(Hypertension. 1995;25:854-859.)
© 1995 American Heart Association, Inc.
Articles |
Identification of Arachidonate P-450 Metabolites in Human Platelet Phospholipids
From the Department of Pharmacology, New York Medical College, Valhalla, NY.
Correspondence to Michael Balazy, PhD, New York Medical College, Department of Pharmacology, Valhalla, NY 10595.
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Abstract Phospholipase A2 (Naja mocambique) catalyzed release of epoxyeicosatrienoic acids (EETs) and 20-hydroxyeicosatetraenoic acid (20-HETE) from phospholipids of isolated human platelets. The amount of EETs released by phospholipase A2 measured by gas chromatography/mass spectrometry (GC/MS) was 4.3±0.9 pmol/106 platelets. No EETs were detected when phospholipase A2 was omitted from the incubations. The relative abundance of EET isomers (14,15-EET, 11,12-EET, 8,9-EET, and 5,6-EET) from human platelets was 5.4:4.5:3.7:1, respectively, as established by a new method based on particle-beam liquid chromatography/mass spectrometry (LC/MS). Fractionation of platelet phospholipids by normal-phase high-performance liquid chromatography followed by hydrolysis and GC/MS analyses indicated that the amount of EETs was highest in fractions containing phosphatidylinositol and phosphatidylserine (142 and 61 pmol/nmol of phosphorus, respectively) while low in phosphatidylcholine and phosphatidylethanolamine (19 and 11 pmol/nmol of phosphorus, respectively). The majority of EETs associated with phosphatidylcholine was found in fractions containing 1-O-alkyl-phosphatidylcholine. Human platelet phospholipids also released 20-HETE on phospholipase treatment (9.7±1.6 fmol/106 cells) and at least three other HETEs, one of which was tentatively identified as 16-HETE. Activation of human platelets by thrombin or platelet-activating factor released 5 to 7 fmol EET/106 cells. Receptor-mediated hydrolysis of phospholipids containing EETs and 20-HETE may play a role in stimulus-response coupling in platelets.
Key Words: platelets • phospholipids • cytochrome P450 • arachidonic acid • mass spectrometry • epoxides
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
O xygenation of arachidonic acid by cytochrome P-450 NADPH-dependent mixed function monooxygenase is known to generate four cis-epoxyeicosatrienoic acids (EETs) and 20-hydroxyeicosatetraenoic acid (20-HETE) and other subterminal HETEs with regioselectivity and stereoselectivity dependent on the source of the enzyme.1 2 3 4 EETs and 20-HETE display a wide range of biological effects1 2 3 4 and have been implicated to play a role in hypertension.4 In particular, EETs and 20-HETE are potent platelet antagonists.5 6 Whereas all four EETs inhibit platelet aggregation induced by arachidonic acid, some of the EETs [eg, 14(S),15(R)-EET and racemic 11,12-EET] inhibit platelet aggregation without alteration of thromboxane A2 levels originating from exogenous arachidonic acid.5 20-HETE inhibits platelet aggregation predominantly through antagonism of the prostaglandin H2/thromboxane A2 receptor.6 Although EETs and 20-HETE are strong antiplatelet agents, their physiological role is unclear, partly because they have not been found in incubations of platelets with arachidonic acid. Carbon monoxide and other inhibitors of cytochrome P-450 can inhibit platelet aggregation induced by arachidonic acid.7 8 The antiaggregatory effect of carbon monoxide on platelets also occurs through elevation of cyclic GMP.9 Thromboxane synthase, a microsomal enzyme that converts arachidonate endoperoxide into thromboxane A2, shows some of the characteristics of cytochrome P-450 but is not inhibited by carbon monoxide.9 It is unclear whether platelets contain "epoxygenase"-type cytochrome P-450 distinct from thromboxane synthase. A recent report by Ballou et al10 suggesting that human platelets may contain an enzyme system that catalyzes formation of a polar metabolite identified as 14,15-EET from 2-arachidonyl-phosphatidylinositol, combined with the objection raised about the identity of this metabolite,3 prompted us to examine a complex platelet lipid mixture using sensitive mass spectrometric techniques to detect endogenous P-450 arachidonic acid metabolites. In this study we analyzed human platelet phospholipids, and we found that epoxides of arachidonic acid and 20-HETE are preformed, integral components of normal human platelet membrane, which can be released during receptor-mediated hydrolysis of platelet phospholipids.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Isolation of EETs and 20-HETE From Human Platelets
Fresh human platelet concentrate from healthy donors who had not received medication was purchased from Hudson Valley Blood Bank. Platelets were isolated as described11 by sequential centrifugation at 200g (10 minutes) and at 1100g (15 minutes) and were finally suspended in phosphate-buffered saline (PBS) at 1x109 to 2x109 platelets per milliliter. The platelets were counted using a Coulter T 540 cell sorter (Coulter Electronics) and contained <0.1% leukocytes and 3% to 5% erythrocytes. Platelet suspension was extracted by the modified method of Bligh and Dyer (Broekman12 ) with 5 nCi of 2-[3H]arachidonyl-phosphatidylcholine (specific activity, 139 Ci/mmol) as a recovery tracer. The final phospholipid extract, containing 70% to 80% of radioactivity added, was suspended in 1 mL chloroform/methanol (2:1). Aliquots (10 to 500 µL) of the phospholipid extract were incubated with 2 to 10 U of phospholipase A2 (Naja mocambique, Sigma Chemical Co) in PBS containing 1 mmol/L of Ca2+ and 0.8 mmol/L of Mg2+ for 10 minutes at 37°C. The lipids were extracted with ethyl acetate and purified by reversed-phase high-performance liquid chromatography (HPLC; Ultrasphere ODS column, 250x4.6 mm, Beckman Instruments) with acetonitrile/water/acetic acid (62.5:37.5:0.05) increased to acetonitrile by 1.88%/min at 1 mL/min. Before extraction, octadeuterium-labeled 14,15-EET (14,15-EET-d8, 10 ng) and trideuterium-labeled 19-HETE (19-HETE-d3, 10 ng) were added as internal standards. The fractions containing HETEs and EETs were esterified to the pentafluorobenzyl ester,13 and, finally, fractions containing HETEs were reacted with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) to protect hydroxyl groups. After derivatization, the samples were dissolved in decane and analyzed by gas chromatography/mass spectrometry (GC/MS). In experiments with antagonists, platelets (4 to 5x108 cells/mL; total volume, 5 mL) were incubated with
-thrombin (0.2 U/mL) or platelet-activating factor (2 µmol/L) for
3 minutes at 37°C. The incubations were terminated by addition of 20
mL cold methanol. The methanolic solutions (supplemented with 50 ng of
14,15-EET-d8) were centrifuged, and the
supernatant was evaporated to near dryness. The residue was dissolved
in water, extracted with ethyl acetate, and, after HPLC purification
and derivatization, analyzed by GC/MS.
Gas Chromatography/Mass Spectrometry
Mass spectrometry was performed on an HP 5989A mass spectrometer
interfaced to an HP 5890 gas chromatograph (Hewlett-Packard)
essentially as described.13 Gas chromatographic analyses
were carried out on a DB-1 fused-silica capillary column (15 m, 0.25-mm
ID, 0.25-µm film thickness, J&W Scientific), and 1 µL of the sample
was injected using the splitless mode with an injector temperature of
250°C. The column was temperature programmed from 170°C to 300°C
at 25°C/min for analysis of EETs, or from 150°C to 300°C at
15°C/min for analysis of HETEs. All four EETs eluted as a single
chromatographic peak, whereas the HETE isomers (16-, 17-, 18-, 19-, and
20-HETE) were fully separated one from another. Helium was used as a
carrier gas with a linear velocity of 0.4 m/s. Electron-capture
ionization was carried out using methane as a moderating gas at a flow
resulting in ion source pressure of approximately 1.5 mm Hg at ion
source temperature of 200°C and electron energy of 180 eV. Selected
ion monitoring was used to record ion abundances at m/z 319
and m/z 391 (endogenous EETs and HETEs, respectively) and
m/z 327 and m/z 394 (internal standards,
14,15-EET-d8 and
19-HETE-d3, respectively). Standard
curves were prepared by addition of 14,15-EET (2 to 100 ng) to constant
amounts of 14,15-EET-d8 (10 or 50 ng) before
derivatization. The areas under the chromatographic peaks defined for
ions m/z 319 and 327 were obtained from GC/MS analyses;
their ratio was plotted against the amount of 14,15-EET added, which
resulted in a linear relation. The amount of endogenous EETs was
calculated from a regression line (r>.997). An identical
approach was taken to quantify the amounts of HETEs using
19-HETE-d3 as internal standard and ions
m/z 391 and 394.
Liquid Chromatography/Mass Spectrometry
Aliquots (200 µL) of platelet phospholipid extracts from four
healthy donors were hydrolyzed with phospholipase
A2, and the hydrolysates (containing 100 ng of
14,15-EET-d8) were combined and esterified with
pentafluorobenzyl bromide.14 The esterified samples were
injected using a Rheodyne injector into an HPLC silica column (100x4.6
mm, 5 µm, Hypersil, Hewlett-Packard) and eluted with
hexane/2-propanol (100:0.1 vol/vol) at a flow of 1 mL/min. The effluent
from the column was mixed with helium (50 psi) in a desolvation chamber
(temperature, 55°C) of the particle beam interface (Hewlett-Packard),
which was connected to the mass spectrometer operating in
electron-capture mode with methane flow into the ion source giving a
pressure of 1.6 mm Hg.15 The epoxides were detected by
monitoring ions m/z 319 and 327.
Analysis of Phospholipids
Aliquots of human platelet phospholipid extract were injected
into a silica column (150x4.6 mm, 3 µm, Alltech Associates) and
eluted with hexane/2-propanol/ethanol/sodium acetate (50 mmol/L)/acetic
acid (400:260:100:30:1, pH 6.7) at 1 mL/min, which was increased to 1.5
mL/min at 20 minutes. The elution of phospholipids was monitored by UV
absorbance at 205 nm using a photodiode array detector.15
The retention time of individual phospholipids was established by
coelution with phospholipid standards obtained from Sigma. Phosphorus
content was measured spectrophotometrically from the phosphomolybdate
formed by reaction of phospholipids with perchloric acid followed by
treatment with molybdate as described.12 16
Materials
HETE standards (16-, 17-, 18-, 19-, and 20-HETE) and
19-HETE-d3 (99+ atom % deuterium) were gifts
from Dr J.R. Falck (University of Texas, Southwest Medical Center,
Dallas, Tex). 14,15-EET-d8 was prepared by
reaction of [2H8]arachidonic acid (98
atom % deuterium, Biomol) with 3-chloroperoxybenzoic
acid.14 All solvents used were HPLC grade (JT
Baker), and other reagents were the highest grade commercially
available. BSTFA was from Aldrich, and
2-[3H]arachidonyl-phosphatidylcholine was from
DuPont–New England Nuclear.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Hydrolysis of platelet phospholipids by cobra venom phospholipase A2 followed by GC/MS analysis revealed the presence of endogenous EETs and HETEs (Table). The total amount of EETs released from human platelet phospholipids was 4.3±0.9 pmol/106 cells (n=4). In the absence of phospholipase, the EETs were not detected. Phospholipase A2 also released 20-HETE (Figs 1 and 2; retention time, 8.6 minutes) from platelet phospholipids in amounts approximately 200-fold lower than those found for EETs (9.7±1.6 fmol/106 cells, n=3). At least three other HETEs were also detected (Fig 1), one of which coeluted with standard 16-HETE (Fig 2; retention time, 7.9 minutes). The concentration of 16-HETE was 8.8±0.6 fmol/106 cells (n=3). The majority of 12-HETE was separated from samples containing 16-HETE and 20-HETE during HPLC purification. Any unseparated 12-HETE eluted during GC/MS analysis at 8.0 minutes (Fig 1). The amount of 12-HETE in analyzed samples was comparable to that of 16-HETE. The amount of material eluting at 7.85 minutes, the occurrence of which was phospholipase dependent (Fig 1), was insufficient for full structural identification. We have not detected any EETs or 20-HETE in human platelets that were free or unbound to phospholipids. The generation of these arachidonate metabolites was dependent on the presence of phospholipase, which was free of these compounds. The control incubations of phospholipase A2 with standard 2-arachidonyl-phosphatidylcholine did not result in detectable amounts of EETs or 20-HETE. The EETs were also detected when platelet phospholipids were saponified with sodium hydroxide.
|
|
|
Since the complete resolution of EETs by capillary column gas
chromatography was not possible, we used normal-phase HPLC
chromatography coupled to a mass spectrometer via particle-beam LC/MS
to separate and detect isomers of EETs. LC/MS analysis revealed
that human phospholipids contained all four EETs regioisomers in
proportions of (as percentage of total EET) 37.1:30.8:25.3:6.8
(14,15-/11,12-/8,9-/5,6-EET, respectively). Fractionation of platelet
phospholipids, using normal-phase HPLC followed by phospholipase
A2 hydrolysis and GC/MS quantitation, revealed that, when
normalized to 1 nmol of phosphorus, 87% of the EETs were esterified in
phosphatidylinositol and phosphatidylserine (61% and 26%,
respectively), whereas the remaining 13% were in phosphatidylcholine
and phosphatidylethanolamine (8% and 5%, respectively). We further
investigated the agonist-induced release of EETs from isolated human
platelets (Fig 3). Treatment of platelets with
-thrombin (0.2 U/mL) or platelet-activating factor (2 µmol/L) for
3 minutes resulted in the release of EETs in the amount of 7.1±2.5 and
5.7±2.3 fmol/106 cells (n=3), respectively. No EETs
were detected when the agonist was absent (Fig 3).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Platelets play an important role in the events that follow blood vessel injury.17 18 Platelets that have come in contact with collagen in the injured vessel wall rapidly aggregate. Proaggregatory mediators released from these platelets cause further aggregation, and eventually a hemostatic platelet plug forms. Membrane lipoprotein of the aggregated platelets serves as a catalytic surface for the interaction of coagulation factors. Eventually, this results in formation of thrombin, which produces fibrin from fibrinogen, causing further platelet aggregation and "consolidation" of the hemostatic platelet plug. It is reasonable to assume that the membrane of the platelets plays an important role in these events. For example, oxidative modification of platelet membrane phospholipids (eg, during prolonged storage) is known to alter platelet function.19
In this study we analyzed a complex mixture of human platelet phospholipids, and we found that normal human platelets contained a pool of phospholipids having endogenous, oxidatively modified, arachidonic acid metabolites esterified in sn-2 position, which suggests involvement of cytochrome P-450 in their formation. Naja mocambique venom phospholipase A2 released 4.3±0.9 pmol EETs per 106 platelets, which indicates that about 0.5% of total platelet phospholipids contained EETs (assuming 4.28 mg lipid per 1010 platelets and 80 mg of phospholipid per 100 mg of total platelet lipid20 ). The amounts of 20-HETE were approximately 200-fold lower than those found for EETs. The generation of these metabolites was strictly phospholipase A2 dependent. When calculated on a molar basis, 61% of EETs were esterified in phosphatidylinositol and 26% in phosphatidylserine. These two phospholipids constituted 5.7% and 12.5% of platelet lipid phosphorus, respectively. The remaining 13% of EETs were esterified in phosphatidylethanolamine and phosphatidylcholine, which, combined, constituted 81.8% of total lipid phosphorus. These data indicate that, taking into account an asymmetrical distribution of phospholipids in the platelet membrane, the overwhelming proportion of EETs (93%) was esterified in phospholipids that compose the inner leaflet of the platelet membrane.20 The occurrence of EETs in phosphatidylinositol suggests that these epoxides may play a role in signal transduction in platelets. These data refer to phospholipids extracted from whole platelets; at the present time, it is not known whether EETs are uniformly distributed among phospholipids of membranes and subcellular granules.
Although in this study we have not investigated the origin of phospholipid-bound EETs and HETEs, it seems unlikely that these metabolites originate from spontaneous oxidation during isolation and handling of phospholipids. Should this autooxidation occur, the distribution of EETs among phospholipids should parallel the distribution of arachidonic acid among phospholipids. That was not the case. Marcus et al21 have demonstrated that arachidonic acid comprises 41, 32, 24, and 12 mol% of total fatty acids associated with phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine, respectively. Our results indicate that the distribution of EETs does not parallel this trend (61, 5, 26, and 8 pmol% of total EETs associated with phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine, respectively).
To hydrolyze phospholipids, we used phospholipase A2 (from cobra venom Naja mocambique), which is known to activate platelets through the release of arachidonic acid.22 This phospholipase displays substrate specificity. It has been reported that phosphatidylcholine is a good substrate, whereas phosphatidylethanolamine is not efficiently hydrolyzed by phospholipase A2 from Naja venom.22 We have tested this and found that Naja venom phospholipase efficiently releases fatty acids from phosphatidylinositol and phosphatidylserine. This indicates that the hydrolysis rates for phosphatidylinositol, phosphatidylserine, and phosphatidylcholine are similar while those for phosphatidylethanolamine are lower; consequently, the amounts of EETs released from phosphatidylethanolamine may be underestimated.
The phospholipid-bound EETs may originate from enzymatic or nonenzymatic processes. Human platelets were shown to contain cytochromes P-450 and b5 and their respective reductases, NADPH-cytochrome c reductase and NADPH-cytochrome b5 reductase,7 but no data are available regarding the metabolism of arachidonic acid by platelet cytochrome P-450. The formation of 14,15-EET described by Ballou et al10 did not require NADPH, suggesting that cytochrome P-450 was not involved. Platelet thromboxane A2 synthase shares some properties of cytochrome P-450 enzyme; however, it is not known whether it can act as a monooxygenase. Platelets can generate superoxide anion23 and nitric oxide,24 which may react together to produce a strong short-lived oxidant, peroxynitrite. We have shown that treatment of arachidonic acid with peroxynitrite generates EETs.25 The identification of 20-HETE in human platelet phospholipids strongly indicates involvement of cytochrome P-450 hydroxylase in its formation, since this metabolite is unlikely to be formed via autooxidation of arachidonic acid under normal conditions. It remains to be established whether 20-HETE is biosynthesized by platelets or originates from another biochemical source. There is a precedent for our findings. Capdevila and coworkers (Karara et al26 ) have found that EETs are esterified in phospholipids of rat liver. The liver phosphatidylinositol contained the highest amount of EETs (106 µmol) per mole of phospholipid.26 Also, rat plasma lipoprotein phospholipids contain EETs.27
Our results have two major implications. First, we found that during activation of platelets, EETs were released from platelet phospholipids. Assuming that the average platelet volume is 5 fL,28 the amount of EETs released by thrombin or platelet-activating factor can reach an intracellular concentration in the range of 1 µmol/L. At these concentrations, EETs reveal biological activity in human platelets. Fitzpatrick et al3 5 have shown that EETs can inhibit platelet aggregation (IC50, 1 to 10 µmol/L), platelet cyclooxygenase (IC50, 1 to 50 µmol/L), calcium entry, and 40-kD protein phosphorylation. 20-HETE inhibits platelet aggregation (IC50, 5 to 16 µmol/L) predominantly through antagonism of the prostaglandin H2/thromboxane A2 receptor.6 EETs and 20-HETE do not alter the levels of cyclic nucleotides.5 6 In addition, the EETs and 20-HETE also display vascular activity.29 5,6-EET is a potent vasorelaxant,30 whereas 20-HETE is a renal vasoconstrictor.31 The hydrolysis of EET-phosphatidylinositol by phospholipase C raises the possibility of release of EET-containing diglycerides during platelet stimulation. Such new diglycerides may alter signaling mediated by protein kinase C; this possibility is currently under investigation.
Second, unlike hydroperoxy-modified fatty acids, which activate cyclooxygenase, the EETs and 20-HETE display inhibitory properties toward cyclooxygenase. It is attractive to speculate that enrichment of platelets with phospholipids containing 20-HETE or 14,15-EET (a major human platelet EET) may yield platelets with diminished capability to synthesize thromboxane A2, since activation of these platelets should result in release of larger amounts of cyclooxygenase inhibitors. Platelets are capable of phagocytosing liposomes without significant alteration of platelet function and morphology.32 Much further work is needed to establish the role and potential therapeutic implications of cytochrome P-450 metabolites of arachidonic acid stored in cellular phospholipids.
|
Acknowledgments |
|---|
This research was supported by grants 92013280 from the American Heart Association, Dallas, Tex (Dr Balazy) and HL-34300 from the National Institutes of Health. We thank Melody Steinberg for editorial assistance.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Oliw EH. Oxygenation of polyunsaturated fatty acids by cytochrome P450 monooxygenases. Prog Lipid Res. 1994;33:329-354. [Medline] [Order article via Infotrieve]
2. Capdevila JH, Falck JR, Estabrook RW. Cytochrome P450 and the arachidonic acid cascade. FASEB J. 1992;6:731-736. [Abstract]
3. Fitzpatrick FA, Murphy RC. Cytochrome P450 metabolism of arachidonic acid: formation and biological actions of `epoxygenase'-derived eicosanoids. Pharmacol Rev. 1989; 40:229-241.
4. McGiff JC. Cytochrome P-450 metabolism of arachidonic acid. Annu Rev Pharmacol Toxicol. 1991;31:339-369. [Medline] [Order article via Infotrieve]
5. Fitzpatrick FA, Ennis MD, Baze ME, Wynalda MA, McGee JE, Liggett WF. Inhibition of cyclooxygenase activity and platelet aggregation by epoxyeicosatrienoic acids. J Biol Chem. 1986; 261:15334-15338.
6. Hill E, Fitzpatrick F, Murphy RC. Biological activity of 20-hydroxyeicosatetraenoic acid in the human platelet. Br J Pharmacol. 1992;106:267-274. [Medline] [Order article via Infotrieve]
7. Cinti DL, Feinstein MB. Platelet cytochrome P-450: a possible role in arachidonate-induced aggregation. Biochem Biophys Res Commun. 1976;73:171-178. [Medline] [Order article via Infotrieve]
8. Parnham MJ, Bragt PC, Bast A, Zulstra FJ. Comparison of the effects of inhibitors of cytochrome P450-mediated reactions on human platelet aggregation and arachidonic acid metabolism. Biochim Biophys Acta. 1981;677:165-173. [Medline] [Order article via Infotrieve]
9.
Haurand M, Ullrich V. Isolation and characterization of
thromboxane synthase from human platelets as a cytochrome P450 enzyme.
J Biol Chem. 1985;260:15059-15067.
10.
Ballou LR, Lam BK, Wong PYK, Cheung WY. Formation of
cis-14,15-oxido-5,8,11-eicosatrienoic acid from
phosphatidylinositol in human platelets. Proc Natl Acad Sci
U S A. 1987;84:6990-6994.
11. Mustard JF, Kinlough-Rathbone RL, Packham MA. Isolation of human platelets from plasma by centrifugation and washing. Methods Enzymol. 1989;169:3-11. [Medline] [Order article via Infotrieve]
12. Broekman MJ. Endogenous phosphatidylinositol in stimulated human platelets. Methods Enzymol. 1989;169:415-430. [Medline] [Order article via Infotrieve]
13.
Balazy M. Metabolism of 5,6-epoxyeicosatrienoic acid by the
human platelet. J Biol Chem. 1991;266:23561-23567.
14. Balazy M, Nies AS. Characterization of epoxides of polyunsaturated fatty acids by mass spectrometry. Biomed Environ Mass Spectrom. 1989;18:328-336. [Medline] [Order article via Infotrieve]
15. Balazy M, Schieber EB, McGiff JC. Identification of arachidonate epoxides in human platelets. Adv Prostaglandin Thromboxane Leukot Res. 1995;23:199-201. [Medline] [Order article via Infotrieve]
16. Chen PS, Toribara TY, Warner H. Microdetermination of phosphorus. Anal Chem. 1956;28:1756-1758.
17. Baumgartner HR, Sakariassen KS. Factors controlling thrombus formation on arterial lesions. Ann N Y Acad Sci. 1985;454:162-177. [Medline] [Order article via Infotrieve]
18. Peterson SN, Lapetina EG. Platelet activation and inhibition: novel signal transduction mechanisms. Ann N Y Acad Sci. 1994;714:53-63. [Medline] [Order article via Infotrieve]
19. Fagiolo E, Lippa S, Mores N, Oradei A, Aureli V. Peroxidative events in stored platelet concentrations. Vox Sang. 1989;56:32-36. [Medline] [Order article via Infotrieve]
20. Koerner TA, Cunningham MT, Zhang DS. The role of membrane lipid in the platelet storage lesion. Blood Cells. 1992;18:481-497. [Medline] [Order article via Infotrieve]
21. Marcus AJ, Ullman HL, Safir LB. Lipid composition of subcellular particles of human blood platelets. J Lipid Res. 1969;10:108-114. [Abstract]
22. Mounier C, Vargaftig BB, Franken PA, Verheij HM, Bon C, Touqui L. Platelet secretory phospholipase A2 fails to induce rabbit platelet activation and to release arachidonic acid in contrast with venom phospholipases A2. Biochim Biophys Acta. 1994;1214:88-96. [Medline] [Order article via Infotrieve]
23. Marcus AJ, Slik ST, Safier LB, Ullman HL. Superoxide production and reducing activity in human platelets. J Clin Invest. 1977;59: 149-158.
24. Radomski MW, Moncada S. Biological role of nitric oxide in platelet function. In: Moncada S, Higgs EA, Berrazueta JR, eds. Clinical Relevance of Nitric Oxide in the Cardiovascular System. Madrid, Spain: Edicomplet; 1991.
25. Balazy M. Peroxynitrite and arachidonic acid: identification of arachidonate epoxides. Pol J Pharmacol. In press.
26.
Karara A, Dishman E, Falck JR, Capdevila JH. Endogenous
epoxyeicosatrienoyl-phospholipids. J Biol Chem. 1991;266:7561-7569.
27. Karara A, Wei S, Spady D, Swift L, Capdevila JH, Falck JR. Arachidonic acid epoxygenase: structural characterization and quantification of epoxyeicosatrienoates in plasma. Biochem Biophys Res Commun. 1992;182:1320-1325. [Medline] [Order article via Infotrieve]
28. Bessman D. Determination of platelet volume and number. Methods Enzymol. 1989;169:164-172. [Medline] [Order article via Infotrieve]
29. Carroll MA, Balazy M, McGiff JC. Tubular and vascular actions of cytochrome P450-arachidonate metabolites. J Physiol Pharmacol. 1993;44:37-49.
30.
Carroll MA, Balazy M, Margiotta P, Falck JR, McGiff JC. Renal
vasodilator activity of 5,6-epoxyeicosatrienoic acid depends upon
conversion by cyclooxygenase and release of prostaglandins. J
Biol Chem. 1993;268:12260-12266.
31. Escalante B, Sessa WC, Falck JR, Yadagiri P, Schwartzman ML. Vasoactivity of 20-hydroxyeicosatetraenoic acid depends on metabolism by cyclooxygenase. J Pharmacol Exp Ther. 1989;248: 229-232.
32.
Male R, Vannier WE, Baldeschwieler JD. Phagocytosis of
liposomes by human platelets. Proc Natl Acad Sci U S A. 1992;89:9191-9195.
This article has been cited by other articles:
 |
|
 |
|
  A. A. Spector and A. W. Norris Action of epoxyeicosatrienoic acids on cellular function Am J Physiol Cell Physiol, March 1, 2007; 292(3): C996 - C1012. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Fang, S. Hu, B. Xu, G. D. Snyder, S. Harmon, J. Yao, Y. Liu, B. Sangras, J. R. Falck, N. L. Weintraub, et al. 14,15-Dihydroxyeicosatrienoic acid activates peroxisome proliferator-activated receptor-{alpha} Am J Physiol Heart Circ Physiol, January 1, 2006; 290(1): H55 - H63. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Lu, M.-P. Hong, and H.-C. Lee Molecular Determinants of Cardiac KATP Channel Activation by Epoxyeicosatrienoic Acids J. Biol. Chem., May 13, 2005; 280(19): 19097 - 19104. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Krotz, T. Riexinger, M. A. Buerkle, K. Nithipatikom, T. Gloe, H.-Y. Sohn, W. B. Campbell, and U. Pohl Membrane Potential-Dependent Inhibition of Platelet Adhesion to Endothelial Cells by Epoxyeicosatrienoic Acids Arterioscler Thromb Vasc Biol, March 1, 2004; 24(3): 595 - 600. [Abstract] [Full Text]
|
|
|
|
 |
|
 R. J. Roman P-450 Metabolites of Arachidonic Acid in the Control of Cardiovascular Function Physiol Rev, January 1, 2002; 82(1): 131 - 185. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Lu, T. Hoshi, N. L Weintraub, A. A Spector, and H.-C. Lee Activation of ATP-sensitive K+ channels by epoxyeicosatrienoic acids in rat cardiac ventricular myocytes J. Physiol., December 15, 2001; 537(3): 811 - 827. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Fleming Cytochrome P450 and Vascular Homeostasis Circ. Res., October 26, 2001; 89(9): 753 - 762. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Lu, P. V G Katakam, M. VanRollins, N. L Weintraub, A. A Spector, and H.-C. Lee Dihydroxyeicosatrienoic acids are potent activators of Ca2+-activated K+ channels in isolated rat coronary arterial myocytes J. Physiol., August 1, 2001; 534(3): 651 - 667. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Rastaldo, N. Paolocci, A. Chiribiri, C. Penna, D. Gattullo, and P. Pagliaro Cytochrome P-450 metabolite of arachidonic acid mediates bradykinin-induced negative inotropic effect Am J Physiol Heart Circ Physiol, June 1, 2001; 280(6): H2823 - H2832. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. A Carroll and J. C McGiff A new class of lipid mediators: cytochrome P450 arachidonate metabolites Thorax, October 1, 2000; 55(90002): 13S - 16. [Full Text]
|
|
|
|
|
|
N. L. Weintraub, X. Fang, T. L. Kaduce, M. VanRollins, P. Chatterjee, and A. A. Spector Epoxide hydrolases regulate epoxyeicosatrienoic acid incorporation into coronary endothelial phospholipids Am J Physiol Heart Circ Physiol, November 1, 1999; 277(5): H2098 - H2108. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. C. McGiff and J. Quilley 20-HETE and the kidney: resolution of old problems and new beginnings Am J Physiol Regulatory Integrative Comp Physiol, September 1, 1999; 277(3): R607 - R623. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H.-C. Lee, T. Lu, N. L Weintraub, M. VanRollins, A. A Spector, and E. F Shibata Effects of epoxyeicosatrienoic acids on the cardiac sodium channels in isolated rat ventricular myocytes J. Physiol., August 15, 1999; 519(1): 153 - 168. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Fang, S. A. Moore, L. L. Stoll, G. Rich, T. L. Kaduce, N. L. Weintraub, and A. A. Spector 14,15-Epoxyeicosatrienoic acid inhibits prostaglandin E2 production in vascular smooth muscle cells Am J Physiol Heart Circ Physiol, December 1, 1998; 275(6): H2113 - H2121. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. L. Ivey, A. H. Stephenson, and M. I. Townsley Involvement of cytochrome P-450 enzyme activity in the control of microvascular permeability in canine lung Am J Physiol Lung Cell Mol Physiol, October 1, 1998; 275(4): L756 - L763. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. S. Keeney, C. Skinner, S. Wei, T. Friedberg, and M. R. Waterman A Keratinocyte-specific Epoxygenase, CYP2B12, Metabolizes Arachidonic Acid with Unusual Selectivity, Producing a Single Major Epoxyeicosatrienoic Acid J. Biol. Chem., April 10, 1998; 273(15): 9279 - 9284. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. R. Harder, N. J. Alkayed, A. R. Lange, D. Gebremedhin, and R. J. Roman Functional Hyperemia in the Brain : Hypothesis for Astrocyte-Derived Vasodilator Metabolites Stroke, January 1, 1998; 29(1): 229 - 234. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. L. Weintraub, X. Fang, T. L. Kaduce, M. VanRollins, P. Chatterjee, and A. A. Spector Potentiation of Endothelium-Dependent Relaxation by Epoxyeicosatrienoic Acids Circ. Res., August 19, 1997; 81(2): 258 - 267. [Abstract] [Full Text]
|
|
|
|
|
|
X. Fang, T. L. Kaduce, N. L. Weintraub, M. VanRollins, and A. A. Spector Functional Implications of a Newly Characterized Pathway of 11,12-Epoxyeicosatrienoic Acid Metabolism in Arterial Smooth Muscle Circ. Res., October 1, 1996; 79(4): 784 - 793. [Abstract] [Full Text]
|
|
|
|
|
|
M. VanRollins, T. L. Kaduce, X. Fang, H. R. Knapp, and A. A. Spector Arachidonic Acid Diols Produced by Cytochrome P-450 Monooxygenases Are Incorporated into Phospholipids of Vascular Endothelial Cells J. Biol. Chem., June 14, 1996; 271(24): 14001 - 14009. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||