thromboxane-a2 and 12-hydroxy-5-8-10-heptadecatrienoic-acid

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

Despite an almost total suppression of platelet cyclooxygenase (COX) by aspirin, as monitored ex vivo, incomplete suppression of thromboxane (Tx)A(2) metabolite excretion has been detected in some patients with unstable angina treated with low doses of aspirin. A plausible explanation for this finding is the transcellular formation of TxA(2) by platelets from prostaglandin H(2) released by endothelial cells. We recently reported that probably only COX and PGI-synthase (PGIS) are involved in the biosynthesis of prostanoids in endothelial cells. The present work was thus focused to ascertain the dependence of the transcellular biosynthesis of TxA(2), by endothelial cells and aspirin-treated platelets, on the relative activity of these enzymes. Synthesis of eicosanoids from exogenous and endogenous arachidonic acid (AA) by mixed incubations of human umbilical vein endothelial cells (HUVEC) in culture and aspirin-treated platelets were determined by HPLC and enzyme immune assay. The ratio of COX to PGIS activities was modified in HUVEC by treatment with interleukin-1beta (IL-1beta). Transcellular formation of TxA(2) was only relevant when HUVEC overexpressed COX-2 (monitored by RT-PCR and Western blotting), and in these conditions TxA(2) formation started 2 minutes after substrate addition. Progression curves showed that half-times (t(1/2)) of the COX and PGIS activity were 2.73 and 0.47 minutes, respectively, in resting HUVEC, whereas these values for IL-1beta-treated cells were 1.33 and 0.07 minutes, respectively, indicating that expression of COX-2 increased the rate of PGIS "suicide" inactivation. Collectively, these results indicated that not only enhanced COX activity but also substantial PGIS inactivation was required for significant transcellular biosynthesis of TxA(2).

Topics: Aspirin; Blood Platelets; Coculture Techniques; Cyclooxygenase 1; Cyclooxygenase 2; Cytochrome P-450 Enzyme System; Endothelium, Vascular; Fatty Acids, Unsaturated; Humans; Interleukin-1; Intramolecular Oxidoreductases; Isoenzymes; Membrane Proteins; Prostaglandin-Endoperoxide Synthases; Prostaglandins; RNA, Messenger; Thromboxane A2

Both polymorphonuclear (PMN) leukocytes and metabolites of arachidonic acid, especially lipoxygenase products, have been reported to contribute to myocardial damage after coronary artery occlusion and reperfusion. While canine models of myocardial ischemia were used in many of these studies, very little is known about arachidonic acid metabolism by canine PMNs. Moreover, it is unclear whether arachidonic acid metabolites released by canine PMNs affect vascular tone. Therefore, we characterized arachidonic acid metabolism by canine PMNs and determined the effect of these metabolites on vascular tone of isolated canine coronary arteries. Suspensions of canine PMNs were incubated with [14C]arachidonic acid and the calcium ionophore A23187. The incubation media was extracted, and the metabolites resolved by HPLC. 20-Hydroxy-leukotriene B4 (LTB4), 12,20-dihydroxyeicosatetraenoic acid (diHETE), LTB4, 12-hydroxyheptadeclatrienoic acid (HHT), and 12-(S)-hydroxyeicosatetraenoic acid (HETE) were isolated, and their structures confirmed by gas chromatography/mass spectrometry. There was also evidence for the formation of 20-HETE, thromboxane B2 (TXB2), 5-HETE, and several isomers of LTB4. None of the arachidonic acid metabolites that were isolated from incubates of canine PMNs augmented vascular tone, but material migrating with 12,20-diHETE relaxed canine coronary arteries. Authentic 12(S),20-diHETE also produced a concentration-related relaxation of canine coronary artery. 12(R), 20-diHETE was inactive. 20-HETE inhibited A23187-induced PMN aggregation. Thus, arachidonic acid is metabolized in canine PMNs through the cyclooxygenase, lipoxygenases and cytochrome P-450 pathways. Whether these metabolites contribute to myocardial injury remains to be determined.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Arachidonic Acid; Arteries; Cell Aggregation; Chromatography, High Pressure Liquid; Coronary Vessels; Dogs; Fatty Acids, Unsaturated; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Leukotriene B4; Mass Spectrometry; Neutrophils; Prostaglandin Endoperoxides, Synthetic; Thromboxane A2; Vasoconstriction; Vasoconstrictor Agents; Vasodilation

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

An intraperitoneal (rat) cytomegalovirus (RCMV) infection in the rat caused an influx of mononuclear cells, which have been altered in functions and arachidonic acid (AA) metabolism. Phagocytosis has been increased considerably 3 days postinfection (p.i.), whereas the release of prostacyclin, thromboxane A2, 12-hydroxyheptadecatrienoic acid (HHT), 5-hydroxyeicosatetraenoic acid (5-HETE), and leukotriene B4 (LTB4) was inhibited for more than 80%. The release of superoxide anions and the chemiluminescence response (CL) upon opsonized zymosan stimulation did not differ from those observed in resident peritoneal macrophages. Additionally, the levels of cyclic nucleotides (cAMP and cGMP) were low in both resident and influx macrophages (day 3 p.i.). In contrast, peritoneal macrophages harvested on day 10 p.i. still showed a high level of phagocytosis. However, the intracellular level of cyclic AMP had decreased fivefold, whereas CL response and superoxide anion release were inhibited significantly. Moreover, the production of prostacyclin, LTB4, and 5-HETE was still suppressed in contrast to thromboxane synthesis, which has selectively been restored in these macrophages. A direct regulatory role of AA metabolites in changes in macrophage functions that were due to a RCMV infection could not be demonstrated.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Cyclic AMP; Cyclic GMP; Cytomegalovirus Infections; Epoprostenol; Fatty Acids, Unsaturated; In Vitro Techniques; Luminescent Measurements; Macrophages; Male; Rats; Rats, Inbred Strains; Superoxides; Thromboxane A2

The leukotactic responsiveness of human peripheral blood monocytes is regulated by the cell-directed inhibitor of monocyte leukotaxis, CDI-MLx. The actions of CDI-MLx on normal monocytes in vitro were abrogated by co-incubation with inhibitors of cyclooxygenase and thromboxane synthetase with indomethacin and dazmegrel (UK-38,485) being most active. The actions of CDI-MLx were mimicked by the thromboxane A2 analogue, U-46619, and by 12-HHT with half-maximal inhibition observed at 10(-10) M; PGE2 was 1000-fold less active. SQ 29,548, a thromboxane A2 receptor antagonist, blocked the effects of CDI-MLx, U-46619, and 12-HHT. Production of PGE2 and thromboxane B2 by purified monocytes was stimulated by CDI-MLx and this effect was also blocked by indomethacin, dazmegrel, and dazoxiben. These data suggest a major regulatory role for thromboxane synthetase products in human monocyte leukotaxis.

Topics: Bridged Bicyclo Compounds, Heterocyclic; Chemotactic Factors; Chemotaxis, Leukocyte; Dinoprostone; Fatty Acids, Unsaturated; Humans; Hydrazines; In Vitro Techniques; Lymphokines; Monocytes; Prostaglandin Endoperoxides, Synthetic; Prostaglandins E; Radioimmunoassay; Thromboxane A2

Normal hemostasis depends in part on the balance achieved between proaggregatory and prothrombotic platelet thromboxane A2, measured as its stable end-product thromboxane B2 (TXB2), and vascular prostacyclin (PGI2), which inhibits platelet aggregation and is antithrombotic. Cystathionine-beta-synthase deficiency is characterized by a high frequency of thromboembolic disease. We therefore studied, in vitro, the effects of homocysteine and related compounds on platelet TXB2 and vascular PGI2 formation. In paired samples of platelet rich plasma, which had been preincubated with L-homocystine (1 mM), mean production of the two platelet cyclooxygenase products, TXB2 and 12-hydroxy-5, 8,10-heptadecatrienoic acid increased significantly from control levels [13.6% +/- 1.9 to 19.8% +/- 2.1 (P less than 0.02) TXB2 and 29.8% +/- 4.2 to 39.4% +/- 4.1 (P less than 0.01) HHT]. In the presence of D,L-homocysteine (1 mM), mean TXB2 and 12-hydroxy-5,8,10-heptadecatrienoic acid production was also significantly increased [12.7% +/- 1.5 to 16.9% +/- 1.5 (P less than 0.01) TXB2 and 27% +/- 4 to 31% +/- 4.1 (P less than 0.02) HHT]. Cystine, cysteine, or methionine (1 mM) did not have similar effects in this test system. Homocysteine and homocystine were without effect on the synthesis of vascular PGI2 by umbilical artery segments [control, 0.22 +/- 0.03 to 0.21 +/- 0.03 ng/mg with D,L-homocysteine and 0.20 +/- 0.04 control to 0.19 +/- 0.04 ng/mg with D,L-homocystine]. A homocyst(e)ine-induced increase in platelet thromboxane production in the absence of an increase in vascular prostacyclin, if present in vivo, may contribute to the vascular thromboses characteristic of human homocystinemias (homocystinurias).

Topics: Arachidonic Acid; Arachidonic Acids; Blood Platelets; Epoprostenol; Fatty Acids, Unsaturated; Homocysteine; Homocystine; Humans; Hydroxy Acids; Hydroxyeicosatetraenoic Acids; Methionine; Thromboxane A2; Umbilical Arteries

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

Topics: Arachidonic Acids; Blood Platelets; Chromatography, Thin Layer; Collagen; Fatty Acids, Unsaturated; Humans; Hydroxy Acids; Imidazoles; Male; Platelet Aggregation; Prostaglandins; Prostaglandins E; Prostaglandins H; Thromboxane A2; Thromboxanes