 6) and 22 : 4(n - 6) was observed, with no changes in 22 : 5( n - 6) or the shorter (n - 6) fatty acids. However, when 22 : 4( n - 6) or 22 : 5( n - 6) were added, only these fatty acids increased, whereas the shorter acids of the (n - 6) family actually were reduced. When equimolar supplements of 20 : 4(n - 6) and 22 : 4(n - 6) were used in the medium, both fatty acids increased in the total phospholipids, but the increase of 22 : 4(n - 6) was less than when this fatty acid was supplemented separately. 22 : 6( n - 3) supplement induced a drastic increase of this fatty acid in endothelial cell phospholipids, without an increase of the other (n - 3) fatty acids and with a significant decrease of 20 : 4( n - 6). This effect on 20 : 4(n - 6) was abolished when equimolar concentrations of 22 : 6( n - 3) and 20 : 4( n - 6) were used in the medium, even if this supplement also markedly increased the content of 22 : 6( n - 3).
TABLE I FREE FATTY ACID COMPOSITION (PERCENTAGE) IN HUMAN ENDOTHELIAL CELLS INCUBATED FOR 24 h WITH MEDIUM (SEE METHODS) SUPPLEMENTED WITH FATTY ACIDS BOUND TO ALBUMIN, RATIO 2 : 1 Mean f S.D. of three experiments. HSA, human serum albumin, u.d., unidentified. Fatty acid (mol%)
14:o 16:0 16:l 18:0 18:l 18:2 18:3+20:1 20:o 20:2 20:3(n 20:4(n 20:5(n 22:o 22:l u.d. 22:4(n 22:6(n 24:l
-6) -6) -3)+24:0
-6) -3)
HSA
4.17 f 1.82 30.83 f 1.51 5.91+ 1.39 8.89+0.43 13.96kO.67 3.17 + 1.19 1.14*0.47 1.15 *0.15 _ 1.64 f 0.66 7.77 + 1.20 2.39 k 0.56 18.92 + 3.23 _ _ _
Fatty acid supplement 20:4(n -6)
22:4(n -6)
22:6(n -3)
(50 PM)
(50 PM)
(50 PM)
3.32 +0.51 28.21 k 0.98 3.58+0.44 8.58k1.51 16.09 + 2.65 4.36 & 2.47 1.66 * 0.35 1.33k0.83
3.37 * 0.68 21.22 + 0.20 * 4.53 * 0.13 6.98 & 1.23 11.48+0.67 2.88k1.51 1.42k 1.36 0.76 f 0.04
3.50 + 0.60 24.67 + 1.72 2.63 k 0.22 8.87 k 0.64 12.81 k 2.13 3.61 f 1.94 2.77 + 0.32 0.90 + 0.15
_ 4.99 f 3.05 * 7.89 f 3.42 3.24 f 2.14 21.03 f 2.67 _ _
3.86 f 1.65 * 4.90k0.67 * 4.60k0.57 * 2.86 f 6.23 17.21 f 3.34 10.96k2.15 * 0.97 f 0.80 * 2.58+0.12 *
* Significance of difference to human serum albumin; P -c 0.05.
3.60 + 0.45 * 1.81+0.18 5.68 f 1.02 3.18+0.08 0.38 5 0.08 28.13 f 3.80 1.32k0.53 * -
20:4(n -6) +22:4(n -6) (50pM+50pM)
20:4(n -6) +22:6(n -3) (50pM+50pM)
4.04 29.08 4.95 7.12 14.19 4.60 2.23 1.22 _
4.61+ 1.83 26.26 + 4.56 5.88 + 2.68 6.78 k 2.31 15.90+1.97 5.28kO.15 2.49 * 1.08 0.79 * 0.20 _
1.92 2.61 4.14 1.82
2.33 k 0.65 * 5.42 k 4.05 * 4.85 + 1.00 2.31+0.6
19.86 1.12
16.63k5.13 2.24 f 0.26 * 3.14*0&l * 1.16*0.16
_
34 TABLE 11 FATTY COMPOSITION (PERCENTAGE) OF ENDOTHELIAL FOR 24 h WITH MEDIUM (SEE METHODS) SUPPLEMENTED
CELL TOTAL PHOSPHOLIPIDS AFTER INCUBATION WITH FATTY ACIDS BOUND TO ALBUMIN, RATIO 2 : 1
Mean f S.D. of three experiments. HSA, human serum albumin. Fatty acid (moI%)
14:o 16:0 16:l 18:0 18:l 18:2 18:3+20:1 20:2 20:3(n -6) 20:4(n -6) 20:5(n -3)+ 24:0 22:o 22:5(n-3) 22:4(n -6) 22:6(n-3) 24:l 22:5(n -6)
Fatty acid supplement
HSA
1.21+ 0.09 22.87 f 1.73 2.44kO.56 17.97 f 1.67 20.87 + 0.68 3.71+ 1.66 0.68 f 0.31 0.43 * 0.04 2.61+0.26 14.29 f 0.59 2.27 + 0.29 050~0.11 4.43 kO.58 3.42 f 0.69 3.88+0.18 0.84+0&l
20:4(n-6)
22:4(n -6)
22:5(n -6)
22:6(n -3)
20:4(n-6)
(50 IBM)
(50 PM)
(50 PM)
**
(50 PM)
+22:4(n-6) (50pM+50/tM)
20:4(n -6) +22:6(n -3) (50pM+50pM)
1.14kO.48 21.38k1.47 2.90 & 0.51
1.35kO.12 23.86 k 2.86 3.1OkO.48
2.00k0.18 19.20 f 0.61 3.43 +0.36
0.90~0.10 22.45 f 0.36 4.04 f 0.27
1.36k0.35 21.64 f 0.69 2.49k 1.17
23.67 + 2.16 _
14.79 + 1.23 17.10+2.10 19.10 * 1.88 19.21 k1.51 2.60 f 0.34 2.75 k 0.24 _ 0.69 k 0.22 0.46 * 0.04 0.57 + 0.05 2.62 f 0.08 2.64 i 0.20 18.84k1.56 * 12.405 1.39
19.85kO.20 19.14k2.03 2.11*0.05 0.67 + 0.06 0.33 f 0.21 1.66 + 0.10 10.94+0.70 0.34 * 0.09 1.46+0.22 1.83 + 0.19 0.22 * 0.08 0.44kO.13 0.71* 0.11 1.66k0.12 3.74 f 0.28 3.00 k 0.16 3.32 + 0.32 7.76kO.80 * 13.10*2.43 * 0.89rtO.11 3.92 k 0.35 3.52 k 0.40 0.85 * 0.43 2.19+ 1.06
17.55kO.44 14.70+ 1.40 20.23 + 0.36 19.04 k 0.88 2.50k0.21 3.04 * 0.71 0.40 + 0.08 0.69 _) 0.50 0.43 f 0.03 0.42kO.12 2.41 k 0.07 1.81 kO.35 10.04+0.59 * 17.29k1.75 * 1.72kO.44 0.72+ 0.10 2.47 k 0.15 2.70+0.15 * 9.74kO.66 * 1.54kO.90
16.44 + 3.03 20.05 f 1.74 3.05 kO.35 0.74 + 0.37 0.43kO.12 1.91*0.11 15.55 f 1.57
1.27k0.36 3.5 f 0.61 8.85f1.53 * 3.82kO.60 1.04+0.30
1.62+0.48 0.55 + 0.12 2.85 *0.57 4.37 * 1.19 6.48 k 1.94 * 1.34 * 0.20
13.46 * 1.82
* Significance of difference to human serum albumin; P -C0.05. ** Mean k range of two experiments.
TABLE III 6-KETOPROSTAGLANDIN F,, RELEASE FROM ENDOTHELIAL CELLS INTO GROWTH MEDIUM (1) DURING 24 h INCUBATION WITH LONG-CHAIN POLYUNSATURATED FATTY ACIDS AFTER SUBSEQUENT 5 min INCUBATION WITH TRIS BUFFER (2), AFTER 5 min INCUBATION WITH THROMBIN (0.5 U/ml) (3) AND FINALLY AFTER FREEZING (f) AND THAWING (t) THE CELLS x 3 (4) Mean and S.D. of 5-14 experiments, HSA, human serum albumin. Source
6-Ketoprostaglandin F,, (ng/ml) HSA
1. Mediumafter24h14.34* 2. After Tris buffer, 5 min 1.59+ 3. After thrombin, 5 min 8.05 f 4. After (f + t) 14.22 *
20:4(n-6)
22:4(n
(50 PM)
(50 PM)
-6)
22:5(n
-6)
(50 PM)
6.131.30~6.03**7.50~2.66**2.80f0.6* 2.030.38kO.48 *
0.80 + 0.72
0.55 f 0.20
20:4(n -6) +22:4(n -6) (50+50 PM)
22:6(n -3) (50 PM)
20:4(n -6) +22:6(n -3) rM)(50+50 /.tM)
20:4(n
-6)
22:5(n
-6)
(50+50
5.65 +2.46 ** 19.85 * 5.55 _
8.65 + 2.65
0.31 f0.20
0.31 kO.10 *
*
0.43 kO.46
4.87 3.60* 2.77 ** 6.59 * 3.34 3.45 + 0.80 ** 10.36 f 3.61 4.13f2.68 12.17.32 + 4.68 * 1.34+1.08 **5.40*1.82 * 0.44kO.12 ** 6.38k4.70 _
* Significance of difference to human serum albumin, P c 0.05; ** P < 0.01.
**2.40*0.85 * 9.32k4.20
24.28 f 4.25 *
*
0.89+ 1.06 4.6653.12 * 8.01 f 3.85 *
35
6-Ketoprostaglandin F,, synthesis The release of 6-ketoprostaglandin F,, into the medium was, as shown earlier, [12] stimulated by 20 : 4(n - 6) (Table III). 22 : 4(n - 6) 22 : 5(n - 6) and 22 : 6( n - 3) all significantly reduced the release of 6-ketoprostaglandin Fi,, and they also suppressed the prostacylin synthesis stimulated by exogenous 20 : 4( n - 6). When cells were preincubated for 24 h with the separate or combined fatty acids and then stimulated with Tris buffer, they regularly showed a reduced release of 6-ketoprostaglandin F,, compared with cells incubated with medium supplemented with albumin only; however, the reductions were not significant. When the cells were subsequently stimulated with thrombin for 5 min (Table III), the release of 6-ketoprostaglandin F,, was always reduced in cells preincubated with 20 : 4( n - 6) or 22 : 5( n - 6) or the combination of 20 : 4(n - 6) with one of the docosaenoic fatty acids. 22 : 4(n - 6) and 22 : 6(n - 3) separately, did not significantly reduce the release. Finally, when cells were frozen and thawed three times to induce total release of 6-ketoprostaglandin Fi,, all cells preincubated with single or combined fatty acids contained reduced amounts compared with cells incubated with only albumin. Cells preincubated with 22 : 4( n - 6), 22 : 5( n - 6) or 22 : 6( n - 3) only had significantly (P < 0.01) lower release than any of the other cell cultures. Discussion Human endothelial cell phospholipids and triacylglycerols contain approximately 15 mol% docosapolyenoic fatty acids, whereas the other neutral lipids have been reported to contain only trace amounts of these fatty acids [3]. The present study has confirmed these observations; however, both in cells collected directly from umbilical cord veins and in cells grown in culture for 5 days, the free fatty acid fraction contained about 10% of an unidentified fatty acid. This fatty acid was only observed in trace amounts (less than 0.1%) in the other endothelial cell lipid fractions. Work is in progress to identify this peak. Earlier studies [3] have reported about 12% of 24 : 1 in endothelial cell free fatty acids. The present study could not confirm this observation.
The function of the docosaenoic fatty acids in endothelial cells is unknown. Recent studies indicate that the 22 fatty acids of the (n - 3) family may be related to the possible antithrombotic effect of fish oils [6,12]. Docosahexaenoic acid (22 : 6(n - 3)) stimulates a platelet aggregation inhibitory substance in endothelial cells different from prostacylin [6]. In fact, it reduces the synthesis of prostacylin, an observation confirmed in this study. This reduction may be directly related to a reduction of 20 : 4(n - 6) in the phospholipids. However, the analysis of fatty acids and 6-ketoprostaglandin F,, production were performed on separate cultures in the present study due to limited number of cells, and thus no definite conclusions can be made at this point. Recent reports [13] have shown that 22 : 6(n - 3) is also a strong competitive inhibitor of the conversion of arachidonate by prostaglandin synthetase. This mechanism, and the fact that 22 : 6(n - 3) is also a substrate, however poor, for cyclooxygenase [14], may, in addition to effects of the 14- and ll-isomers of hydroxydocosaenoic acid produced by the lipoxygenase pathway [14], modulate the prostacylin synthesis. There is no indication for a desaturation and de-elongation of 22 : 6( n - 3) to 20 : 5(n - 3) by endothelial cells. When endothelial cells were incubated simultaneously with 22 : 6( n - 3) and 20 : 4( n - 6) in equimolar concentrations, the incorporation of 20 : 4(n - 6) into phospholipids was not significantly lower than when 20 : 4(n - 6) was supplemented alone. This has been reported previously for human platelets [4,8]. At present we do not know if this incorporation into endothelial cell phospholipids shows the same variation for the individual phospholipids as seen in platelets [7,8]. 22 : 4(n - 6) was incorporated both into free fatty acids and total phospholipids of endothelial cells. Simultaneously, 20 : 4(n - 6) in the phospholipids was reduced. However, when endothelial cell monolayers were incubated with 20 : 4( n - 6) a significant increase of 22 : 4(n - 6) occurred, indicating an active chain elongation of 20 : 4( n 6) in these cells. In parallel experiments 6-ketoprostaglandin F,, synthesis was markedly reduced by 22 : 4(n - 6). When the endothelial cells were incubated simultaneously with 22 : 4(n - 6) and 20 : 4(n - 6) the synthesis was still significant less
36
than when incubated with 20 : 4(n - 6) only. However, the 20 : 4( n - 6) content of the phospholipids was not significantly reduced. This observation may indicate that 22 : 4(n - 6) or one of its metabolites suppresses the synthesis of 6-ketoprostaglandin Ft, and thus represents a modulator to 20 : 4( n - 6) in the stimulation of prostacylin production. A similar counterbalance between the effects of 20 : 4(n - 6) and 22 : 4( n - 6) has been reported in platelets [4]. 22 : 5(n - 6) was also incorporated into endothelial cell phospholipids, with subsequent relative reduction of 20 : 4( n - 6) content and a reduced prostacylin synthesis. This may indicate that the two long-chain fatty acids of the (n - 6) family act similarly with regard to 20 : 4( n - 6) and prostacylin synthesis. This study has shown that 22 : 4(n - 6) and 22 : 6(n - 3) both of which are present in human endothelial cells, are incorporated into the cell phospholipids and thus modulate the membrane lipid composition. One of the consequences is a reduction of prostacylin synthesis. Earlier studies in rats have shown that the synthesis of 22 : 4( n 6) and 22 : 6(n - 3) in platelets is stimulated by dietary supplement of 18 : 2( n - 6) and 20 : 5( n 3) respectively [15]. Together, these studies may indicate that dietary polyunsaturated fatty acids may modify platelet and endothelial cell function partly by effects on the cellular content of docosapolyenoic fatty acids. This may represent another
link between dietary fatty acid composition thrombogenesis.
and
References 1 Mead, J.F. (1971) in Progress in the Chemistry of Fats and Other Lipids (Holman, R.T., ed.), Vol. IX, pp. 161-192, Pergamon Press, Oxford 2 Nordoy, A. and Lund, S. (1968) Stand. J. Clin. Lab. Invest. 22, 328-338 3 Rastogi, B. and Nordoy, A. (1980) Thromb. Res. 18. 629-641 4 Van Rollins, M., Horrocks, L. and Sprecher, H. (1985) Biochim. Biophys. Acta 833, 272-280 5 Milks, M.M. and Sprecher, H. (1985) Biochim. Biophys. Acta 835, 29-25 6 Dyerberg, J., Bang, H.O., Stoffersen, E., Moncada, S. and Vane, J.R. (1978) Lancet ii, 117-119 7 Fischer, S., Schacky, C.V., Siess, W., Strasser, T. and Weber, P.C. (1984) Biochem. Biophys. Res. Commun. 120,907-918 8 Weiner, T.W. and Sprecher, H. (1985) J. Biol. Chem. 260, 6032-6038 9 Jaffe, E.A., Nachman, R.L., Becker, C.G. and Minick. C.R. (1973) J. Clin. Invest. 52, 2745-2753 10 Chervionke, R.L., Hoak, J.C. and Fry, G.L. (1978) J. Clin. Invest. 62, 847-856 11 Salmon, J.A. (1979) Prostaglandins 15, 383-397 12 Brox, J.H. and Nordoy, A. (1984) Thromb. Haemostas. 50, 762-767 13 Corey, E.J., Shih, C. and Cashman, J.R. (1983) Proc. Natl. Acad. Sci. USA 80, 3581-3584 14 Alvadano, M.I. and Sprecher, H. (1983) J. Biol. Chem. 258, 9339-9343 15 Nordoy, A., Davenas, E., Ciavatti, M. and Renaud, S. (1985) Biochim. Biophys. Acta 835, 491-500
31
Elsevier
BBA 52246
Docosapolyenoic fatty acids and human endothelial cells
A. Nordoy *, V. Lyngmo, A. V&m Department
ofMedicine,University Hospital, 9012 (Received
Key words:
and B. Svensson
Lipid metabolism;
December
Tromso (Norway)
2nd, 1985)
Prostacyclin
synthesis;
(Endothelial
cell)
The total fatty acids in human endothelial cells include approximately 5% each of 22: 4(n - 6), 22 : 5(n - 3) and 22: 6(n - 3), whereas 22: 5(n - 6) is present only in trace amounts. This study evaluates the effect of three of these fatty acids bound to albumin on lipid composition and prostacyclin (prostaglandin I*) synthesis in primary cultures of endothelial cell monolayers. 22: 4(n - 6), 22: 5(n - 6) and 22: 6(n - 3) were all incorporated into total phospholipids. 20: 4(n - 6) was reduced in phospholipids in all cells incubated with the three different docosaenoic fatty acids. This reduction was abolished when equimolar concentrations of 20: 4(n - 6) and the separate docosaenoic fatty acid were added to the medium simultaneously. 22: 4(n - 6) incorporation into the free fatty acids was associated with an increase of 20: 4(n - 6) in this fraction. 22 : 4( n - 6), 22 : S(n - 6) and 22 : 5(n - 3) all reduced the synthesis of prostacylin measured as 6-ketoprostaglandin F,,. These effects were reversed by simultaneous incubation with 20: 4(n - 6). This study shows that three of the docosaenoic fatty acids present in human endothelial cells of the (n - 6) and (n - 3) family were all incorporated into endothelial cells with a simultaneous reduction in 20: 4(n - 6). The three fatty acids reduced the synthesis of prostacylin. Introduction
Dietary linoleic (n - 6) and linolenic (n - 3) acids are metabolized by desaturation and elongation to two series of very-long-chain polyunsaturated fatty acids [l]. The docosaenoic fatty acids of the (n - 6) family, 22 : 4 (7,10,13,16) and 22 : 5 (4,7,10,13,16) are present in human platelets and endothelial cells [2,3] and may be further metabolized by platelets into dihomothromboxane B, and 4-dihomothromboxane B,, respectively [4]. Also, hydroxyacids are formed by the lipoxygenase pathway [4,5]. The docosaenoic fatty acids of the (n - 3) family 22 : 5 (7,10,13,16,19) and 22 : 6 (4,7,10,13,16,19) are present in marine oils and fish. Recent studies have indicated that particularly 20 : 5 (5,8,11,14,17) of the (n - 3)
family may reduce platelet aggregation when it is incorporated into platelet phospholipids [6]. Small amounts of (n - 3) docosaenoic acids are also present in human platelets and endothelial cells [2,3]. They may be further metabolized by platelets into hydroxy acid isomers and may depress thromboxane A 2 production [7,8]. In the present study we have examined the content of docosaenoic acids in human endothelial cells and investigated the metabolism of 22: 4 (7,10,13,16), 22 : 5 (4,7,10,13,16) and 22 : 6 (4,7,10,13,16,19) and their effects on prostacyclin production. Methods Cell
human cell monolayers were Jaffe et al. [9] modifications [lo]. The cells were in 35 x 10 umbillical vein
* To whom correspondence
0005-2760/86/$03.50
should be addressed.
1986 Elsevier Science Publishers
B.V. (Biomedical
Division)
32
mm culture dishes in modified medium E 199 (medium E 199) containing 20% of foetal calf serum until confluency (4-5 days). Test procedure. At the time of confluence the medium was pipetted off, and the cells were washed twice with 2 ml medium E 199 without foetal calf serum. The endothelial cells were then incubated with 2 ml medium E 199 containing 2.5% foetal calf serum and supplemented with the following fatty acids (NuCheck Prep. Elysian MN) bound to human serum albumin (essential fatty acid-free, Sigma Chem. Corp.) in mol ratio 2 : 1, 20 : 4(n - 6) 22 : 4(n - 6) 22 : 5(n - 6) 22 : 6(n 3) and 20 : 4( n - 6) combined with the other in equal concentrations. 22 : 5( n - 6) was a generous gift from Dr. Howard Sprecher, University of Ohio, Columbus, OH, U.S.A. Each fatty acid was in a final cont. of 50 PM and was incubated with endothelial cells for 24 h at 37°C. At the end of the incubation the medium was pipetted off, and the cells were washed twice with 2 ml medium E 199 without supplement. Lipid analysis. Endothelial cells were dislodged from the dishes and extracted as described earlier [3]. The extract was used for lipid separation by thin-layer chromatography on 0.5 mm thick silica gel plates (Type D.O. without CaSO, binder) (Camag, mutterz, Switzerland) using light petroleum (40-60”(Z)/ diethyl ether/ acetic acid (82 : 18 : 1, v/v). Reference standards (The Hormel Institute, Austin, MN) were run in parallel to the sample. Spots corresponding to cholesterol ester triacylglycerols, cholesterol, free fatty acids and total phospholipids were visualized, scraped off and methylated by BF,/methanol as described earlier [3] after addition of internal standard, heptadecaenoic acid (17 : 0). Analysis of fatty acid methyl esters were carried out with a Hewlett Packard 5830 A gas chromatograph with single column and flame detector. The glass column (8.0 m x 2 mm i.d.) was packed with 8% SP-2340 on chromosorb W AW (Supelco Inc., Bellefonte, PA). Each run was programmed from 160 to 200°C. The carrier gas was argon at a flow rate of 30 ml/min. Peaks were identified by known fatty acid methyl ester mixtures (Supelco Inc., Bellefonte PA) and purified 22 : 5( n - 6). Peak areas were measured by an H-P 18850 A GC terminal. CKetoprostaglandin F,, measurement. When the
endothelial cells had been incubated with medium supplemented with the various fatty acids for 24 h, the medium (I) was removed for further analysis. The cells were washed twice with 2 ml Tris buffer (140 mM NaCl/6 mM KCl/ 3 mM CaCl,/16 mM Tris-HCl, pH 7.4) and once with 2 ml of this buffer supplemented with 0.125 mM bovine serum albumin (buffer A). The washing solutions were preheated to 37°C. The cells were then incubated with 1.5 ml buffer A for 5 min at 37°C. The incubation medium (II) was collected for further analysis. The cells were then incubated with 1.5 ml Tris-buffered saline with albumin containing human thrombin (Sigma) (1.0 U/ml final cont.) for 5 min at 37°C (III). Finally, the cells were washed once with buffer A before they were frozen and thawed three times (IV). 6-Ketoprostaglandin Fi,, the stable metabolite of prostacylin was measured by radioimmunoassay following the method of Salmon [ll] as described in detail recently [12]. The cross reactivity has been given before [12] and the detection limit was 50 pg. The antibodies against 5-ketoprostaglandin F,, were a generous gift from the Wellcome Research Laboratories, Beckenham, U.K. Statistics. Student’s t-test was used to determine the significance of differences. Results Endothelial cell free fatty acids The distribution of fatty acids in the free fatty acids of endothelial cells is given in Table I. In cells grown for the last 24 h in growth medium containing human serum albumin as the only supplement, the fatty acid composition was, with one exception, similar to that reported earlier [3]. Consistently, we observed an unidentified peak in this fraction, containing more than 10% of the total free fatty acids and with a retention time in our system about 1 min longer than that registered for 22 : 5( n - 6). In endothelial cell monolayers incubated with 20 : 4(n - 6), a significant increase of this fatty acid was observed; however, no increase of the other fatty acids of the (n - 6) family was registered. However, when cells were grown with 22 : 4( n - 6) the relative contents of both 20 : 4( n - 6) and 20 : 3( n - 6) increased in addition to
33
22 : 4( n - 6). When equimolar supplements of 20 : 4(n - 6) and 22 : 4( n - 6) were used, only a moderate increase of 22 : 4( n - 6) was observed (only one experiment). When 22 : 6(n - 3) was added to the medium, a significant, but very modest increase of this acid was seen in the free fatty acids, with no increase of the other (n - 3) fatty acids. When equimolar supplements of 20 : 4( n - 6) and 22 : 6( n - 3) were used, an increase of the n - 6 fatty acids was observed, similar to that registered when 20 : 4( n - 6) was given separately. Endothelial cell total phospholipids The distribution of the fatty acids in total phospholipids is given in Table II. The composition in cells grown in medium supplemented only with human serum albumin was similar to that reported earlier [3]. When cells were incubated with 20 : 4( n - 6) a significant increase of both 20 : 4( n
- 6) and 22 : 4(n - 6) was observed, with no changes in 22 : 5( n - 6) or the shorter (n - 6) fatty acids. However, when 22 : 4( n - 6) or 22 : 5( n - 6) were added, only these fatty acids increased, whereas the shorter acids of the (n - 6) family actually were reduced. When equimolar supplements of 20 : 4(n - 6) and 22 : 4(n - 6) were used in the medium, both fatty acids increased in the total phospholipids, but the increase of 22 : 4(n - 6) was less than when this fatty acid was supplemented separately. 22 : 6( n - 3) supplement induced a drastic increase of this fatty acid in endothelial cell phospholipids, without an increase of the other (n - 3) fatty acids and with a significant decrease of 20 : 4( n - 6). This effect on 20 : 4(n - 6) was abolished when equimolar concentrations of 22 : 6( n - 3) and 20 : 4( n - 6) were used in the medium, even if this supplement also markedly increased the content of 22 : 6( n - 3).
TABLE I FREE FATTY ACID COMPOSITION (PERCENTAGE) IN HUMAN ENDOTHELIAL CELLS INCUBATED FOR 24 h WITH MEDIUM (SEE METHODS) SUPPLEMENTED WITH FATTY ACIDS BOUND TO ALBUMIN, RATIO 2 : 1 Mean f S.D. of three experiments. HSA, human serum albumin, u.d., unidentified. Fatty acid (mol%)
14:o 16:0 16:l 18:0 18:l 18:2 18:3+20:1 20:o 20:2 20:3(n 20:4(n 20:5(n 22:o 22:l u.d. 22:4(n 22:6(n 24:l
-6) -6) -3)+24:0
-6) -3)
HSA
4.17 f 1.82 30.83 f 1.51 5.91+ 1.39 8.89+0.43 13.96kO.67 3.17 + 1.19 1.14*0.47 1.15 *0.15 _ 1.64 f 0.66 7.77 + 1.20 2.39 k 0.56 18.92 + 3.23 _ _ _
Fatty acid supplement 20:4(n -6)
22:4(n -6)
22:6(n -3)
(50 PM)
(50 PM)
(50 PM)
3.32 +0.51 28.21 k 0.98 3.58+0.44 8.58k1.51 16.09 + 2.65 4.36 & 2.47 1.66 * 0.35 1.33k0.83
3.37 * 0.68 21.22 + 0.20 * 4.53 * 0.13 6.98 & 1.23 11.48+0.67 2.88k1.51 1.42k 1.36 0.76 f 0.04
3.50 + 0.60 24.67 + 1.72 2.63 k 0.22 8.87 k 0.64 12.81 k 2.13 3.61 f 1.94 2.77 + 0.32 0.90 + 0.15
_ 4.99 f 3.05 * 7.89 f 3.42 3.24 f 2.14 21.03 f 2.67 _ _
3.86 f 1.65 * 4.90k0.67 * 4.60k0.57 * 2.86 f 6.23 17.21 f 3.34 10.96k2.15 * 0.97 f 0.80 * 2.58+0.12 *
* Significance of difference to human serum albumin; P -c 0.05.
3.60 + 0.45 * 1.81+0.18 5.68 f 1.02 3.18+0.08 0.38 5 0.08 28.13 f 3.80 1.32k0.53 * -
20:4(n -6) +22:4(n -6) (50pM+50pM)
20:4(n -6) +22:6(n -3) (50pM+50pM)
4.04 29.08 4.95 7.12 14.19 4.60 2.23 1.22 _
4.61+ 1.83 26.26 + 4.56 5.88 + 2.68 6.78 k 2.31 15.90+1.97 5.28kO.15 2.49 * 1.08 0.79 * 0.20 _
1.92 2.61 4.14 1.82
2.33 k 0.65 * 5.42 k 4.05 * 4.85 + 1.00 2.31+0.6
19.86 1.12
16.63k5.13 2.24 f 0.26 * 3.14*0&l * 1.16*0.16
_
34 TABLE 11 FATTY COMPOSITION (PERCENTAGE) OF ENDOTHELIAL FOR 24 h WITH MEDIUM (SEE METHODS) SUPPLEMENTED
CELL TOTAL PHOSPHOLIPIDS AFTER INCUBATION WITH FATTY ACIDS BOUND TO ALBUMIN, RATIO 2 : 1
Mean f S.D. of three experiments. HSA, human serum albumin. Fatty acid (moI%)
14:o 16:0 16:l 18:0 18:l 18:2 18:3+20:1 20:2 20:3(n -6) 20:4(n -6) 20:5(n -3)+ 24:0 22:o 22:5(n-3) 22:4(n -6) 22:6(n-3) 24:l 22:5(n -6)
Fatty acid supplement
HSA
1.21+ 0.09 22.87 f 1.73 2.44kO.56 17.97 f 1.67 20.87 + 0.68 3.71+ 1.66 0.68 f 0.31 0.43 * 0.04 2.61+0.26 14.29 f 0.59 2.27 + 0.29 050~0.11 4.43 kO.58 3.42 f 0.69 3.88+0.18 0.84+0&l
20:4(n-6)
22:4(n -6)
22:5(n -6)
22:6(n -3)
20:4(n-6)
(50 IBM)
(50 PM)
(50 PM)
**
(50 PM)
+22:4(n-6) (50pM+50/tM)
20:4(n -6) +22:6(n -3) (50pM+50pM)
1.14kO.48 21.38k1.47 2.90 & 0.51
1.35kO.12 23.86 k 2.86 3.1OkO.48
2.00k0.18 19.20 f 0.61 3.43 +0.36
0.90~0.10 22.45 f 0.36 4.04 f 0.27
1.36k0.35 21.64 f 0.69 2.49k 1.17
23.67 + 2.16 _
14.79 + 1.23 17.10+2.10 19.10 * 1.88 19.21 k1.51 2.60 f 0.34 2.75 k 0.24 _ 0.69 k 0.22 0.46 * 0.04 0.57 + 0.05 2.62 f 0.08 2.64 i 0.20 18.84k1.56 * 12.405 1.39
19.85kO.20 19.14k2.03 2.11*0.05 0.67 + 0.06 0.33 f 0.21 1.66 + 0.10 10.94+0.70 0.34 * 0.09 1.46+0.22 1.83 + 0.19 0.22 * 0.08 0.44kO.13 0.71* 0.11 1.66k0.12 3.74 f 0.28 3.00 k 0.16 3.32 + 0.32 7.76kO.80 * 13.10*2.43 * 0.89rtO.11 3.92 k 0.35 3.52 k 0.40 0.85 * 0.43 2.19+ 1.06
17.55kO.44 14.70+ 1.40 20.23 + 0.36 19.04 k 0.88 2.50k0.21 3.04 * 0.71 0.40 + 0.08 0.69 _) 0.50 0.43 f 0.03 0.42kO.12 2.41 k 0.07 1.81 kO.35 10.04+0.59 * 17.29k1.75 * 1.72kO.44 0.72+ 0.10 2.47 k 0.15 2.70+0.15 * 9.74kO.66 * 1.54kO.90
16.44 + 3.03 20.05 f 1.74 3.05 kO.35 0.74 + 0.37 0.43kO.12 1.91*0.11 15.55 f 1.57
1.27k0.36 3.5 f 0.61 8.85f1.53 * 3.82kO.60 1.04+0.30
1.62+0.48 0.55 + 0.12 2.85 *0.57 4.37 * 1.19 6.48 k 1.94 * 1.34 * 0.20
13.46 * 1.82
* Significance of difference to human serum albumin; P -C0.05. ** Mean k range of two experiments.
TABLE III 6-KETOPROSTAGLANDIN F,, RELEASE FROM ENDOTHELIAL CELLS INTO GROWTH MEDIUM (1) DURING 24 h INCUBATION WITH LONG-CHAIN POLYUNSATURATED FATTY ACIDS AFTER SUBSEQUENT 5 min INCUBATION WITH TRIS BUFFER (2), AFTER 5 min INCUBATION WITH THROMBIN (0.5 U/ml) (3) AND FINALLY AFTER FREEZING (f) AND THAWING (t) THE CELLS x 3 (4) Mean and S.D. of 5-14 experiments, HSA, human serum albumin. Source
6-Ketoprostaglandin F,, (ng/ml) HSA
1. Mediumafter24h14.34* 2. After Tris buffer, 5 min 1.59+ 3. After thrombin, 5 min 8.05 f 4. After (f + t) 14.22 *
20:4(n-6)
22:4(n
(50 PM)
(50 PM)
-6)
22:5(n
-6)
(50 PM)
6.131.30~6.03**7.50~2.66**2.80f0.6* 2.030.38kO.48 *
0.80 + 0.72
0.55 f 0.20
20:4(n -6) +22:4(n -6) (50+50 PM)
22:6(n -3) (50 PM)
20:4(n -6) +22:6(n -3) rM)(50+50 /.tM)
20:4(n
-6)
22:5(n
-6)
(50+50
5.65 +2.46 ** 19.85 * 5.55 _
8.65 + 2.65
0.31 f0.20
0.31 kO.10 *
*
0.43 kO.46
4.87 3.60* 2.77 ** 6.59 * 3.34 3.45 + 0.80 ** 10.36 f 3.61 4.13f2.68 12.17.32 + 4.68 * 1.34+1.08 **5.40*1.82 * 0.44kO.12 ** 6.38k4.70 _
* Significance of difference to human serum albumin, P c 0.05; ** P < 0.01.
**2.40*0.85 * 9.32k4.20
24.28 f 4.25 *
*
0.89+ 1.06 4.6653.12 * 8.01 f 3.85 *
35
6-Ketoprostaglandin F,, synthesis The release of 6-ketoprostaglandin F,, into the medium was, as shown earlier, [12] stimulated by 20 : 4(n - 6) (Table III). 22 : 4(n - 6) 22 : 5(n - 6) and 22 : 6( n - 3) all significantly reduced the release of 6-ketoprostaglandin Fi,, and they also suppressed the prostacylin synthesis stimulated by exogenous 20 : 4( n - 6). When cells were preincubated for 24 h with the separate or combined fatty acids and then stimulated with Tris buffer, they regularly showed a reduced release of 6-ketoprostaglandin F,, compared with cells incubated with medium supplemented with albumin only; however, the reductions were not significant. When the cells were subsequently stimulated with thrombin for 5 min (Table III), the release of 6-ketoprostaglandin F,, was always reduced in cells preincubated with 20 : 4( n - 6) or 22 : 5( n - 6) or the combination of 20 : 4(n - 6) with one of the docosaenoic fatty acids. 22 : 4(n - 6) and 22 : 6(n - 3) separately, did not significantly reduce the release. Finally, when cells were frozen and thawed three times to induce total release of 6-ketoprostaglandin Fi,, all cells preincubated with single or combined fatty acids contained reduced amounts compared with cells incubated with only albumin. Cells preincubated with 22 : 4( n - 6), 22 : 5( n - 6) or 22 : 6( n - 3) only had significantly (P < 0.01) lower release than any of the other cell cultures. Discussion Human endothelial cell phospholipids and triacylglycerols contain approximately 15 mol% docosapolyenoic fatty acids, whereas the other neutral lipids have been reported to contain only trace amounts of these fatty acids [3]. The present study has confirmed these observations; however, both in cells collected directly from umbilical cord veins and in cells grown in culture for 5 days, the free fatty acid fraction contained about 10% of an unidentified fatty acid. This fatty acid was only observed in trace amounts (less than 0.1%) in the other endothelial cell lipid fractions. Work is in progress to identify this peak. Earlier studies [3] have reported about 12% of 24 : 1 in endothelial cell free fatty acids. The present study could not confirm this observation.
The function of the docosaenoic fatty acids in endothelial cells is unknown. Recent studies indicate that the 22 fatty acids of the (n - 3) family may be related to the possible antithrombotic effect of fish oils [6,12]. Docosahexaenoic acid (22 : 6(n - 3)) stimulates a platelet aggregation inhibitory substance in endothelial cells different from prostacylin [6]. In fact, it reduces the synthesis of prostacylin, an observation confirmed in this study. This reduction may be directly related to a reduction of 20 : 4(n - 6) in the phospholipids. However, the analysis of fatty acids and 6-ketoprostaglandin F,, production were performed on separate cultures in the present study due to limited number of cells, and thus no definite conclusions can be made at this point. Recent reports [13] have shown that 22 : 6(n - 3) is also a strong competitive inhibitor of the conversion of arachidonate by prostaglandin synthetase. This mechanism, and the fact that 22 : 6(n - 3) is also a substrate, however poor, for cyclooxygenase [14], may, in addition to effects of the 14- and ll-isomers of hydroxydocosaenoic acid produced by the lipoxygenase pathway [14], modulate the prostacylin synthesis. There is no indication for a desaturation and de-elongation of 22 : 6( n - 3) to 20 : 5(n - 3) by endothelial cells. When endothelial cells were incubated simultaneously with 22 : 6( n - 3) and 20 : 4( n - 6) in equimolar concentrations, the incorporation of 20 : 4(n - 6) into phospholipids was not significantly lower than when 20 : 4(n - 6) was supplemented alone. This has been reported previously for human platelets [4,8]. At present we do not know if this incorporation into endothelial cell phospholipids shows the same variation for the individual phospholipids as seen in platelets [7,8]. 22 : 4(n - 6) was incorporated both into free fatty acids and total phospholipids of endothelial cells. Simultaneously, 20 : 4(n - 6) in the phospholipids was reduced. However, when endothelial cell monolayers were incubated with 20 : 4( n - 6) a significant increase of 22 : 4(n - 6) occurred, indicating an active chain elongation of 20 : 4( n 6) in these cells. In parallel experiments 6-ketoprostaglandin F,, synthesis was markedly reduced by 22 : 4(n - 6). When the endothelial cells were incubated simultaneously with 22 : 4(n - 6) and 20 : 4(n - 6) the synthesis was still significant less
36
than when incubated with 20 : 4(n - 6) only. However, the 20 : 4( n - 6) content of the phospholipids was not significantly reduced. This observation may indicate that 22 : 4(n - 6) or one of its metabolites suppresses the synthesis of 6-ketoprostaglandin Ft, and thus represents a modulator to 20 : 4( n - 6) in the stimulation of prostacylin production. A similar counterbalance between the effects of 20 : 4(n - 6) and 22 : 4( n - 6) has been reported in platelets [4]. 22 : 5(n - 6) was also incorporated into endothelial cell phospholipids, with subsequent relative reduction of 20 : 4( n - 6) content and a reduced prostacylin synthesis. This may indicate that the two long-chain fatty acids of the (n - 6) family act similarly with regard to 20 : 4( n - 6) and prostacylin synthesis. This study has shown that 22 : 4(n - 6) and 22 : 6(n - 3) both of which are present in human endothelial cells, are incorporated into the cell phospholipids and thus modulate the membrane lipid composition. One of the consequences is a reduction of prostacylin synthesis. Earlier studies in rats have shown that the synthesis of 22 : 4( n 6) and 22 : 6(n - 3) in platelets is stimulated by dietary supplement of 18 : 2( n - 6) and 20 : 5( n 3) respectively [15]. Together, these studies may indicate that dietary polyunsaturated fatty acids may modify platelet and endothelial cell function partly by effects on the cellular content of docosapolyenoic fatty acids. This may represent another
link between dietary fatty acid composition thrombogenesis.
and
References 1 Mead, J.F. (1971) in Progress in the Chemistry of Fats and Other Lipids (Holman, R.T., ed.), Vol. IX, pp. 161-192, Pergamon Press, Oxford 2 Nordoy, A. and Lund, S. (1968) Stand. J. Clin. Lab. Invest. 22, 328-338 3 Rastogi, B. and Nordoy, A. (1980) Thromb. Res. 18. 629-641 4 Van Rollins, M., Horrocks, L. and Sprecher, H. (1985) Biochim. Biophys. Acta 833, 272-280 5 Milks, M.M. and Sprecher, H. (1985) Biochim. Biophys. Acta 835, 29-25 6 Dyerberg, J., Bang, H.O., Stoffersen, E., Moncada, S. and Vane, J.R. (1978) Lancet ii, 117-119 7 Fischer, S., Schacky, C.V., Siess, W., Strasser, T. and Weber, P.C. (1984) Biochem. Biophys. Res. Commun. 120,907-918 8 Weiner, T.W. and Sprecher, H. (1985) J. Biol. Chem. 260, 6032-6038 9 Jaffe, E.A., Nachman, R.L., Becker, C.G. and Minick. C.R. (1973) J. Clin. Invest. 52, 2745-2753 10 Chervionke, R.L., Hoak, J.C. and Fry, G.L. (1978) J. Clin. Invest. 62, 847-856 11 Salmon, J.A. (1979) Prostaglandins 15, 383-397 12 Brox, J.H. and Nordoy, A. (1984) Thromb. Haemostas. 50, 762-767 13 Corey, E.J., Shih, C. and Cashman, J.R. (1983) Proc. Natl. Acad. Sci. USA 80, 3581-3584 14 Alvadano, M.I. and Sprecher, H. (1983) J. Biol. Chem. 258, 9339-9343 15 Nordoy, A., Davenas, E., Ciavatti, M. and Renaud, S. (1985) Biochim. Biophys. Acta 835, 491-500