thromboxane-b2 and 12-hydroxy-5-8-10-heptadecatrienoic-acid

For more than 25 years 12-S-hydroxyheptadecatrienoic acid (HHT) has been known to be a product of thromboxanesynthase (TX-Syn) when synthesized with thromboxane A2 (TXA2). Although there are some hints that HHT has anti-aggregatory effects, to date, it has neither been shown to have any specific pathological relevance nor is there much information about its physiological role. This review presents a summary of the physicochemical properties of HHT, its chemical synthesis, the impact of various biological systems on its enzymatic and non-enzymatic production and its physiological function and metabolization, as well as a survey of the most important methods for analyzing this unsaturated hydroxy-fatty acid. Due to the low antibody-raising potency expected in HHT, no immunological system for HHT quantification has been developed so far. In our report we present the development and validation of a sensitive and reliable, competitive radioimmunoassay (RIA) suitable for the quantitative determination of HHT. HHT was produced by an enhanced enzymatic method using platelet-rich plasma (PRP). With an effective and modified liquid-liquid and solid-phase extraction method we were able to produce highly purified HHT (97% purity by GC/MS) in sub-milligram ranges. These fractions were used for the synthesis of BSA-antigen-conjugates and for immunization of rabbits. The tritiated tracer was synthesized using prostaglandin H synthase for the production of prostaglandin H2 (PGH2) followed by an aqueous reaction with Fe(2+)-solution to rear-range PGH2 to HHT. The dynamic range of the assay was from 30-400 pg/tube, with a sensitivity of approximately 40 pg/tube. The evaluation of the assay was performed by a HPLC-RIA method as well as by correlation with a quantitative HPLC method and correlation with TXB2 concentrations in a blood coagulation study. The assay may be useful for the quantification of HHT in several tissues and body fluids under various physiological conditions and may also help to understand the possible physiological role of HHT in biological processes.

Topics: Animals; Blood Platelets; Chemical Phenomena; Chemistry, Physical; Fatty Acids, Unsaturated; Humans; Radioimmunoassay; Thromboxane B2

Elderly people ingested 150 mg/day of icosapentaenoic acid (20:5n-3) or a placebo for one month. Platelet aggregation, platelet arachidonate metabolism and the fatty acid composition of both plasma and platelet lipids were investigated before and after the intake. Platelet aggregation induced by collagen, epinephrine or low concentrations of ADP was significantly reduced after 20:5n-3 intake. Besides, the main oxygenated product formation from endogenous platelet arachidonate under thrombin stimulation was markedly decreased after the 20:5n-3 supplementation. Such a decrease was absent after placebo. Moreover, no modification in the fatty acid composition of both plasma lipids and platelet phosphatidylcholine could be observed. We conclude that intake of low amounts of 20:5n-3 by elderly people, is able to lower their platelet sensitivity to aggregating agents, probably by decreasing the endogenous formation of platelet thromboxane A2, although no modification in the fatty acid composition was detected.

Topics: Adenosine Diphosphate; Aged; Arachidonic Acids; Blood Platelets; Chromatography, Gas; Chromatography, High Pressure Liquid; Collagen; Eicosapentaenoic Acid; Epinephrine; Fatty Acids, Unsaturated; Humans; Hydroxyeicosatetraenoic Acids; Lipids; Male; Middle Aged; Phosphatidylcholines; Phospholipids; Platelet Aggregation; Thromboxane A2; Thromboxane B2; Thromboxanes

In this study, a combined targeted/untargeted UHPLC-ESI-QTOF-MS/MS method for the targeted quantitative analysis of the primary platelet lipid mediators thromboxane B2 (TXB2), 12S-hydroxy-5Z,8E,10E-heptadecatrienoic acid (HHT) and its oxidation product 12-keto-5Z,8E,10E-heptadecatrienoic acid (KHT) was developed, which allowed simultaneous untargeted profiling for the detection of other lipid biomarkers such as other oxylipins and fatty acids (FAs) in platelet releasates. A general procedure for the synthesis of keto-analogs from hydroxylated polyunsaturated FAs (PUFAs) using Dess-Martin periodinane oxidation reagent was proposed for the preparation of KHT standard. MS detection was performed in data independent acquisition (DIA) mode with sequential window acquisition of all theoretical fragment ion mass spectra (SWATH) in the range of 50-500 Da with variable window sizes. The LC-MS/MS assay was validated for the targeted analytes and applied for analysis of supernatants derived from resting platelets and from platelets treated with thrombin. The targeted analytes KHT, HHT and TXB2 were found at highly elevated levels in the activated platelet releasates. On average, 13 ± 7, 15 ± 9, and 0.6 ± 0.2 attomols per platelet were released upon thrombin-activation. Furthermore, the simultaneous untargeted profiling (n = 8 in each group) revealed that these oxylipins are released with a pool of other (significantly upregulated) oxidized (12-HETE, 12-HEPE) and non-oxidized PUFAs. All these compounds can be considered additional biomarkers of platelet activation complementing the primary platelet activation marker thromboxane B2. The other lipids may support platelet activation or trigger other biological actions with some potential implications in thromboinflammation.

Topics: Blood Platelets; Chromatography, High Pressure Liquid; Fatty Acids; Fatty Acids, Unsaturated; Humans; Limit of Detection; Platelet Activation; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry; Thrombin; Thromboxane B2

12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT) has long been considered a by-product of thromboxane A₂ (TxA₂) biosynthesis with no biological activity. Recently, we reported 12-HHT to be an endogenous ligand for BLT2, a low-affinity leukotriene B4 receptor. To delineate the biosynthetic pathway of 12-HHT, we established a method that enables us to quantify various eicosanoids and 12-HHT using LC-MS/MS analysis. During blood coagulation, 12-HHT levels increased in a time-dependent manner and were relatively higher than those of TxB₂, a stable metabolite of TxA₂. TxB₂ production was almost completely inhibited by treatment with ozagrel, an inhibitor of TxA synthase (TxAS), while 12-HHT production was inhibited by 80-90%. Ozagrel-treated blood also exhibited accumulation of PGD₂ and PGE₂, possibly resulting from the shunting of PGH₂ into synthetic pathways for these prostaglandins. In TxAS-deficient mice, TxB₂ production during blood coagulation was completely lost, but 12-HHT production was reduced by 80-85%. HEK293 cells transiently expressing TxAS together with cyclooxygenase (COX)-1 or COX-2 produced both TxB₂ and 12-HHT from arachidonic acid, while HEK293 cells expressing only COX-1 or COX-2 produced significant amounts of 12-HHT but no TxB₂. These results clearly demonstrate that 12-HHT is produced by both TxAS-dependent and TxAS-independent pathways in vitro and in vivo.

Topics: Animals; Blood Coagulation; Blood Platelets; Cyclooxygenase 1; Cyclooxygenase 2; Enzyme Inhibitors; Fatty Acids, Unsaturated; Gene Knockout Techniques; HEK293 Cells; Humans; Ligands; Mice; Mice, Inbred C57BL; Prostaglandin H2; Receptors, Leukotriene B4; Thromboxane B2; Thromboxane-A Synthase

In the present study, the effects of hypochlorous acid (HOCl), monochloramine (NH(2)Cl), glutamine-chloramine (Glu-Cl) and taurine-chloramine (Tau-Cl) on the formation of 12-lipoxygenase (LOX) metabolite, 12-HETE, and cyclooxygenase (COX) metabolites, TXB(2), and 12-HHT, from exogenous arachidonic acid (AA) in rat platelets were examined. Rat platelets (4x10(8)/ml) were preincubated with drugs for 5min at 37 degrees C prior to the incubation with AA (40microM) for 2min at 37 degrees C. HOCl (50-250microM) showed an inhibition on the formation of LOX metabolite (12-HETE, 5-67% inhibition) and COX metabolites (TXB(2), 33-73% inhibition; 12-HHT, 27-74% inhibition). Although Tau-Cl and Glu-Cl up to 100microM were without effect on the formation of 12-HETE, TXB(2) and 12-HTT, NH(2)Cl showed a strong inhibition on the formation of all three metabolites (10-100microM NH(2)Cl, 12-HETE, 21-92% inhibition; TXB(2), 58-94% inhibition; 12-HHT, 36-92% inhibition). Methionine reversed a reduction of formation of LOX and COX metabolites induced by NH(2)Cl, and taurine restoring that induced by both NH(2)Cl and HOCl. These results suggest that NH(2)Cl is a more potent inhibitor of COX and LOX pathways in platelets than HOCl, and taurine and methionine can be modulators of NH(2)Cl-induced alterations in the COX and LOX pathways in vivo.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acid; Blood Platelets; Chloramines; Cyclooxygenase Inhibitors; Enzyme Inhibitors; Fatty Acids, Unsaturated; Glutamine; Hypochlorous Acid; Lipoxygenase Inhibitors; Prostaglandin-Endoperoxide Synthases; Rats; Taurine; Thromboxane B2

Microsomal prostaglandin E synthase (mPGES)-1 is one of several prostaglandin E synthases involved in prostaglandin H2 (PGH2) metabolism. In the present report, we characterize the contribution of mPGES-1 to cellular PGH2 metabolism in murine macrophages by studying the synthesis of eicosanoids and expression of eicosanoid metabolism enzymes in wild type and mPGES-1-deficient macrophages. Thioglycollate-elicited macrophages isolated from mPGES-1-/- animals and genetically matched wild type controls were stimulated with diverse pro-inflammatory stimuli. Prostaglandins were released in the following order of decreasing abundance from wild type macrophages stimulated with lipopolysaccharide: prostaglandin E2 (PGE2)>thromboxane B2 (TxB2)>6-keto prostaglandin F1alpha (PGF1alpha), prostaglandin F(2alpha) (PGF2alpha), and prostaglandin D2 (PGD2). In contrast, we detected in mPGES-1-/- macrophages a >95% reduction in PGE2 production resulting in the following altered prostaglandin profile: TxB2>6-keto PGF1alpha and PGF2alpha>PGE2, despite the comparable release of total prostaglandins. No significant change in expression pattern of key prostaglandin-synthesizing enzymes was detected between the genotypes. We then further profiled genotype-related differences in the eicosanoid profile using macrophages pre-stimulated with lipopolysaccharide followed by a 10-min incubation with 10 microm [3H]arachidonic acid. Eicosanoid products were subsequently identified by reverse phase high pressure liquid chromatography. The dramatic reduction in [3H]PGE2 formation from mPGES-1-/- macrophages compared with controls resulted in TxB2 and 6-keto PGF1alpha becoming the two most abundant prostaglandins in these samples. Our results also suggest a 5-fold increase in 12-[3H]hydroxyheptadecatrienoic acid release in mPGES-1-/- samples. Our data support the hypothesis that mPGES-1 induction in response to an inflammatory stimulus is essential for PGE2 synthesis. The redirection of prostaglandin production in mPGES-1-/- cells provides novel insights into how a cell processes the unstable endoperoxide PGH2 during the inactivation of a major metabolic outlet.

Topics: Animals; Arachidonic Acid; Blotting, Western; Chromatography, High Pressure Liquid; Dose-Response Relationship, Drug; Eicosanoids; Fatty Acids, Unsaturated; Genotype; Imidazoles; Inflammation; Intramolecular Oxidoreductases; Kinetics; Lipopolysaccharides; Macrophages; Mice; Mice, Transgenic; Microsomes; Prostaglandin-E Synthases; Prostaglandins; Thioglycolates; Thromboxane B2; Time Factors

To explore the possible actions of endocrine disruptors on the autacoid synthesis in the body, we investigated the effects of nonylphenol (NP), bisphenol A (BPA), di-n-butyl phthalate (DBP), benzyl-n-butyl phthalate (BBP), and di-2-ethylhexyl phthalate (DEHP) on the formation of 12-lipoxygenase metabolite, 12-HETE, and cyclooxygenase metabolites, TXB(2) and 12-HHT, from exogenous arachidonic acid (AA) in rabbit platelets. NP (10-50 microM) showed strong inhibition on the formation of cyclooxygenase metabolites (TXB(2), 34-95% inhibition; 12-HHT, 13-78% inhibition) and weaker inhibition on the formation of 12-HETE (0-49% inhibition). BPA, DBP, BBP, DEHP, and 17beta-estradiol (endogenous estrogen) failed to show any effect on the formation of cyclooxygenase and 12-lipoxygenase metabolites at concentrations up to 100 microM. These results suggest that NP inhibits AA metabolism in platelets and that its effects on the cyclooxygenase pathway predominate over those exerted via the 12-lipoxygenase pathway.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acid; Blood Platelets; Endocrine System; Enzyme Inhibitors; Fatty Acids, Unsaturated; In Vitro Techniques; Lipoxygenase Inhibitors; Male; Prostaglandin-Endoperoxide Synthases; Rabbits; Thromboxane B2

Recent studies have shown that long-term in vivo exposure of dogs to neutral sulfur(IV)/sulfite aerosols induces mild inflammatory reactions, whereas the combination of neutral sulfite with acidic sulfur(VI)/sulfate aerosols evokes less pronounced effects. To understand underlying mechanisms, we studied in vitro the role of lipid mediators in the responses of alveolar macrophages (AMs) to sulfur-related compounds under neutral (pH 7) or moderate acidic (pH 6) conditions. Canine AMs incubated with sulfite at pH 7 released threefold higher amounts of platelet-activating factor than control (P < 0.005). Generation of arachidonic acid, leukotriene B4, 5-hydroxy-eicosatetraenoic acid, prostaglandin E2, thromboxane B2 and 12-hydroxyheptadecatrienoic acid increased twofold (P < 0.0005). However, these metabolites remained unchanged following incubation of AMs with sulfite at pH 6 or with sulfate at pH 7 or pH 6. Mediator release by sulfite-treated AMs at pH 7 stimulated respiratory burst activity of neutrophils. Inhibition of MAPK pathway by PD 98059, of cytosolic (cPLA2) and secretory phospholipases A2 by AACOCF3 and thioetheramide-PC, respectively, reduced sulfite-induced eicosanoid formation in AMs. Sulfite activated cPLA2 activity twofold at pH 7. This mechanism of sulfite-stimulated responses in phospholipid metabolism predicts that chronic exposure to sulfur(IV)/sulfite is associated with a considerable health risk.

Topics: Air Pollutants; Animals; Arachidonate 5-Lipoxygenase; Arachidonic Acid; Autoradiography; Carbon Radioisotopes; Cells, Cultured; Chromatography, Thin Layer; Dinoprostone; Dogs; Enzyme Activation; Fatty Acids, Unsaturated; Hydrogen-Ion Concentration; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Macrophages, Alveolar; Phospholipases A; Platelet Activating Factor; Prostaglandin-Endoperoxide Synthases; Sulfates; Sulfites; Sulfur; Thromboxane B2

Platelets of patients with uremia develop a defective platelet function and have a decreased production of thromboxane B2 (TxB2). Activated platelets generate thromboxane from free arachidonate that is previously released from the membrane phospholipids (PLs) by phospholipases. Phospholipase A2 (PLA2) release up to 70% of the arachidonate in normal platelets, and to date, the activity of this enzyme in uremia is unknown. This work studied the PLA2 activity in the platelets of nine uremic patients and nine healthy volunteers. Washed platelets were labelled with [(14)C]arachidonic acid and activated with calcium ionophore A-23187 (4 microgr/ml). Lipids were resolved by TLC and identified by autoradiography. The distribution of [(14)C]arachidonic acid in the five major platelet phospholipids was found to be normal. Uremic platelets released more radioactivity than normal platelets (19.0 +/- 5.2% versus 11.3 +/- 1.6%, P = 0.001). The production of both, radioactive thromboxane B2 and hydroxyheptadecatrienoic acid was normal (2.6 +/- 1.2% and 3.5 +/- 1.6% of total radioactivity respectively), but the formation of the lipoxygenase metabolite hydroxyeicosatetraenoic acid was increased with respect to the controls (12.9 +/- 4.6% vs 7.0 +/- 1.3% of total radioactivity, P = 0002). In conclusion, platelets of patients with uremia have an increased activity of phospholipase A2 and produce increased amounts of hydroxyeicosatetraenoic acid, an inhibitor of the platelet function.

Topics: Arachidonic Acid; Blood Platelets; Calcimycin; Case-Control Studies; Fatty Acids, Unsaturated; Humans; Kidney Failure, Chronic; Phospholipases A; Phospholipases A2; Platelet Function Tests; Thromboxane B2; Uremia

Endothelial cells secrete large amounts of 5,6,7,8-tetrahydrobiopterin (BH(4)) in septic conditions. BH(4) is a cofactor for nitric oxide (NO) synthase and an essential regulator of its activity. We recently showed that NO can be a modulator of both platelet 12-lipoxygenase and cyclooxygenase activities. In the present study, we investigated the effect of BH(4) on the activities of 12-lipoxygenase and cyclooxygenase in rabbit platelets. The influence of BH(4) on NO-induced modulation of these enzyme activities was investigated. Exogenous BH(4) did not affect platelet 12-lipoxygenase and cyclooxygenase activities. The modulatory effects of NO on the two enzymatic pathways were reversed by addition of BH(4) but not by reduced glutathione. These results suggest that exogenous BH(4) is not essential for NO synthase activity of platelets, but that it is an important regulator of the action of NO released from other sources on platelet 12-lipoxygenase and cyclooxygenase activities.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonate 12-Lipoxygenase; Arachidonic Acids; Biopterins; Blood Platelets; Cyclooxygenase Inhibitors; Fatty Acids, Unsaturated; Glutathione; Lipoxygenase Inhibitors; Nitric Oxide; Nitric Oxide Donors; Prostaglandin-Endoperoxide Synthases; Rabbits; Thromboxane B2; Triazenes

Agglomerates of ultrafine particles (AUFPs) may cause adverse health effects because of their large surface area. To evaluate physiologic responses of immune cells, we studied whether agglomerates of 77-nm elemental carbon [(EC); specific surface area 750 m2/g] and 21 nm titanium dioxide (TiO(2) particles (specific surface area 50 m(2)/g) affect the release of lipid mediators by alveolar macrophages (AMs). After 60-min incubation with 1 microg/mL AUFP-EC (corresponding to 7.5 cm(2) particle surface area), canine AMs (1 x 10(6) cells/mL) released arachidonic acid (AA) and the cyclooxygenase (COX) products prostaglandin E(2) (PGE(2), thromboxane B(2), and 12-hydroxyheptadecatrienoic acid but not 5-lipoxygenase (5-LO) products. AUFP-TiO(2) with a 10-fold higher mass (10 microg/mL) than AUFP-EC, but a similar particle surface area (5 cm(2) also induced AMs to release AA and COX products. Agglomerates of 250 nm TiO(2) particles (specific surface area 6.5 m(2)/g) at 100 microg/mL mass concentration (particle surface area 6.5 cm(2) showed the same response. Interestingly, 75 cm(2)/mL surface area of AUFP-EC and 16 cm(2)/mL surface area of AUFP-TiO(2) additionally induced the release of the 5-LO products leukotriene B(4) and 5-hydroxyeicosatetraenoic acid. Respiratory burst activity of stimulated canine neutrophils was partially suppressed by supernatants of AMs treated with various mass concentrations of the three types of particles. Inhibition of neutrophil activity was abolished by supernatants of AMs treated with COX inhibitors prior to AUFP-incubation. This indicates that anti-inflammatory properties of PGE(2) dominate the overall response of lipid mediators released by AUFP-affected AMs. In conclusion, our data indicate that surface area rather than mass concentration determines the effect of AUFPs, and that activation of phospholipase A(subscript)2(/subscript) and COX pathway occurs at a lower particle surface area than that of 5-LO-pathway. We hypothesize a protective role of PGE(2) in downregulating potential inflammatory reactions induced by ultrafine particles.

Topics: Air Pollutants; Animals; Arachidonic Acid; Carbon; Dinoprostone; Dogs; Fatty Acids, Unsaturated; Macrophages, Alveolar; Particle Size; Thromboxane B2; Titanium

Morphological and functional alterations of platelets in migraineurs may be linked to the development of migraine. We examined the eicosanoid synthesis of platelets of untreated female migraineurs in a headache-free period and compared it to that of age- and blood group-matched healthy female volunteers. In the platelets of headache-free migraineurs significantly less amounts of anti-aggregatory prostaglandin D2 and prostacyclin, as well as of 12-L-hydroxy-5,8,10-heptadecatrienoic acid (a potent endogenous inducer of endothelial prostacyclin production) were produced, while the synthesis of platelet aggregatory thromboxane did not differ when compared to that of healthy women. These results suggest that the platelet eicosanoids of migraineurs in the headache-free period might promote the development of cellular, vascular and neurological events inducing headache.

Topics: Adult; Arachidonic Acid; Blood Platelets; Case-Control Studies; Eicosanoids; Epoprostenol; Fatty Acids, Unsaturated; Female; Humans; Lipoxygenase; Middle Aged; Migraine Disorders; Prostaglandin D2; Prostaglandin-Endoperoxide Synthases; Thromboxane B2

The effect of sphingomyelin on the formation of 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE), thromboxane B2 and 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) in washed rabbit platelets was examined. Sphingomyelin had a powerful inhibitory effect on 12-HETE formation, while it produced only a small increase in thromboxane B2 and HHT formation. The sphingomyelin metabolite ceramide did not affect the formation of 12-HETE, thromboxane B2 and HHT. These results suggest that sphingomyelin is a selective inhibitor of platelet 12-lipoxygenase and may have functional effects in platelets.

Topics: Animals; Arachidonate 12-Lipoxygenase; Arachidonic Acid; Blood Platelets; Ceramides; Fatty Acids, Unsaturated; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Male; Rabbits; Sphingomyelins; Thromboxane B2

The effects of a new type of nitric oxide (NO)-releasing compound, 1-hydroxyl-2-oxo-3-(N-methyl-3-aminopropyl)-3-methyl-1-triazene (NOC7), and peroxynitrite (ONOO-) on the formation of 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE), thromboxane (TX) B2 and 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) from exogenous arachidonic acid in washed rabbit platelets have been compared. At concentrations of 5 microM and below, NOC7 inhibited 12-HETE formation (56.5-98.8% inhibition). Moreover, NOC7 inhibited TXB2 and HHT formation at concentrations ranging from 5 to 20 microM (TXB2, 62.2-88.1% inhibition; HHT, 11.6-62.2% inhibition). ONOO- had little or no effect on the production of these three metabolites at concentrations of up to 50 microM. Experiments utilizing a new class of NO antidote, carboxy-2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide, revealed that the observed effects of NOC7 are caused by NO. The effects of NO were reversed by addition of the superoxide generating system (xanthine plus xanthine oxidase and catalase), indicating that superoxide is a vital modulator of the action of NO. These results suggest that NO, but not ONOO- (up to 50 microM), can be a potent dual inhibitor of the 12-lipoxygenase and cyclooxygenase activities in platelets and that superoxide is an important regulator of the action of NO.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonate 12-Lipoxygenase; Arachidonic Acid; Blood Platelets; Catalase; Cyclooxygenase Inhibitors; Fatty Acids, Unsaturated; Lipoxygenase Inhibitors; Male; Nitrates; Nitric Oxide; Prostaglandin-Endoperoxide Synthases; Rabbits; Superoxides; Thromboxane B2; Triazenes; Xanthine; Xanthine Oxidase

There is mounting evidence that lipid peroxides contribute to pathophysiological processes and can modulate cellular functions. The aim of the present study was to investigate the effects of lipid hydroperoxides on platelet aggregation and arachidonic acid (AA) metabolism. Human platelets, isolated from plasma, were incubated with subthreshold (i.e. non-aggregating) concentrations of AA in the absence or presence of hydroperoxyeicosatetraenoic acids (HPETEs). Although HPETEs alone had no effect on platelet function, HPETEs induced the aggregation of platelets co-incubated with non-aggregating concentrations of AA, HPETEs being more potent than non-eicosanoid peroxides. The priming effect of HPETEs on platelet aggregation was associated with an increased formation of cyclo-oxygenase metabolites, in particular thromboxane A2, and was abolished by aspirin, suggesting an activation of cyclo-oxygenase by HPETEs. It was not receptor-mediated because the 12-HPETE-induced enhancement of AA metabolism was sustained in the presence of SQ29, 548 or RGDS, which blocked the aggregation. These results indicate that physiologically relevant concentrations of HPETEs potentiate platelet aggregation, which appears to be mediated via a stimulation of cyclo-oxygenase activity.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonic Acid; Aspirin; Blood Platelets; Bridged Bicyclo Compounds, Heterocyclic; Deferoxamine; Enzyme Activation; Fatty Acids, Unsaturated; Humans; Hydrazines; Hydrogen Peroxide; Leukotrienes; Lipid Peroxides; Oligopeptides; Peroxides; Platelet Aggregation; Prostaglandin-Endoperoxide Synthases; tert-Butylhydroperoxide; Thromboxane B2; Vitamin E

The effect of 15-hydroperoxy-5,8,11,13,15-eicosapentaenoic acid (15-HPEPE), a hydroperoxy adduct of eicosapentaenoic acid (EPA), on the formation of 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE), thromboxane (TX) B2 and 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) from exogenous arachidonic acid in washed rabbit platelets was examined. 15-HPEPE inhibited 12-HETE, TXB2 and HHT formation at concentrations ranging from 2 to 8 microM. The inhibitory effect of 15-HPEPE was dose-dependent (12-HETE, 16.0-82.9% inhibition; TXB2, 16.7-57.2% inhibition; HHT, 4.6-52.0% inhibition). EPA inhibited the production of these three metabolites, but the inhibitory effect was kept low (20-100 microM: 12-HETE, 8.3-31.1% inhibition; TXB2, 18.9-49.5% inhibition; HHT, 12.5-41.7% inhibition) as compared with 15-HPEPE. Experiments utilizing 15-hydroxy-5,8,11,13,15-eicosapentaenoic acid and hydroxyl radical scavengers (dimethyl sulfoxide and mannitol) revealed that 15-HPEPE exerted its effect in the form of the hydroperoxy adduct. These results suggest that 15-HPEPE has the potential to modulate the activities of the cyclo-oxygenase and 12-lipoxygenase in platelets. This may also be one convincing mechanism for the anti-thrombotic and anti-atherosclerotic actions of EPA.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acid; Blood Platelets; Eicosapentaenoic Acid; Fatty Acids, Unsaturated; Free Radical Scavengers; Hydroxyeicosatetraenoic Acids; Hydroxyl Radical; Lipoxygenase; Male; Prostaglandin-Endoperoxide Synthases; Rabbits; Thromboxane B2

The addition of a trans isomer of arachidonic acid (20:4 delta 14trans) to rat platelet suspensions inhibited the aggregation induced by 7.5 microM of arachidonic acid. This inhibitory effect of 20:4 delta 14trans was significant at concentrations of 7.5-22.5 microM and the range of inhibition was 20% at an inhibitor/substrate ratio (I/S) 1 to 66% when I/S reached 3. However, the addition of its structural homolog (20:3n-9) or the natural isomer (20:4n-6) did not induce any modification of the platelet aggregation. In parallel, adding 20:4 delta 14trans to the platelet significantly decreased thromboxane B2 and 12-hydroxyheptadecatrienoic acid production. In contrast, the 12-lipoxygenase pathway was stimulated, as 12-hydroxyeicosatetraenoic acid production increased up to 55% when the I/S reached 3. 20:3n-9, not being a substrate of the cyclooxygenase, did not induce any significant modification in the formation of thromboxane B2 and 12-hydroxyheptadecatrienoic acid. 20:4 delta 14t alone did not induce any platelet aggregation. However, this fatty acid was metabolized to a limited extent into two products that have still to be identified. One of them would be a product of the 12-lipoxygenase pathway.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonate 12-Lipoxygenase; Arachidonic Acid; Eicosanoids; Fatty Acids, Unsaturated; Leukotrienes; Male; Platelet Aggregation; Platelet Aggregation Inhibitors; Rats; Rats, Wistar; Stereoisomerism; Thromboxane B2

The effects of fatty acyl coenzyme A (CoA) esters (palmitoyl-, stearoyl-, oleoyl-, linoleoyl- and arachidonoyl--CoA) on the activities of lipoxygenase and cyclooxygenase in rabbit platelets were examined. Palmitoyl-, stearoyl-, oleoyl- and linoleoyl- CoA were potent inhibitors of platelet lipoxygenase activity. In addition to the lipoxygenase, the four fatty acyl-CoA esters elicited inhibitory activity on platelet cyclooxygenase, although the inhibition was a little weaker. The CoA derivative of the icosanoid precursor arachidonic acid (AA) showed little inhibition on lipoxygenase and cyclooxygenase. Palmitic, stearic and oleic acids had little or no effect on lipoxygenase and cyclooxygenase, in contrast with their CoA derivatives. Linoleic acid was more potent than linoleoyl-CoA as an inhibitor of the cyclooxygenase, but it was a weak inhibitor of the lipoxygenase. These results suggest that the CoA derivatives of palmitic, stearic, oleic and linoleic acids have the potential to modulate both platelet lipoxygenase and cyclooxygenase activities and may have functional effects within platelets.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Acyl Coenzyme A; Animals; Arachidonic Acid; Blood Platelets; Cyclooxygenase Inhibitors; Esters; Fatty Acids, Unsaturated; Hydroxyeicosatetraenoic Acids; Linoleic Acid; Linoleic Acids; Lipoxygenase; Lipoxygenase Inhibitors; Oleic Acid; Oleic Acids; Palmitic Acid; Palmitic Acids; Prostaglandin-Endoperoxide Synthases; Rabbits; Stearic Acids; Thromboxane B2

The effects of high density lipoprotein (HDL) and low density lipoprotein (LDL) on the formation of 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE), thromboxane (TX) B2 and 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) in washed rabbit platelets were examined. HDL had a powerful inhibitory effect on 12-HETE formation, while it produced only a small increase in TXB2 and HHT formation. LDL did not affect the formation of 12-HETE, TXB2 and HHT. These results suggest that HDL is a selective inhibitor of platelet 12-lipoxygenase and may play a protective role in atherogenesis by preventing the generation of 12-HETE.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Blood Platelets; Fatty Acids, Unsaturated; Hydroxyeicosatetraenoic Acids; Lipoproteins, HDL; Lipoproteins, LDL; Lipoxygenase Inhibitors; Rabbits; Thromboxane B2

Arachidonic acid (AA) metabolism in nuclei of human pro-myelocytic leukemia (HL-60) cells was investigated during retinoic acid (RA)-induced granulocytic differentiation and 1 alpha, 25 dihydroxy-vitamin D3-induced monocytic differentiation. The whole control HL-60 cells and their nuclei hardly converted [1-14C]-AA to any metabolites comigrating with authentic prostaglandins (PGs). On the other hand, RA-treated HL-60 cells acquired the ability to convert [1-14C]-AA to PGE2 predominantly and thromboxane B2 (TXB2) to a small degree, whereas the nuclei of the differentiated cells acquired the ability to convert predominantly to TXB2. In contrast, 1 alpha, 25-dihydroxy-vitamin D3-treated HL-60 cells acquired the ability to convert [1-14C]-AA to PGE2, PGF2 alpha, TXB2 and 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT), whereas the nuclei of the differentiated cells acquired the ability to convert to PGF2 alpha, TXB2 and HHT. The significance of the acquisition of cyclooxygenase and TX synthetase by the nucleus is unclear, but there may be a specific relationship between the specific PGs formed by the nuclear membrane and nuclear events during HL-60 cell differentiation.

Topics: Arachidonic Acid; Calcitriol; Cell Differentiation; Cell Nucleus; Dinoprost; Dinoprostone; Fatty Acids, Unsaturated; Granulocytes; Humans; Leukemia, Promyelocytic, Acute; Monocytes; Thromboxane B2; Tretinoin; Tumor Cells, Cultured

Effects of three sulfonylurea agents on arachidonic acid metabolism of platelet homogenates were evaluated using HPLC. Gliclazide had no significant inhibitory effects on arachidonic acid metabolism. Glibenclamide and glimepiride both inhibited the production of cyclooxygenase-related metabolites, thromboxane B2 (TXB2) and 12-hydroxy 5,8,10-heptadecatrienoic acid (HHT), whereas 12-hydroxy 5,8,10,14-eicosatetraenoic acid (12-HETE), a 12-lipoxygenase-related product, was unaffected. These findings confirmed part of our previous report using intact platelets, except that we found in the present study that glibenclamide had no inhibitory effect on 12-lipoxygenase.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonic Acid; Blood Platelets; Chromatography, High Pressure Liquid; Fatty Acids, Unsaturated; Gliclazide; Humans; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Sulfonylurea Compounds; Thromboxane B2

1. The effect of 13-hydroperoxy-9,11-octadecadienoic acid (13-HPODE) on the formation of thromboxane (TX) B2, 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) from exogenous arachidonic acid in washed rabbit platelets was examined. 2. 13-HPODE inhibited TXB2 and HHT formation without affecting 12-HETE production. 3. 13-Hydroxy-9,11-octadecadienoic acid which was produced rapidly from 13-HPODE, did not suppress the formation of TXB2 and HHT, indicating the requirement of the hydroperoxy moiety for the inhibitory effect of 13-HPODE on TXB2 and HHT formation. 4. Experiments utilizing mannitol and dimethyl sulfoxide (hydroxy radical scavengers) revealed that the action of 13-HPODE is not due to hydroxy radicals which are expected to be formed from 13-HPODE. 5. These results suggest that 13-HPODE is a selective inhibitor of platelet cyclo-oxygenase and may have functional effects within platelets.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acid; Blood Platelets; Fatty Acids, Unsaturated; Hydroxyeicosatetraenoic Acids; Linoleic Acids; Lipid Peroxides; Rabbits; Thromboxane B2

Rabbit platelets were exposed to a reactive oxygen species (ROS) generating system (xanthine plus xanthine oxidase) to explore the effect of ROS on the formation of 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE), thromboxane (TX) B2, and 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) from exogenous arachidonic acid. Xanthine plus xanthine oxidase suppressed the production of 12-HETE, TXB2, and HHT by 65-69%. This effect was reversed by addition of catalase to the ROS generating system but not by superoxide dismutase, mannitol, or dimethylsulfoxide, indicating that H2O2 is the responsible metabolite. These results suggest that H2O2 plays an important role in the regulation of platelet cyclo-oxygenase and lipoxygenase activities.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acid; Blood Platelets; Catalase; Fatty Acids, Unsaturated; Hydrogen Peroxide; Hydroxyeicosatetraenoic Acids; Male; Rabbits; Reactive Oxygen Species; Superoxide Dismutase; Thromboxane B2; Xanthine; Xanthine Oxidase; Xanthines

Sipid mediators of inflammation have been implicated in the pathogenesis of Pseudomonas aeruginosa (PA) related pulmonary damage in patients with cystic fibrosis. We studied the role of these mediators in a rat model of PA endobronchitis using essential fatty acid deficient (EFAD) animals. Whole blood from EFAD animals produced significantly less leukotriene B4 (LTB4) and hydroxyheptadecatrienoic acid when stimulated ex vivo than did whole blood from control animals (p less than 0.005). Similarly, lung lavage fluid from EFAD animals infected with PA contained less LTB4 and thromboxane B2 (TXB2) than that from control animals. Despite these differences, cellular infiltration of airways in response to PA infection was virtually identical in animals from the regular diet and the EFAD groups. Both EFAD and control animals had a significant increase in white blood cells (WBC) in lung lavage fluid at 1, 3 and 6 days following infection with PA when compared to animals receiving sterile beads. Localized areas of consolidation and nodularity were grossly evident in the lungs of all PA infected animals irrespective of their ability to generate the lipid inflammatory mediators. Microscopic examination of lung sections demonstrated similar changes in all infected animals. We conclude that LTB4 and TXB2 production occurs early in the course of PA pulmonary infection in rats. This early rise in lipid mediators is temporally associated with an influx of WBC into the airways. However, attenuation of eicosanoid production by use of an EFAD diet does not lead to a reduction in the inflammatory response to PA infection.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Bronchoalveolar Lavage Fluid; Dietary Fats, Unsaturated; Eicosanoids; Fatty Acids, Essential; Fatty Acids, Unsaturated; Inflammation; Leukotriene B4; Male; Pneumonia; Pseudomonas Infections; Rats; Rats, Inbred Strains; Thromboxane B2

Density-defined macrophages isolated from fluids of patients with liver cirrhosis mainly generated the 5-lipoxygenase products leukotriene B4 (LTB4, 16%) and 5-hydroxy-eicosatetraenoic acid (5-HETE, 24%) and the cyclooxygenase products 12-hydroxyheptadecatrienoic acid (HHT, 22%) and thromboxane B2 (TXB2, 18%). The synthesis of eicosanoids was linear with the maturity of the macrophage subpopulations, suggesting that eicosanoid production is increased in in-vivo activated macrophages.

Topics: Eicosanoids; Fatty Acids, Unsaturated; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Lipoxygenase; Liver Cirrhosis; Macrophages; Peritoneal Cavity; Prostaglandin-Endoperoxide Synthases; Thromboxane B2

The effects of a new chalcone derivative, xanthoangelol E, isolated from Angelica keiskei Koidzumi, on arachidonic acid metabolism in the gastric antral mucosa and platelet of the rabbit were examined. When gastric antral mucosal slices were incubated with xanthoangelol E (0.05-1.0 mM), there was no significant effect on the production of prostaglandin (PG) E2, PGF2 alpha and their metabolites. On the other hand, this compound inhibited effectively the production of thromboxane B2 and 12-hydroxy-5,8,10-heptadecatrienoic acid from exogenous arachidonic acid in platelets, and the concentration required for 50% inhibition (IC50) was approximately 5 microM. The formation of 12-hydroxy-5,8,10,14-eicosatetraenoic acid was also reduced by this drug (IC50, 50 microM). These results suggest that xanthoangelol E has the potential to modulate arachidonic acid metabolism in platelets and that this action may participate in some pharmacological effect of the plant.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Adenosine Triphosphatases; Animals; Arachidonic Acid; Blood Platelets; Chalcone; Fatty Acids, Unsaturated; Gastric Mucosa; Hydroxyeicosatetraenoic Acids; Male; Pyloric Antrum; Rabbits; Thromboxane B2

The effect of tert-butyl hydroperoxide (t-BOOH) on the formation of thromboxane (TX) B2, 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) from exogenous arachidonic acid (AA) in washed rabbit platelets was examined. t-BOOH enhanced TXB2 and HHT formation at concentrations of 8 microM and below, and at 50 microM it inhibited the formation, suggesting that platelet cyclooxygenase activity can be enhanced or inhibited by t-BOOH depending on the concentration. t-BOOH inhibited 12-HETE production in a dose-dependent manner. When the platelets were incubated with 12-hydroperoxy-5,8,10,14-eicosatetraenoic acid (12-HPETE) instead of AA, t-BOOH failed to inhibit the conversion of 12-HPETE to 12-HETE, indicating that the inhibition of 12-HETE formation by t-BOOH occurs at the lipoxygenase step. Studies utilizing indomethacin (a selective cyclooxygenase inhibitor) and desferrioxamine (an iron-chelating agent) revealed that the inhibitory effect of t-BOOH on the lipoxygenase is not mediated through the activation of the cyclooxygenase and that this effect of t-BOOH is due to the hydroperoxy moiety. These results suggest that hydroperoxides play an important role in the control of platelet cyclooxygenase and lipoxygenase activities.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acid; Blood Platelets; Deferoxamine; Dose-Response Relationship, Drug; Fatty Acids, Unsaturated; Ferrous Compounds; Hydroxyeicosatetraenoic Acids; Indomethacin; Leukotrienes; Lipoxygenase; Male; Oxidants; Peroxides; Platelet Aggregation Inhibitors; Prostaglandin-Endoperoxide Synthases; Quercetin; Rabbits; tert-Butylhydroperoxide; Thromboxane B2

The mechanism was studied by which isoliquiritigenin, a new aldose reductase inhibitor purified from licorice (Glycyrrhizae radix), inhibits platelet aggregation. This new agent significantly inhibited the phosphorylation of 40,000- and 20,000-dalton proteins, and inhibited the formation of 12 (S)-hydroxy-5,8,10-heptadecatrienoic acid, 12-hydroxyeicosatetraenoic acid and thromboxane B2. The inhibitory effect of isoliquiritigenin on platelet aggregation in vitro was comparable to that of aspirin. Our findings may indicate that isoliquiritigenin elicits an anti-platelet action by inhibiting not only cyclooxygenase but also lipoxygenase or peroxidase activity in platelets. Isoliquiritigenin also showed an anti-platelet action in vivo. Isoliquiritigenin appears to be the only aldose reductase inhibitor with a significant anti-platelet action. Since the hyperaggregability of platelets has been implicated in the pathogenesis of diabetic complications, isoliquiritigenin may offer a unique benefit as an aldose reductase inhibitor.

Topics: Aldehyde Reductase; Blood Platelets; Blood Proteins; Chalcone; Chalcones; Fatty Acids, Unsaturated; Glycyrrhiza; Humans; Hydroxyeicosatetraenoic Acids; Molecular Weight; Phosphorylation; Plants, Medicinal; Platelet Aggregation Inhibitors; Thromboxane B2

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acid; Arachidonic Acids; Chromatography, High Pressure Liquid; Dinoprostone; Fatty Acids, Unsaturated; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Lipoxygenase; Male; Neutrophils; Peritoneal Cavity; Prostaglandin-Endoperoxide Synthases; Rats; Rats, Inbred Lew; Thromboxane B2

An in vitro system designed to mimic the effect of various plasma nonesterified (polyunsaturated) fatty acids on platelet function and metabolism was employed. Human platelet aggregation induced by submaximal (1.8 micrograms/ml) collagen stimulation was significantly inhibited by 2 min preincubation with 20 microM albumin-bound docosahexaenoic acid (22:6n-3) (DHA), but not by the other fatty acids tested. [3H]Phosphatidic acid (PA) formation, an indicator of phospholipase C activation following platelet stimulation, was moderately inhibited by eicosapentaenoic acid (20:5n-3), 11,14,17-eicosatrienoic acid (20:3n-3), dihomo-gamma-linolenic acid (20:3n-6), as well as DHA, but not by arachidonic acid (20:4n-6); this inhibition of phospholipase C activation could not explain the differential effect of DHA on platelet aggregation. The decreased production of thromboxane A2 (TxA2), as assessed by [3H]12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) formation, may account for the inhibition of collagen-induced aggregation by 20 microM DHA. Surprisingly, preincubation with 40 microM albumin-bound DHA, even though resulting in greater inhibition of collagen-induced aggregation, had less impact on HHT formation. A small but significant increase in [3H]prostaglandin D2 (PGD2) levels following 3-min collagen stimulation may have contributed to the greater antiaggregatory effect of 40 muM DHA. It is concluded that increased plasma nonesterified DHA may contribute to the dampened platelet activation and altered metabolism following fish oil supplementation of the diet.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Blood Platelets; Collagen; Docosahexaenoic Acids; Eicosapentaenoic Acid; Enzyme Activation; Fatty Acids, Nonesterified; Fatty Acids, Unsaturated; Humans; Hydroxyeicosatetraenoic Acids; Phosphatidic Acids; Platelet Aggregation; Platelet Aggregation Inhibitors; Prostaglandin D2; Serum Albumin; Thromboxane A2; Thromboxane B2; Type C Phospholipases

N-3 trans geometrical isomers of 20:5 n-3 and 22:6 n-3 were isolated from rats fed heated linseed oil. The ability of these acids to inhibit 20:4 n-6 metabolism by human platelets was examined. The concentrations required to inhibit 50% of platelet aggregation (IC50) induced by 2.5 microM 20:4 n-6 were higher for the 20:5 delta 17t isomer compared to all cis 20:5 n-3; means 29.2 and 7.6 microM, respectively (P less than 0.05). There were no significant differences in IC50 between 22:6 delta 19t and all cis 22:6 n-3; means 4.3 and 5.6 microM, respectively (P greater than 0.05). Inhibition of action of cyclooxygenase on 20:4 n-6 was similar for 20:5 delta 17t and 20:5 n-3 when examined at their IC50s, but comparison at equal concentrations indicated that 20:5 n-3 was a significantly better inhibitor (P less than 0.05). The ability to inhibit platelet aggregation was paralleled by cyclooxygenase inhibition as determined by thromboxane B2 and 12-hydroxyheptadecatrienoic acid formation. 22:6 delta 19t appeared to inhibit cyclooxygenase more completely than 22:6 n-3, examined at their IC50s or at similar concentrations (P less than 0.05). Isomers of 20:5 n-3 and 22:6 n-3 having an n-3 cis or trans bond appear to have similar modes of action, although levels required for effectiveness are different for the C20 acids.

Topics: Arachidonic Acids; Blood Platelets; Chromatography, High Pressure Liquid; Docosahexaenoic Acids; Eicosapentaenoic Acid; Fatty Acids, Unsaturated; Humans; Platelet Aggregation; Stereoisomerism; Thromboxane B2

The effects of dauricine (Dau), an isoquinoline alkaloid and anti-arrhythmic agent used in China recently, on the biosynthesis of metabolites of arachidonic acid in rat pleural neutrophils, comparing with dazoxiben, indomethacin and BW-755 c, were studied. The major products of metabolism by 5-lipoxygenase (5-LPO), measured by HPLC, were LTB4 and 5-HETE, whereas the major cyclooxygenase products measured by HPLC and RIA, were HHT and TXB2. The formation of all products by neutrophils was significantly depressed by Dau in a dose-dependent manner. The concentration of Dau required to obtain 50% inhibition (IC50) of formation of HHT, TXB2, LTB4 and 5-HETE was 25.3, 59.3, 31.8 and 59.5 mumol/L, respectively. These results indicate that the two major metabolic pathways of arachidonic acid in rat pleural neutrophils are inhibited by Dau.

Topics: Alkaloids; Animals; Arachidonic Acids; Benzylisoquinolines; Dose-Response Relationship, Drug; Fatty Acids, Unsaturated; Hydroxyeicosatetraenoic Acids; Isoquinolines; Leukotriene B4; Male; Neutrophils; Rats; Rats, Inbred Strains; Tetrahydroisoquinolines; Thromboxane B2

Dauricine (Dau), an isoquinoline alkaloid extracted from the roots of Menispermum dauricum D. C. and used as an antiarrhythmic agent in China recently, was shown to inhibit rat platelet aggregation induced by arachidonic acid (AA) and ADP, as well as human platelet aggregation induced by AA, ADP and adrenaline (Adr) in vitro in a dose-dependent manner. The concentration of Dau required for 50% inhibition (IC50) of rat platelet aggregation induced by AA and ADP was 26 and 37 mumol/L, respectively. For human platelet aggregation induced by AA, ADP and Adr the IC50 of Dau was found to be 39, 55 and 43 mumol/L, respectively. Dau inhibited the cyclooxygenase pathway metabolites of AA (TXB2 and HHT) in washed intact rat platelets. The production of TXB2 and HHT was reduced by 26% and 19%, respectively, when the Dau concentration was 50 mumol/L and by 46 and 45%, respectively, when the concentration of Dau was 100 mumol/L. The formation of 12-HETE was also inhibited at 100 mumol/L of Dau. The inhibitory effect of Dau on AA metabolism may be one of the mechanisms related to its inhibition of platelet aggregation.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Alkaloids; Animals; Arachidonic Acids; Benzylisoquinolines; Fatty Acids, Unsaturated; Hydroxyeicosatetraenoic Acids; Isoquinolines; Male; Platelet Aggregation; Platelet Aggregation Inhibitors; Rats; Rats, Inbred Strains; Tetrahydroisoquinolines; Thromboxane B2

In previous studies on arachidonic acid (AA) metabolism by pulmonary macrophages in vitro, we observed that the presence of serum in the culture medium influenced the profile of AA metabolites released. To further characterize this phenomenon, rat alveolar macrophages were placed in plastic tissue culture dishes and allowed to adhere in the presence or absence of 7.5% fetal bovine serum (FBS) for 1 h. Adherent cells were then maintained in medium (equilibration) with or without FBS for 3.5 h before stimulation with the calcium ionophore A23187. The release of thromboxane B2 (TXB2) (the stable metabolite of TXA2) and leukotriene B4 (LTB4) during culture was measured by radioimmunoassay and reverse-phase high pressure liquid chromatography, respectively, at the end of each culture step. Cell adhesion to the plastic substrate in FBS-free medium induced an intense stimulation of AA metabolism, with the release of both TXB2 and LTB4. Adhesion and the accompanying TXB2 release appear to be mediated by trypsin-sensitive components since trypsin-pretreated macrophages showed a dramatic reduction in both adherence and TXB2 synthesis. The presence of FBS during the attachment phase of culture reduced both adhesion and release of TXB2 and LTB4 by more than 50%. On the other hand, addition of FBS to cells that had completed adhesion in serum-free medium stimulated release of both metabolites. When challenged with calcium ionophore after 4.5 h of culture, macrophages that had adhered in FBS-free medium released a much smaller amount of TXB2 than did macrophages that had been cultured in the presence of FBS.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Blood; Calcimycin; Cattle; Cell Adhesion; Cells, Cultured; Fatty Acids, Unsaturated; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Macrophages; Plastics; Pulmonary Alveoli; Rats; Thromboxane B2

Topics: Animals; Arachidonic Acids; Fatty Acids, Unsaturated; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Male; Neutrophils; Pleura; Rats; Rats, Inbred Strains; Solanaceous Alkaloids; Thromboxane B2

Platelet functions were investigated in sixteen old (78-94 years) and eight young (25-35 years) subjects. Whole blood platelet aggregation induced by collagen was higher in the elderly. Similarly, aggregation of platelet rich plasma and plasma-free platelets induced by various agents was increased but the collagen-induced release of ATP was reduced. In agreement with the enhanced platelet aggregability, the increase of thromboxane formation (under thrombin stimulation) was also noted in platelets from elderly people. To further assess platelet and vascular function in vivo, we measured the excretion of urinary TXB2, 2,3-dinor TXB2, 6-keto-PGF1 alpha and 2,3-dinor-6-keto-PGF1 alpha. The four metabolites were all increased in the elder population. In addition, a significant reduction of platelet vitamin E was observed in the elderly people, although the plasma content was normal. These results indicate numerous modifications of platelet behaviour with aging. They include the increased platelet susceptibility to aggregation, and the depletion of ATP granule content, which could reflect an activation in vivo in agreement with the enhanced urinary excretion of thromboxane and prostacyclin metabolites. We hypothesize that platelet hyperactivity associated with the enhanced oxygenated metabolism of arachidonic acid could be linked to vitamin E depletion. These changes may reveal a prethrombotic state in the elderly population.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 6-Ketoprostaglandin F1 alpha; Adult; Aged; Aged, 80 and over; Aging; Arachidonic Acid; Arachidonic Acids; Blood Platelets; Fatty Acids, Unsaturated; Humans; Hydroxyeicosatetraenoic Acids; Platelet Aggregation; Thromboxane B2; Vitamin E

The in vitro effect of trichosanic acid (TCA; C18:3, omega-5), a major component of Trichosanthes japonica, on platelet aggregation and arachidonic acid (AA) metabolism in human platelets was studied. TCA dose-dependently suppressed platelet aggregation of platelet rich plasma and washed platelets. TCA decreased collagen (50 micrograms/ml)-stimulated production of thromboxane B2 (TXB2) and 12-hydroxyhepta-decatrienoic acid (HHT) in a dose-dependent manner, while that of 12-hydroxyeicosatetraenoic acid (12-HETE) was rather enhanced. The conversion of exogenously added [14C]AA to [14C]TXB2 and [14C]HHT in washed platelets was dose-dependently reduced by the addition of TCA, while that to [14C]12-HETE was increased. Similar observations were obtained when linolenic acid (LNA; C18:3, omega-3) was used. These results suggest that TCA may decrease TXA2 formation in platelets, probably due to the inhibition of cyclooxygenase pathway, and thereby reduce platelet aggregation.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonic Acids; Blood Platelets; Chromatography, High Pressure Liquid; Collagen; Fatty Acids, Unsaturated; Humans; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Linolenic Acids; Platelet Aggregation; Platelet Aggregation Inhibitors; Thromboxane B2

Topics: Animals; Arachidonic Acids; Blood Platelets; Drugs, Chinese Herbal; Fatty Acids, Unsaturated; Hydroxyeicosatetraenoic Acids; Rabbits; Thromboxane B2

The endogenous arachidonic acid metabolism was investigated ex vivo, in separated serum from clotted whole blood, soon after the onset of acute myocardial infarction (3.3 +/- 0.7 hr). A group of eight consecutive male patients was selected, since no evidence was obtained of any associated disease known to increase platelet activity or any recent exposure to cyclo-oxygenase inhibitors. This group of patients compared to an age and sex matched control group showed a large decrease in the platelet cyclo-oxygenase end-products in whole blood: thromboxane B2 (TXB2), 12-hydroxy-5-cis, 8-cis, 10-trans-heptadecatrienoic acid (HHT) and 6-keto-PGF1 alpha (p less than .01). In addition, platelet lipoxygenase produced an increased amount of 12-hydroperoxy-5,8,10,14-eicosatetraenoic acid (12-HPETE) as measured by its reduced metabolite 12-HETE (p less than .05). Furthermore, the TXB2 plasma concentration was significantly elevated in patients (p less than .01), confirming the enhanced platelet reactivity during the early stages of acute myocardial infarction. These results point out that a decreased level of cyclo-oxygenase end-products and an increased level of lipoxygenase end-product in serum is consistent with a previous in vivo cyclo-oxygenase hyperactivity.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 6-Ketoprostaglandin F1 alpha; Arachidonic Acid; Arachidonic Acids; Blood Platelets; Fatty Acids, Unsaturated; Humans; Hydroxyeicosatetraenoic Acids; Lipoxygenase; Male; Middle Aged; Myocardial Infarction; Prostaglandin-Endoperoxide Synthases; Thromboxane B2

Although HHT accounts for approximately one third of the arachidonic acid (AA) metabolites produced by stimulated platelets, no well defined function has been attributed to this product. We report that HHT stimulates prostacyclin production by endothelial cells, and have identified the mechanism for this effect. In human umbilical venous endothelial cells, HHT (0.5 and 1 microM) stimulated prostacyclin (RIA for 6KPGF1 alpha) by 32 +/- 22% (1SD) and 42 +/- 38% (P less than 0.05 and less than 0.01). Similar changes were observed when the effect of HHT on exogenous [1-14C] AA metabolism in fetal bovine aortic endothelial cells (FBAECs) was studied. Kinetic analyses revealed that HHT affected vascular cyclooxygenase. HHT (1 microM) increased Vmax in test microsomes (706 +/- 21 pmol/mg/min, mean +/- 1SE) when compared to controls (529 +/- 20; P less than 0.02). No concomitant effect on Km was observed. A further effect of HHT on AA release from endothelial cell membrane phospholipids was noted. Prelabeling experiments revealed that HHT (1 microM) increased the ionophore stimulated release of AA from FBAECs (20952 +/- 555 cpm/well control mean +/- 1SE vs 25848 +/- 557 for paired HHT treated cells; P less than 0.05). The effect of HHT on platelet AA metabolism was next studied. Preincubation of washed platelets with HHT (1 microM) did not enhance thrombin or arachidonic acid induced platelet TXB2 formation. In platelets prelabelled with [1-14C]AA, HHT (1 microM) had no effect on AA release post thrombin stimulation. Conversion to cyclooxygenase metabolites was also not enhanced. HHT stimulates vascular prostacyclin without a concomitant effect on platelet AA metabolism. HHT may thus be an important local modulator of platelet plug formation.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Blood Platelets; Cattle; Endothelium, Vascular; Epoprostenol; Fatty Acids, Unsaturated; Hemostasis; Humans; In Vitro Techniques; Kinetics; Thrombin; Thromboxane B2

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acid; Blood Platelets; Chromatography, High Pressure Liquid; Fatty Acids, Unsaturated; Hydroxyeicosatetraenoic Acids; Influenza A virus; Leukotrienes; Male; Mice; Orthomyxoviridae Infections; Thromboxane B2

Washed human platelets prelabeled with [14C]arachidonic acid and then exposed to the Ca2+ ionophore A23187 mobilized [14C]arachidonic acid from phospholipids and formed 14C-labeled thromboxane B2, 12-hydroxy-5-8,10-heptadecatrienoic acid, and 12-hydroxy-5,8,10,14-eicosatetraenoic acid. Addition of phorbol myristate acetate (PMA) by itself at concentrations from 10 to 1000 ng/ml did not release arachidonic acid or cause the formation of any of its metabolites, nor did it affect the metabolism of exogenously added arachidonic acid. When 1 microM A23187 was added to platelets pretreated with 100 ng of PMA/ml for 10 min, the release of arachidonic acid, and the amount of all arachidonic acid metabolites formed, were greatly increased (average 4.1 +/- 0.5-fold in eight experiments). This effect of PMA was mimicked by other stimulators of protein kinase C, such as phorbol dibutyrate and oleoyl acetoyl glycerol, but not by 4-alpha-phorbol 12,13-didecanoate, which does not stimulate protein kinase C. However, phosphorylation of the cytosolic 47-kDa protein, the major substrate for protein kinase C in platelets, was produced at lower concentrations of PMA and at a much higher rate than enhancement of arachidonic acid release by PMA, suggesting that 47-kDa protein phosphorylation is not directly involved in mobilization of the fatty acid. PMA also potentiated arachidonic acid release when stimulation of phospholipase C by the ionophore (which is due to thromboxane A2 and/or secreted ADP) was blocked by aspirin plus ADP scavengers, i.e. apyrase or creatine phosphate/creatine phosphokinase. Increased release of arachidonic acid was attributable to loss of [14C]arachidonic acid primarily from phosphatidylcholine (79%) with lesser amounts derived from phosphatidylinositol (12%) and phosphatidylethanolamine (8%). Phosphatidic acid, whose production is a sensitive indicator of phospholipase C activation, was not formed. Thus, the potentiation of arachidonic acid release by PMA appeared to be due to phospholipase A2 activity. These results suggest that diacylglycerol formed in response to stimulation of platelet receptors by agonists may cooperatively promote release of arachidonic acid via a Ca2+/phospholipase A2-dependent pathway.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonic Acid; Arachidonic Acids; Blood Platelets; Calcimycin; Diglycerides; Enzyme Activation; Fatty Acids, Unsaturated; Glycerides; Humans; Hydroxyeicosatetraenoic Acids; Kinetics; Phorbols; Phospholipases A; Phospholipases A2; Phospholipids; Phosphoproteins; Protein Kinase C; Tetradecanoylphorbol Acetate; Thromboxane B2

The effects of various alpha-phenylcinnamic acid derivatives (i.e., alpha-(3,4-dihydroxyphenyl)cinnamic acid, alpha-(3,4-dihydroxyphenyl)-3-hydroxycinnamic acid, alpha-(3,4-dihydroxyphenyl)-4-hydroxycinnamic acid and alpha-(3,4-dihydroxyphenyl)-3, 4-dihydroxycinnamic acid) synthesized from 3,4-dihydroxyphenyl acetic acid and hydroxy-benzaldehyde, and 3,3',4-trihydroxystilbene obtained by decarboxylation of alpha-(3,4-dihydroxyphenyl)-3-hydroxycinnamic acid on rat peritoneal polymorphonuclear leukocyte lipoxygenase and cyclooxygenase activities were studied. 3,3',4-Trihydroxystilbene was found to inhibit the 5-lipoxygenase product, 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HETE), and cyclooxygenase products, 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and thromboxane B2; its concentrations for 50% inhibition (IC50) were 0.885 +/- 0.016 microM for the leukocyte lipoxygenase product, 5-HETE, 7.70 +/- 0.104 microM for the formations of HHT and 7.96 +/- 0.143 microM for the formation of thromboxane B2. Alpha-(3,4-Dihydroxyphenyl)cinnamic acid, alpha-(3,4-dihydroxyphenyl)-3-hydroxycinnamic acid and alpha-(3,4-dihydroxyphenyl)-3,4-dihydroxycinnamic acid also inhibited the formations of 5-HETE, HHT and thromboxane B2, although less strongly. Their IC50 values were, respectively, 91.3 +/- 3.62 microM, 947.5 +/- 28.7 microM, 453.3 +/- 229.3 microM and 148.8 +/- 50.6 microM for the formation of 5-HETE, 894.0 +/- 5.57 microM, 792.5 +/- 15.9 microM, greater than 1000 microM and 925.0 +/- 7.64 microM for the formation of HHT and 941.0 +/- 18.0 microM, 825 +/- 14.4 microM, greater than 1000 microM and 932.7 +/- 3.93 microM for the formation of thromboxane B2.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Dose-Response Relationship, Drug; Fatty Acids, Unsaturated; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Leukocytes; Rats; Stilbenes; Thromboxane B2

Organic nitrates exert well known vasodilating effects in coronary arteries, although the exact mechanism of action is not fully understood. In this study the influence of these drugs on the biosynthesis of eicosanoids from labelled exogenous arachidonic acid (AA) in isolated platelets was examined. Platelet malondialdehyde (MDA)-formation in the presence of the substances tested in this system was studied too. The vasodilating drugs were found to reduce the conversion rate to TxB2, hydroxy-5,8,10-heptadecatrienoic acid (HHT) and MDA in platelets dose-dependently. Isosorbid-5-mononitrate (ISMN), nitroglycerin (NG) and SIN 1, the active metabolite of molsidomin, showed their inhibitory effects at concentrations of about 1mM, isosorbid-dinitrate (ISDN) had a much weaker effect on platelet-biosynthesis - a concentration of about 3mM was necessary to inhibit TxB2-formation significantly.

Topics: Arachidonic Acid; Arachidonic Acids; Blood Platelets; Fatty Acids, Unsaturated; Humans; In Vitro Techniques; Malondialdehyde; Nitrates; Nitroglycerin; Thromboxane B2; Vasodilator Agents

Control of pituitary hormone secretion by hypothalamic-releasing peptides appears to involve unidentified products of the cyclooxygenase and lipoxygenase pathways, as well as the adenylate cyclase system. To identify the patterns of arachidonic acid metabolism in specific pituitary cell types, the labeled products formed from [14C]-arachidonic acid were analyzed in rat pituitary cells separated by centrifugal elutriation into fractions enriched in gonadotrophs, somatotrophs and lactotrophs. Gonadotroph-enriched cell fractions metabolized arachidonic acid to 11-, 12- and 15-HETE, HHT, PGD2, PGE2 and TXB2. The products were characterized by high performance liquid and thin-layer chromatography, together with gas chromatographic-mass spectrometric identification of 12- and 15-HETE. In cells preincubated with indomethacin, the formation of 11-HETE, HHT, PGD2, PGE2 and TXB2 was markedly reduced. In gonadotroph-enriched cell fractions, the production of cyclooxygenase metabolites was 3 to 4 times greater than that of lipoxygenase products. The somatotroph- and lactotroph-enriched cell fractions produced only very small amounts of oxygenated arachidonic acid metabolites under the conditions studied, but all cell fractions incorporated [14C]-arachidonate into mono-, di- and tri-glycerides, as well as into phospholipids. These results demonstrate the differential capacities of the individual pituitary cell populations for metabolizing arachidonic acid, and emphasize the relative prominence of the oxidation pathways for arachidonate metabolism in the gonadotroph-enriched cell fraction of the rat pituitary gland.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Fatty Acids, Unsaturated; Gonadotropins, Pituitary; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Pituitary Gland; Prostaglandin-Endoperoxide Synthases; Prostaglandins; Rats; Thromboxane B2

The metabolism of exogenous [1-14C]arachidonic acid in neonatal and maternal peripheral mononuclear leukocytes was studied. Both neonatal and maternal leukocytes converted arachidonic acid to hydroxy acids and to prostaglandin E2, but small amounts of PGF2 alpha and thromboxane B2 were also found. In addition a polar arachidonic acid metabolite with conjugated double bonds was identified in the supernatant from both maternal and neonatal leukocytes. This might be a leukotriene, but further attempts at biochemical characterization are necessary in order to confirm this.

Topics: Arachidonic Acid; Arachidonic Acids; Cells, Cultured; Chromatography, Gas; Chromatography, Thin Layer; Dinoprost; Dinoprostone; Fatty Acids, Unsaturated; Female; Fetal Blood; Humans; Infant, Newborn; Lymphocytes; Phytohemagglutinins; Pregnancy; Prostaglandins E; Prostaglandins F; Thromboxane B2

An assessment of arachidonic acid metabolism in the platelet of the neonate was performed. The uptake of [14C]arachidonic acid into platelets of both the neonate and the adult were similar. Neonatal platelets, however, released a significantly greater amount (P less than .001) of prelabeled arachidonic acid (24.7% +/- 2.8%) in response to the physiologic agent thrombin when compared with platelets from adult control subjects (14.6% +/- 0.8%). When the activities of the lipoxygenase (12-L-hydroxy-5,8,10,14-eicosatetraenoic acid) and cyclooxygenase pathways (12-L-hydroxy-5,8,10-heptadecatrienoic acid and thromboxane B2) were evaluated following incubation of platelets with [14C]arachidonic acid, significant differences were observed between adult and neonatal platelets. Platelets from the neonate produced less (P less than .01) thromboxane B2 (11.1% +/- 1.7%) when compared with platelets from adult control subjects (19% +/- 1.7%). In contrast, the lipoxygenase product 12-L-hydroxy-5,8,10,14-eicostatetraenoic acid was increased (P less than .005) in the platelet from the neonate (41.5% +/- 2%), when compared with the adult (31.2% +/- 2.1%). The observation that the availability of substrate arachidonic acid is increased in the platelet of the neonate may have general implications in neonatal pathophysiologic processes.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Adult; Aging; Arachidonic Acid; Arachidonic Acids; Blood Platelets; Fatty Acids, Unsaturated; Humans; Hydroxy Acids; In Vitro Techniques; Infant, Newborn; Lipoxygenase; Prostaglandin-Endoperoxide Synthases; Thromboxane B2

Feverfew has been used since antiquity to treat fevers and other inflammatory conditions. Feverfew extracts were found to inhibit ADP, thrombin, or collagen-induced aggregation of human platelets, but significantly, did not affect aggregation induced by arachidonic acid. Synthesis of thromboxane B2 from exogenous 14C-arachidonic acid was also not inhibited. Washed platelets prelabelled with 14C-AA responded normally to thrombin by releasing 14C-TXB2. This was completely blocked by feverfew. A purified platelet phospholipase A2 was inhibited by the material with an I50 of 0.1 antiplatelet units. The pharmacological properties of feverfew may thus be due to an inhibitor of cellular phospholipases, which prevents release of arachidonic acid in response to appropriate physiological stimuli.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Adenosine Diphosphate; Arachidonic Acid; Arachidonic Acids; Blood Platelets; Collagen; Fatty Acids, Unsaturated; Humans; Hydroxy Acids; In Vitro Techniques; Magnoliopsida; Phospholipases; Plant Extracts; Plants, Medicinal; Sesquiterpenes; Tanacetum parthenium; Thrombin; Thromboxane B2

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acid; Arachidonic Acids; Bone Marrow Cells; Fatty Acids, Unsaturated; Female; Guinea Pigs; Indomethacin; Male; Megakaryocytes; Thromboxane B2; Time Factors

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonic Acids; Blood Platelets; Fatty Acids, Unsaturated; Humans; Hydroxylation; Thromboxane B2

Three family members from three successive generations presented with a moderate bleeding tendency and a functional platelet defect. They had absent aggregation with arachidonic acid (0.6--3 microM), reversible aggregation with ADP (4 microgram) and cyclic endoperoxide analogues, single wave aggregation only with adrenaline (5.4 microgram) and a prolonged template bleeding time (> min). Malondialdehyde formation was reduced after N-ethylmaleimide stimulation (2--6 nmol/10(9) platelets; control values 8--12 nmol) and serum thromboxane B2 values were reduced (33--101 ng/ml; control values 200--700 ng/ml). When the platelets were incubated with [3H]arachidonic acid the final metabolite of the lipoxygenase pathway (HETE) was produced in normal amounts but the production of thromboxane B2 and HHT was decreased whereas prostaglandin F2a, and E2 and probably D2 were increased. Evidence for enhanced production of prostaglandin D2 was also provided by the rise in the patient's platelet cyclic AMP levels following stimulation with arachidonic acid. The patient's washed platelets stimulated the production of 6-keto PGF 1a by aspirin-pretreated cultured bovine endothelial cells. The plasma levels of 6-keto PGF1a (439--703 pg/ml; normal 181 +/- 46 pg/ml) were raised. The decreased production of thromboxane B2, HHT and malondialdehyde and increased formation of prostaglandin F2a, E2, D2 and of 6-keto PGF1a are compatible with a partial platelet thromboxane synthetase deficiency and reorientation of cyclic endoperoxide metabolism. The markedly prolonged bleeding time would result not only from reduced formation of thromboxane A2 but also from increased production of the aggregation inhibiting prostaglandins PGI2 and PGD2.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Adult; Arachidonic Acids; Blood Coagulation Disorders; Child, Preschool; Cyclic AMP; Fatty Acids, Unsaturated; Humans; Hydroxy Acids; Male; Malondialdehyde; Middle Aged; Oxidoreductases; Pedigree; Platelet Aggregation; Prostaglandin Endoperoxides; Prostaglandins F; Thromboxane B2; Thromboxane-A Synthase

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Adult; Age Factors; Arachidonic Acid; Arachidonic Acids; Blood Platelets; Fatty Acids, Unsaturated; Female; Humans; Hydroxy Acids; In Vitro Techniques; Male; Middle Aged; Myocardial Infarction; Prostaglandin Endoperoxides; Prostaglandins; Sex Factors; Thromboxane B2

Murine macrophage-like cell lines, J774.2, P388D1, RAW264.7 and PU-5-1R, were incubated with exogenous arachidonic acid (AA). The major metabolites were identified by comigration with known standards in TLC and HPLC and by characteristic behavior following reduction. During a 30 min incubation J774.2 cells metabolized exogenous 14C-AA (10 microM) to PGE2 (14.8%), 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) (13.0%), thromboxane B2 (TXB2) (7.4%), PGD2 (4.4%) and PGF2 alpha (3.0%). The remainder was incorporated into phospholipids (39.0%), triglycerides (6.1%), and as yet unidentified metabolites (8.2%). No PGF1 alpha was found. Metabolism of exogenous AA was rapid, being less than 90% completed at 3.5 min. Metabolism of exogenous AA is not increased by the simultaneous addition of macrophage stimuli including the cation ionophore A-23187, particulate phagocytic stimuli and endotoxin. The synthesis of cyclooxygenase products was inhibited by low doses of indomethacin (ID50=0.6 microM) while the synthesis of TXB2 and HHT was selectively inhibited by benzylimidazole (ID50=9.5 microM). Identification of a probable lipoxygenase product is being pursued. The synthesis of this product is not inhibited by indomethacin and migrates with an Rf value close to 5,12-diHETE in TLC. P388D1 and RAW264.7 cells metabolize exogenous AA to the same products as J774.2, but in different proportions, while PU-5-1R does not produce cyclooxygenase metabolites to any appreciable extent.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Cell Line; Cyclooxygenase Inhibitors; Dinoprost; Dinoprostone; Fatty Acids, Unsaturated; Hydroxy Acids; Imidazoles; Indomethacin; Kinetics; Macrophages; Mice; Neoplasms, Experimental; Prostaglandin D2; Prostaglandins D; Prostaglandins E; Prostaglandins F; Thromboxane B2; Thromboxane-A Synthase

Human cortical hydronephrotic microsomes converted [14C] arachidonic acid to [14C] thromboxane B2 as the major metabolic product. Using [14C] PGH2 as substrate, similar enzymatic conversions were noted with HHT greater than TXB2 less than 6KPGF1 alpha greater than PGE2 greater than PGE2 alpha as the major products. Inhibition of thromboxane synthetase with imidazole 5 mM reduced thromboxane B2 production by 60% and the major product then was 6 keto PGF1 alpha. After addition of imidazole, the metabolic profile showed PKPGF1 alpha greater than PGE2 greater than HHT greater than PGF 2 alpha. Control experiments were carried out using normal cortical tissue obtained from kidneys removed surgically for carcinoma of kidney and rejected for transplantation secondary to fracture as a consequence of blunt trauma. These control kidneys, while they demonstrated an ability to generate thromboxane B2 in vitro, had much less activity than hydronephrotic kidneys and with PGH2 as substrate PGE2 greater than TxB2. In addition, inhibition with imidazole produced mainly PGE2. Thus, like the rabbit and rat, there is enhanced thromboxane and prostacyclin synthesis in human ureteral obstruction and are, therefore, potential vasoactive compounds which may in part be responsible for the hemodynamic alterations occurring in human obstructive uropathy.

Topics: 6-Ketoprostaglandin F1 alpha; Arachidonic Acids; Fatty Acids, Unsaturated; Humans; Hydronephrosis; Hydroxy Acids; Imidazoles; Kidney Cortex; Microsomes; Prostaglandins E; Prostaglandins F; Prostaglandins H; Thromboxane A2; Thromboxane B2; Thromboxanes

It has been reported that the prostaglandin (PG) precursor, arachidonic acid, produces divergent hemodynamic responses in the feline pulmonary vascular bed. However, the pattern of arachidonic acid products formed in the lung of this species is unknown. In order to determine the type and activity of terminal enzymes in the lung, prostaglandin biosynthesis by microsomes from cat lung was studied using the prostaglandin endoperoxide, PGH2, as a substrate. The major products of incubations of PGH2 with microsomes were thromboxane (TX) B2 (the major metabolite of TXA2), 6-keto-PGF1 alpha (the breakdown product of PGI2) and 12L-hydroxy-5,8,10-heptadecatrienoic acid (HHT). Formation of TXB2 was markedly reduced by imidazole. Tranylcypromine decreased the formation of TXB2 and HHT and inhibited the formation of 6-keto-PGF1 alpha. At low PGH2 concentrations, equal production of TXB2 and 6-keto-PGF1 alpha was observed. However, as PGH2 concentration increased, 6-keto-PGF1 alpha production approached early saturation while TXB2 production increased in a linear fashion. These results suggest that enzymatic formation of TXA2 and PGI2 is a function of substrate availability in the lung. These findings provide a possible explanation for the divergent hemodynamic responses to arachidonic acid infusions at high and low concentrations in the feline pulmonary vascular bed.

Topics: 6-Ketoprostaglandin F1 alpha; Animals; Cats; Fatty Acids, Unsaturated; Female; Hydroxy Acids; Lung; Male; Microsomes; Prostaglandin Endoperoxides; Prostaglandins F; Prostaglandins H; Thromboxane B2

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonic Acids; Blood Platelets; Chromatography, Thin Layer; Fatty Acids, Unsaturated; Humans; Hydroxy Acids; Thromboxane B2

Separation of the major metabolites of arachidonic acid (AA) produced by the cyclo-oxygenase and the lipoxygenases was achieved by using reverse phase high-pressure liquid chromatography. Prostaglandins (PGs), thromboxane B2 (TXB2), and AA were separated on a C-18 radial compression column. An initial isocratic elution resolved the PGs and TXB2 which was followed by a linear gradient in order to elute AA. Variations of the gradient elution shape were required to permit the separation of 12-L-hydroxy-5,8,10-heptadecatrienoic acid, 5-12 and 15-hydroxy-5,8,11,14-eicosatetraenoic acid. The recovery of the labeled AA and its metabolites was investigated. Use of these separation methods and radiolabeled substrates should permit investigators to obtain reproducibly in one chromatographic run adequate separation and quantitation of both PGs and hydroxy fatty acid systems.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonic Acids; Blood Platelets; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Fatty Acids, Unsaturated; Humans; Hydroxy Acids; Indomethacin; Microsomes; Prostaglandins; Prostaglandins E; Prostaglandins F; Thromboxane B2; Thromboxanes

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Adult; Amnion; Arachidonic Acids; Cesarean Section; Fatty Acids, Unsaturated; Female; Humans; Hydroxy Acids; Labor, Obstetric; Pregnancy; Prostaglandins E; Prostaglandins F; Thromboxane B2

Topics: Blood Platelets; Collagen; Epinephrine; Fatty Acids, Unsaturated; Humans; Hydroxy Acids; Lipid Peroxides; Malondialdehyde; Platelet Aggregation; Thromboxane B2; Thromboxanes

The formation of thromboxane B2 (TxB2), 12L-hydroxy-5,8,10 heptadecatrienoic acid (HHT) and 12L-hydroxy-5,8,10,14 eicosatrienoic acid (HETE) was determined in the platelets of normal human males and of patients with disorders in which an abnormal platelet aggregation occurs. Platelets were labelled with [1-14C] arachidonic acid. After Aggregation and extraction the metabolites wee separated by TLC and determined. In the platelets of normal males, TxB2 values were in the range 4.4-12.4%, expressed as a percentage of total radioactivity. Curves were constructed for the following ratios: TxB2 :HHT, TxB2 :HETE and (TxB2 + HHT) :HETE. These ratios were fairly contant. A comparison was made with the ratios obtained in the platelets of a small number of patients with either an enhanced or a diminished aggregation.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonic Acids; Blood Platelet Disorders; Blood Platelets; Fatty Acids, Unsaturated; Humans; Hydroxy Acids; Male; Thromboxane B2; Thromboxanes

Topics: Arachidonic Acids; Cell Movement; Chemotaxis; Eosinophils; Fatty Acids, Unsaturated; Hydroxy Acids; Neutrophils; Prostaglandins E; Prostaglandins F; Thromboxane B2

Human platelet microsomes convert prostaglandin H2 to thromboxane B2 and 12-L-hydroxy-5,8,10-heptadecatrienoic acid (12OH-17:3) in approximately equimolar amounts. The synthesizing activities of both products appear to go in parallel, both activities gradually decline upon storage and are equally destroyed by heat inactivation. Furthermore imidazole, a potent inhibitor of thromboxane synthetase activity, is an equally effective inhibitor of 12OH-17:3 formation in platelets. These results suggest that both thromboxane B2 and 120H-17:3 are derived from a common intermediate. We propose two alternative pathways for the conversion of prostaglandin H2 to thromboxane A2 and 12OH-17:3 in human platelets. The first pathway depicts thromboxane A2 as the common intermediate for the formation of both thromboxane B2 and 12OH-17:3. In the second pathway, prostaglanding H2 is converted to an activated intermediate which is converted to either thromboxane A2 or 12OH-17:3.

Topics: Arachidonic Acids; Blood Platelets; Fatty Acids, Unsaturated; Hot Temperature; Humans; Hydroxy Acids; Imidazoles; Malondialdehyde; Microsomes; Platelet Aggregation; Prostaglandins; Prostaglandins H; Thromboxane B2; Thromboxane-A Synthase; Thromboxanes