dinoprost and 12-hydroxy-5-8-10-heptadecatrienoic-acid

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

Cells were incubated in the presence of the Ca2+ ionophore A23187 (10 microM) and arachidonic acid (AA, 80 microM). The release of eicosanoids from subcultivated cardiac endothelial and fibroblast-like cells amounted to 23.3 +/- 4.5 and 2.0 +/- 0.4 nmol/mg cellular protein per 30 min, respectively. The release from isolated cardiomyocytes remained below the detection limit of the high-performance liquid chromatography assay (< 0.00015 nmol/assay). When a very sensitive radioimmunoassay was applied, cardiomyocytes released 0.002 +/- 0.0001 nmol prostacyclin per milligram cellular protein per 30 min. Prostaglandin (PG) E2 and PGF2 alpha, 12-hydroxyheptadecatrienoic acid, 11- and 15-hydroxyeicosatetraenoic acid, and thromboxane B2 were the main eicosanoids released by endothelial cells. The stable product of prostacyclin, 6-keto-PGF1 alpha, contributed relatively little to the total amount of eicosanoids formed by endothelial cells. Fibroblast-like cells released predominantly PGE2 and 6-keto-PGF1 alpha and, to a lesser extent, 12-hydroxyheptadecatrienoic and 15-hydroxyeicosatetraenoic acids. Neither endothelial cells nor fibroblast-like cells released leukotrienes. A23187 stimulated eicosanoid release from endothelial cells when exogenous AA was below 40 microM. Addition of albumin reduced the amount of eicosanoids produced. Histamine and bradykinin did not influence 6-keto-PGF1 alpha and PGE2 production in cardiomyocytes. Histamine only gave rise to a slight but significantly higher release of 6-keto-PGF1 alpha in endothelial cells.

Topics: 6-Ketoprostaglandin F1 alpha; Animals; Arachidonic Acid; Calcimycin; Cells, Cultured; Dinoprost; Dinoprostone; Endothelium; Fatty Acids, Unsaturated; Fibroblasts; Heart; Hydroxyeicosatetraenoic Acids; Indomethacin; Male; Microscopy, Electron; Myocardium; Rats; Rats, Inbred Lew; Rats, Wistar

Male weanling Wistar rats were maintained on one of two semisynthetic diets, differing only in the type of oil used: (i) 10% by weight marine fish oil (MFO group) containing 20% eicosapentaenoic acid (EPA) and 17% docosahexaenoic acid (DHA), or (ii) 10% by weight sunflower oil (SFO group). The control group was kept on standard diet for 4 weeks. Blood-free microvessels were isolated from brain cortex by a rapid micromethod, and their fatty acid composition was determined by gas chromatography. It was found that the proportion of n-3 fatty acids (including EPA and DHA) increased significantly in the microvessels of the MFO group, accompanied by a decrease of the n-6 fatty acid series. The changes in fatty acid composition of endothelial cells were not significant in the SFO group in comparison to the control. The amounts of lipoxygenase and cyclooxygenase metabolites were determined. Dietary fish oil decreased the percentage of total products of arachidonate by 50%, while the SFO diet had no effect on it. The amount of lipoxygenase products in the MFO group decreased significantly from 16931 +/- 3131 dpm to 6399 +/- 357 dpm/300 mg wet weight of brain. Significantly less PGF-1 alpha, PGF-2 alpha and 12-hydroxyheptadecatrienoic acid (HHT) were found in the capillaries of MFO treated animals, in comparison to the SFO group. The ratios of vasoconstrictor and vasodilator metabolites of arachidonate cascade were not modified by the diets. Our results suggest that fish oil diet reduces the arachidonate cascade in cerebral microvessels. This effect may explain for the efficiency of n-3 fatty acids in vascular diseases.

Topics: 6-Ketoprostaglandin F1 alpha; Animals; Arachidonic Acid; Capillaries; Cerebral Cortex; Dinoprost; Docosahexaenoic Acids; Eicosapentaenoic Acid; Endothelium, Vascular; Fatty Acids; Fatty Acids, Unsaturated; Fish Oils; Male; Prostaglandin-Endoperoxide Synthases; Rats; Rats, Inbred Strains

Arachidonic acid metabolism produces several biologically important compounds including the leukotrienes and prostaglandins. Prostaglandin H2 (PGH2) is the first metabolite in the arachidonic acid cascade leading to all other prostaglandins. Pivotal to our understanding of PGH2's biology is the ability to separate it in pure form from the numerous other arachidonic acid metabolites produced in a biological milieu. The extensive literature on PGH2 biology and metabolism has relied almost exclusively on the traditional method of separation using gravity flow silicic acid columns. In our hands, such PGH2 preparations were found to contain varying amounts of 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT), PGE2, PGF2 alpha and other minor impurities as determined by further chromatographic and mass spectral analyses. Analytical separation of PGH2 and other arachidonic acid metabolites has been accomplished using reversed-phase HPLC. However, the labile nature of this molecule in aqueous systems makes such techniques unacceptable for preparative isolation of high purity PGH2 and has necessitated the development of a totally nonaqueous separation. To this end, we attempted several stationary phases and found that the cyano-bonded phase showed the best selectivity for resolving PGH2 from its major contaminants. Separations were performed on self-packed columns using a hexane-isopropanol gradient. Peaks were detected both by liquid scintillation counting and uv spectrophotometry (214 nm). Structure assignments were made by chromatographic comparison with authentic standards (PGF2 alpha, PGE2), biological activity (PGH2--platelet aggregation), and by ammonia direct chemical ionization mass spectrometry (HHT, hydroxy-5,8,10,14-eicosatetraenoic acid, PGH2, PGE2, PGF2 alpha). The latter technique, which by its very nature volatilizes all organic material in the sample, was particularly useful in determining not only that the PGH2 preparations were free from the aforementioned side products, but that they were also free from lipid, protein, and other potential residues frequently found in biological preparations.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Chromatography, High Pressure Liquid; Dinoprost; Dinoprostone; Fatty Acids, Unsaturated; Male; Mass Spectrometry; Prostaglandin Endoperoxides; Prostaglandin Endoperoxides, Synthetic; Prostaglandin H2; Prostaglandins; Prostaglandins E; Prostaglandins F; Prostaglandins H; Sheep

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

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