6-ketoprostaglandin-f1-alpha and 12-hydroxy-5-8-10-heptadecatrienoic-acid

The selective inhibition of prostaglandin (PG)E(2) formation via interference with microsomal PGE(2) synthase (mPGES)-1 could have advantages in the treatment of PGE(2)-associated diseases, such as inflammation, fever and pain, compared with a general suppression of all PG biosynthesis, provided by inhibition of cyclooxygenase (COX)-1 and 2. Here, we addressed whether the naturally occurring acylphloroglucinol myrtucommulone (MC) from Myrtus communis L. (myrtle) affected mPGES-1.. The effect of MC on PGE(2) formation was investigated in a cell-free assay by using microsomal preparations of interleukin-1beta-stimulated A549 cells as the source of mPGES-1, in intact A549 cells, and in lipopolysaccharide-stimulated human whole blood. Inhibition of COX-1 and COX-2 activity in cellular and cell-free assays was assessed by measuring 12(S)-hydroxy-5-cis-8,10-trans-heptadecatrienoic acid and 6-oxo PGF(1alpha) formation.. MC concentration-dependently inhibited cell-free mPGES-1-mediated conversion of PGH(2) to PGE(2) (IC(50) = 1 micromol x L(-1)). PGE(2) formation was also diminished in intact A549 cells as well as in human whole blood at low micromolar concentrations. Neither COX-2 activity in A549 cells nor isolated human recombinant COX-2 was significantly affected by MC up to 30 micromol x L(-1), and only moderate inhibition of cellular or cell-free COX-1 was evident (IC(50) > 15 micromol x L(-1)).. MC is the first natural product to inhibit mPGES-1 that efficiently suppresses PGE(2) formation without significant inhibition of the COX enzymes. This provides an interesting pharmacological profile suitable for interventions in inflammatory disorders, without the typical side effects of coxibs and non-steroidal anti-inflammatory drugs.

Topics: 6-Ketoprostaglandin F1 alpha; Anti-Inflammatory Agents, Non-Steroidal; Cell Line, Tumor; Cyclooxygenase 1; Cyclooxygenase 2; Cyclooxygenase Inhibitors; Dinoprostone; Fatty Acids, Unsaturated; Humans; Intramolecular Oxidoreductases; Microsomes; Phloroglucinol; Prostaglandin-E Synthases

We tested the hypothesis that combined cyclooxygenase-1 (COX-1) and prostacyclin synthase (PGIS) gene transfer selectively augments prostacyclin production without a concurrent overproduction of other prostanoids.. ECV304 cells were transfected with bicistronic pCOX-1/PGIS versus pCOX-1 or pPGIS, and prostanoids were analyzed. Contrary to the high prostaglandin E2 synthesis in pCOX-1 transfected cells, selective prostacyclin formation was noted with bicistronic plasmid transfection. Next, we determined the optimal ratio of Ad-COX-1 to Ad-PGIS by transfecting human umbilical vein endothelial cells with various titers of these 2 adenoviral constructs and determined the level of protein expression and prostanoid synthesis. Our results show that optimal ratios of adenoviral titers to achieve a large prostacyclin augmentation without overproduction of prostaglandin E2 or F2alpha were 50 to 100 plaque forming units (pfu) of Ad-COX-1 to 50 pfu of Ad-PGIS per cell. A higher Ad-PGIS to Ad-COX-1 ratio caused a paradoxical decline in prostacyclin synthesis.. Prostacyclin synthesis can be selectively augmented by cotransfecting endothelial cells with an optimal ratio of COX-1 to PGIS. Combined COX-1 and PGIS gene transfer has the potential for therapeutic augmentation of prostacyclin.

Topics: 6-Ketoprostaglandin F1 alpha; Adenoviridae; Arachidonic Acid; Cell Line; Cells, Cultured; Chromatography, High Pressure Liquid; Cyclooxygenase 1; Cytochrome P-450 Enzyme System; Dinoprostone; Dose-Response Relationship, Drug; Endothelium, Vascular; Epoprostenol; Fatty Acids, Unsaturated; Gene Transfer, Horizontal; Genetic Vectors; Humans; Intramolecular Oxidoreductases; Isoenzymes; Membrane Proteins; Plasmids; Prostaglandin-Endoperoxide Synthases; Transfection

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

The synthesis of 14C labelled arachidonic acid metabolites was measured in colonic tissues obtained from mice, rats, guinea pigs, rabbits, piglets and in colonic biopsies from humans during colonoscopy. The main eicosanoids formed after stimulation with calcium ionophore A23187 were: in humans, 15-hydroxy-eicosatetraenoic acid (15-HETE); in mice, 12-HETE; in rats, 12-HETE, 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and 6-keto-prostaglandin F1 alpha (6kPGF1 alpha); in guinea pigs, PGD2; in rabbits, 6kPGF1 alpha, PGE2 and 15-HETE; and in pigs PGE2 and 12-HETE. In inflamed 15-HETE production was increased in man, HHT and 12-HETE production in rats and overall eicosanoid production in mice.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 6-Ketoprostaglandin F1 alpha; Animals; Calcimycin; Colitis, Ulcerative; Colon; Dinoprostone; Eicosanoids; Fatty Acids, Unsaturated; Guinea Pigs; Humans; Hydroxyeicosatetraenoic Acids; Intestinal Mucosa; Mice; Mice, Inbred BALB C; Prostaglandin D2; Rabbits; Rats; Rats, Wistar; Species Specificity

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

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 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

Particulate fractions and slices from fetal calf aorta convert arachidonic acid to 6-oxoprostaglandin F1 alpha (6-oxoPGF1 alpha), 6,15-dioxoPGF1 alpha, 12-hydroxy-5,8,10-heptadecatrienoic acid, 11-hydroxy-5,8,12,14-icosatetraenoic acid (11h-20:4), and 15-hydroxy-5,8,11,13-icosatetraenoic acid (15h-20:4). In some cases, small amounts of 12-hydroxy-5,8,10,14-icosatetraenoic acid (12h-20:4) were also detected. The products were all identified by gas chromatography-mass spectrometry after purification by normal phase and argentation high pressure liquid chromatography. Both 11h-20:4 and 15h-20:4 appeared to be formed by prostaglandin endoperoxide synthetase rather than by lipoxygenases, since their formation was inhibited by indomethacin but not by nordihydroguaiaretic acid. The formation of 12h-20:4, on the other hand, was stimulated by indomethacin, probably due to increased substrate availability. The formation of hydroxyicosatetraenoic acids was markedly stimulated by adrenaline. Substantial amounts of 6,15-dioxoPGF1 alpha were formed from arachidonic acid by particulate fractions from fetal calf blood vessels, especially in the presence of relatively high substrate concentrations. The formation of this product was stimulated by methemoglobin and inhibited by adrenaline, glutathione, and tryptophan. It would appear that particulate fractions from fetal calf aorta convert arachidonic acid to 15-hydroperoxyPGI2, which can either be reduced in the presence of various cofactors to form PGI2 or dehydrated to give 15-oxoPGI2. The formation of hydroperoxides from arachidonic acid could be an important factor in regulating PGI2 synthesis in aorta, since PGI2 synthetase is strongly inhibited by such intermediates.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 6-Ketoprostaglandin F1 alpha; Animals; Aorta; Arachidonic Acids; Cattle; Ductus Arteriosus; Fatty Acids, Unsaturated; Hydroxy Acids; Hydroxyeicosatetraenoic Acids; Kinetics; Lipoxygenase; Prostaglandin-Endoperoxide Synthases; Prostaglandins F, Synthetic

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