thromboxane-b2 and 15-hydroxy-5-8-11-13-eicosatetraenoic-acid

Eicosanoids, active metabolites of arachidonic acid (AA), play an important role in the regulation of renal haemodynamics and glomerular filtration. Our study verified the hypothesis on the positive action of exogenously administered PGE(1) on renal function during an operation with temporary ischaemia of the lower half of the body. Also the effect of alprostadil (prostaglandin E(1) analogue) administered during the operation of an abdominal aorta aneurysm on the postoperative systemic metabolism of AA and the glomerular filtration rate (GFR) was investigated. The study included 42 patients with a diagnosed abdominal aorta aneurysm who have been qualified for the operation of implantation of the aortic prosthesis. The patients were randomly assigned to two groups: the study group (I) receiving alprostadil and the control group (II) without alprostadil. The levels of hydroxyeicosatetraenoic acids (15-HETE, 12-HETE, 5-HETE) were determined by RP-HPLC and the level of thromboxane B(2) (TxB(2)) was determined by ELISA in the plasma of the blood drawn from vena cava superior immediately before aortic clamping (A) and 5 min after aortic declamping (B). The administration of PGE(1) affects the metabolism of 15-HETE in a manner dependent on the baseline value of GFR but does not significantly change the postoperative renal function. The metabolism of 15-HETE is affected by the baseline value of GFR1 and a longer period of ischaemia is correlated with lower concentrations of 5-HETE during reperfusion. The results of our studies indicate that TxB(2) influences the postoperative function of kidneys.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Aged; Alprostadil; Aortic Aneurysm; Eicosanoids; Female; Glomerular Filtration Rate; Humans; Hydroxyeicosatetraenoic Acids; Kidney; Male; Middle Aged; Postoperative Period; Reperfusion Injury; Thromboxane B2

An inherited deficiency of the mitochondrial protein frataxin causes Friedreich's ataxia (FRDA); the mechanism by which this deficiency triggers neuro- and cardio-degeneration is unclear. Microarrays of neural tissue of animal models of the disease showed decreases in antioxidant genes, and increases in inflammatory genes. Cyclooxygenase (COX)-derived oxylipins are important mediators of inflammation. We measured oxylipin levels using tandem mass spectrometry and ELISAs in multiple cell and animal models of FRDA. Mass spectrometry revealed increases in concentrations of prostaglandins, thromboxane B2, 15-HETE and 11-HETE in cerebellar samples of knockin knockout mice. One possible explanation for the elevated oxylipins is that frataxin deficiency results in increased COX activity. While constitutive COX1 was unchanged, inducible COX2 expression was elevated over 1.35-fold (P < 0.05) in two Friedreich's mouse models and Friedreich's lymphocytes. Consistent with higher COX2 expression, its activity was also increased by 58% over controls. COX2 expression is driven by multiple transcription factors, including activator protein 1 and cAMP response element-binding protein, both of which were elevated over 1.52-fold in cerebella. Taken together, the results support the hypothesis that reduced expression of frataxin leads to elevation of COX2-mediated oxylipin synthesis stimulated by increases in transcription factors that respond to increased reactive oxygen species. These findings support a neuroinflammatory mechanism in FRDA, which has both pathomechanistic and therapeutic implications.

Topics: Animals; B-Lymphocytes; Cell Line; Cerebellum; Cyclic AMP Response Element-Binding Protein; Cyclooxygenase 1; Cyclooxygenase 2; Frataxin; Friedreich Ataxia; Gene Expression Regulation; Humans; Hydroxyeicosatetraenoic Acids; Inflammation; Iron-Binding Proteins; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Transgenic; Oxylipins; Prostaglandins; Reactive Oxygen Species; Signal Transduction; Thromboxane B2; Transcription Factor AP-1

Cyclooxygenase (COX)-, lipoxygenase (LOX)-, and cytochrome P450 monooxygenase (CYP)-derived eicosanoids have been implicated in ocular surface inflammation and neovascularization. These eicosanoids are subjected to regulation by enzymes, such as heme oxygenases (HOs) and ferritin.. Quantitative polymerase chain reaction and lipidomics based on liquid chromatography-tandem mass spectrometry were performed on pterygia from patients undergoing surgical pterygium excision. Control tissues consisted of donor corneas. In addition, lipidomics based on liquid chromatography-tandem mass spectrometry was performed on tears collected from patients before the surgery.. Messenger RNA (mRNA) expression of HO-2, the constitutive HO isoform, was upregulated by 40% in pterygia compared with control tissue, whereas the mRNA level of the inducible form, HO-1, was downregulated by more than 50%. Levels of CYP4B1 mRNA showed an approximate 2-fold increase in pterygia compared with control. Lipidomic analysis of tissues indicated a moderate elevation in Prostaglandin E2 and thromboxane B2 levels in pterygia compared with control. Among the LOX-derived metabolites, the antiinflammatory-hydroxyeicosatetraenoic acid (15-HETE) levels were significantly reduced in pterygia (79.3 ± 48.11 pg/mg protein) compared with control (586.2 ± 213.5 pg/mg protein), whereas the proinflammatory LOX- and CYP4B1-derived 12-HETE levels were 10-fold higher in pterygia (2768 ± 832.3 pg/mg protein) compared with control (231.4 ± 87.35 pg/mg protein). Prostaglandin E2 and HETEs were also present in tears from patients with pterygium but were not detected in tears from healthy volunteers. The mRNA expression levels of both light and heavy chain ferritin were 60% and 30% lower, respectively, in pterygia compared with control.. We believe that a dysfunctional HO-ferritin system leads to increased levels of proinflammatory mediators, thus contributing to the inflammation characteristic of pterygia.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Adult; Aryl Hydrocarbon Hydroxylases; Chromatography, High Pressure Liquid; Dinoprostone; Female; Ferritins; Gene Expression Regulation, Enzymologic; Heme Oxygenase (Decyclizing); Heme Oxygenase-1; Humans; Hydroxyeicosatetraenoic Acids; Isoenzymes; Male; Middle Aged; Pterygium; Real-Time Polymerase Chain Reaction; RNA, Messenger; Tears; Thromboxane B2

Novel aspirin (ASA)-triggered 15-epi-lipoxins (ATL) comprise new potent bioactive eicosanoids that may contribute to the therapeutic effect of this drug. ATL biosynthesis is initiated by ASA acetylation of cyclooxygenase (COX)-2 and was originally identified during the interaction of leukocytes with either endothelial or epithelial cells. Here, we examined ATL biosynthesis in rat hepatocytes either alone or in coincubation with nonparenchymal liver cells (NPC) and in liver homogenates from ASA-treated rats. Rat hepatocytes and CC-1 cells, a rat hepatocyte cell line, displayed COX-1 but not COX-2 mRNA expression and predominantly produced thromboxane A(2) (TXA(2)) and 15-hydroxyeicosatetraenoic acid (15-HETE). In these cells, ASA shifted the arachidonic acid metabolism from TXA(2) to 15-HETE in a concentration-dependent manner. In contrast, neither indomethacin, ibuprofen, valeryl salicylate, nor nimesulide was able to trigger 15-HETE biosynthesis. SKF-525A, a cytochrome P-450 inhibitor, significantly reduced the effect of ASA on 15-HETE biosynthesis. Furthermore, phenobarbital, a potent inducer of cytochrome P-450 activity, further increased ASA-induced 15-HETE production. ASA treatment of hepatocyte-NPC coincubations resulted in the generation of significant amounts of ATL. In addition, in vivo experiments demonstrated augmented hepatic levels of 15-epi-lipoxin A(4) in ASA-treated rats. Taken together and considering that ASA is hydrolyzed on its first pass through the portal circulation, these data indicate that, during ASA's consumption, liver tissue generates biologically relevant amounts of ATL by COX-2-independent mechanisms.

Topics: Animals; Aspirin; Cells, Cultured; Cyclooxygenase 1; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Cyclooxygenase Inhibitors; Cytochrome P-450 Enzyme System; Dose-Response Relationship, Drug; Enzyme Activation; Excitatory Amino Acid Antagonists; Gene Expression Regulation, Enzymologic; Hydroxyeicosatetraenoic Acids; Ibuprofen; Indomethacin; Isoenzymes; Lipoxins; Liver; Male; Membrane Proteins; Phenobarbital; Prostaglandin-Endoperoxide Synthases; Rats; Rats, Wistar; RNA, Messenger; Sulfonamides; Thromboxane B2

We tested the hypothesis that aspirin affects trophoblast like other epithelial cells do, by inhibiting prostanoid production, inducing prostaglandin H synthase-2 expression, and enhancing secretion of 15-hydroxyeicosatetraenoic acid.. Cytotrophoblast from placentas (n = 15) of uncomplicated singleton pregnancies were cultured in medium 199 for 4 to 72 hours in the presence or absence of aspirin.. Aspirin (10(-4) M) inhibited (p < 0.01) average trophoblast prostaglandin E2 release by 60% and thromboxane B2 by 86%. Western immunoblotting showed the prostaglandin H synthase-1 was constitutively expressed in cytotrophoblast, and aspirin treatment caused a twofold increase in prostaglandin H synthase-1 expression. Prostaglandin H synthase-2 was also constitutively expressed in untreated cytotrophoblast but at lower levels than prostaglandin H synthase-1. Aspirin enhanced prostaglandin H synthase-2 expression in trophoblast cultures, but prostaglandin H synthase-2 contributed a range of only 10% to 33% (n = 4) of the total cellular prostaglandin H synthase protein pool even after aspirin induction. The increased prostaglandin H synthase expression depended on both transcription and translation because actinomycin D and cycloheximide each inhibited the increased prostaglandin H synthase protein expression after aspirin treatment. The aspirin induction of prostaglandin H synthase was accompanied by decreased release of 15-hydroxyeicosatetraenoic acid.. Trophoblast differs from other cells studied because aspirin enhances expression of both prostaglandin H synthase-1 and prostaglandin H synthase-2 isozymes while decreasing, instead of increasing, the secretion of 15-hydroxyeicosatetraenoic acid. The aspirin effects on prostaglandin H synthase synthesis and 15-hydroxyeicosatetraenoic acid release in trophoblast suggest that the mechanisms of action for aspirin in the prophylaxis of preeclampsia may be more diverse than simply altering platelet thromboxane production.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Aspirin; Blotting, Western; Cells, Cultured; Cycloheximide; Dactinomycin; Dose-Response Relationship, Drug; Female; Gene Expression Regulation, Enzymologic; Humans; Hydroxyeicosatetraenoic Acids; Isomerism; Platelet Aggregation Inhibitors; Pregnancy; Prostaglandin-Endoperoxide Synthases; Prostaglandins; Protein Synthesis Inhibitors; Radioimmunoassay; Thromboxane B2; Time Factors; Trophoblasts

We have studied the liver 15-hydroxyeicosatetraenoic acid (15-HETE) and leukotriene B4 (LTB4) levels in streptozotocin- (ST)-induced diabetes in rats using liquid chromatography and radioimmunological techniques. Diabetic rats showed significant alterations of liver lipoxygenase metabolites when compared to controls. These 15-HETE and LTB4 increases were concomitant with raised levels of plasma and tissue thromboxane B2 (TXB2) and also urinary 2,3-dinor-TXB2 in plasma and urine, respectively. These changes confirm an activation of 5- and 15-lipoxygenase in the liver 3 days after i.p. ST administration.

Topics: Animals; Arachidonate 15-Lipoxygenase; Arachidonate 5-Lipoxygenase; Arachidonic Acid; Blood Glucose; Diabetes Mellitus, Experimental; Hydroxyeicosatetraenoic Acids; Liver; Male; Rats; Rats, Wistar; Streptozocin; Thromboxane B2

Endothelial cells actively regulate their environment by secreting humoral substances, including endothelin-1 and a variety of eicosanoids, that have local actions. To elucidate interactions among these local mediators, we measured release of cyclooxygenase and lipoxygenase pathway products of arachidonate metabolism by human aortic endothelial cells after incubation with endothelin-1. Supernatants were collected, extracted, and fractionated using high-performance liquid chromatography. Radioimmunoassays for eicosanoids were performed on the appropriate fractions. After endothelin stimulation, production of the prostacyclin metabolite 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha), the thromboxane (Tx) metabolite TxB2, and prostaglandin E2 (PGE2) were increased (307 +/- 48, 320 +/- 91, and 315 +/- 74% of control, P < 0.05). Leukotriene B4 (LTB4) release was modestly increased (195 +/- 19% of control, P < 0.05). The release of 5-hydroxyeicosatetraenoic acid (5-HETE) was increased (300 +/- 57% of control, P < 0.05); production of 12-HETE and 15-HETE was unchanged. Production of eicosanoids peaked between 30 and 120 min. Preincubation with pertussis toxin prevented increased production of PGE2, LTB4, and 5-HETE after endothelin-1 stimulation; pretreatment with sphingosine had no effect. Interactions between endothelin and eicosanoids may be important components of the complex network that regulates vascular tone, coagulation, and inflammation at the local level.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 6-Ketoprostaglandin F1 alpha; Aorta; Arachidonic Acid; Cells, Cultured; Dinoprostone; Eicosanoids; Endothelins; Endothelium, Vascular; Gas Chromatography-Mass Spectrometry; Humans; Hydroxyeicosatetraenoic Acids; Kinetics; Leukotriene B4; Pertussis Toxin; Thromboxane B2; Virulence Factors, Bordetella

The differentiation of HL-60 human promyelocytic leukaemia cells into specific monocytic or granulocytic lineage cells depending of the inductor agent is accompanied by selective regulation of several key enzymes involved in the synthesis of eicosanoids. In this communication we have investigated the changes in arachidonic acid metabolic profiles during phorbol 12-myristate 13-acetate (PMA)-induced monocytic differentiation of HL-60 cells. Our results show that HL-60 cells have spontaneous capacity to synthesize large amounts of LTB4, but PMA-differentiated cells lose the ability to release LTB4. Significant differences are found between HL-60 cells and PMA-treated cells in basal conditions and under ionophore stimulation. The addition of LTB4 at the time of PMA differentiation did not have effects on cell proliferation, but nordihydroguaiaretic acid (NDGA), a potent 5-lipoxygenase inhibitor, also inhibited HL-60 cell proliferation and did not have any effect on PMA-differentiated cell proliferation.

Topics: Arachidonic Acids; Cell Differentiation; Cell Division; Cell Line; Dinoprostone; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Lipoxygenase; Macrophages; Masoprocol; Monocytes; Prostaglandin-Endoperoxide Synthases; Tetradecanoylphorbol Acetate; Thromboxane B2

Several studies have reported that prostanoids are involved in many of the physiopathological mechanisms underlying acute pancreatitis but their precise role in this disease remains to be established. The objective of this work is to evaluate the variation of local tissue production of prostanoids and lipoxygenase metabolites of arachidonic acid in acute pancreas inflammation induced by intraductal administration of 3.5% sodium taurocholate (0.1 ml/100 mg body weight) in rats. Pancreatic tissue levels of leukotriene B4 (LTB4), 15 hydroxyeicosatetraenoic acid (15-HETE), 6-keto prostaglandin F1 alpha (6-keto PGF1 alpha), thromboxane B2 (TXB2) and prostaglandin E2 (PGE2) were determined by HPLC-RIA techniques at 5 and 60 minutes after induction of acute pancreatitis (AP). Prostanoids increased significantly at 5 minutes and LTB4 and 15-HETE at 60 minutes. These data confirm that the prostanoid imbalance could be considered as an early specific response of the pancreas to the inflammatory events characteristic of induced AP while the altered levels of the lipoxygenase products (LTB4 and 15-HETE) would be more of a nonspecific organ response associated to the high cellular infiltration rate and necrosis observed in the late phases of acute pancreatitis.

Topics: 6-Ketoprostaglandin F1 alpha; Acute Disease; Animals; Dinoprostone; Hemorrhage; Hydroxyeicosatetraenoic Acids; Kinetics; Leukotriene B4; Lipoxygenase; Male; Pancreas; Pancreatitis; Prostaglandin-Endoperoxide Synthases; Rats; Rats, Sprague-Dawley; Taurocholic Acid; Thromboxane B2

Inflammatory exudates obtained in rats after subcutaneous implantation of carrageenin-soaked sponges were found to contain relatively large amounts of 15-hydroxy-5,8,11, 13-eicosatetraenoic acid (15-HETE) and smaller amounts of cysteinyl-leukotrienes (LT) in addition to LTB4, thromboxane (TX) B2 and prostaglandin (PG)E2. Concentrations of 15-HETE and cysteinyl-LT were high 5 hours after sponge implantation and decreased significantly within 24 hours. This time-course, which is similar to that of TXB2, but differs from that of PGE2, suggests migrating leukocytes as a major source of 15-HETE and cysteinyl-LT. Aspirin, sodium salicylate, dipyrone (100 mg/kg each) and indomethacin (2 and 20 mg/kg) decrease the concentrations of cyclooxygenase products of arachidonate metabolism, but did not significantly affect levels of 15-HETE. Cysteinyl-LT were increased by 20 mg/kg indomethacin, but remained unaffected by 2 mg/kg indomethacin and by the other non-steroidal anti-inflammatory drugs (NSAID) tested. 15-HETE and cysteinyl-LT could play a mediator role in inflammation. In addition, they could modulate the release and effects of other inflammatory mediators.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Carrageenan; Chromatography, High Pressure Liquid; Cyclooxygenase Inhibitors; Dinoprostone; Hydroxyeicosatetraenoic Acids; Inflammation; Leukotriene B4; Lipoxygenase Inhibitors; Male; Radioimmunoassay; Rats; Rats, Inbred Strains; SRS-A; Thromboxane B2

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Blood Platelets; Docosahexaenoic Acids; Eicosapentaenoic Acid; Fatty Acids; Fish Oils; Humans; Hydroxyeicosatetraenoic Acids; Lipids; Lipoxygenase; Lipoxygenase Inhibitors; Neutrophils; Platelet Aggregation; Rats; Thromboxane B2

The effects of high frequency oscillatory ventilation (HFOV) and conventional mechanical ventilation (CMV) on tracheal secretion were compared in 6 anesthetized dogs. Using a double-balloon endotracheal catheter, 5 ml of saline was instilled into an isolated tracheal segment during HFOV and CMV for 10 min respectively. Two eicosanoids, 15-hydroxyeicosatetraenoic acid (15-HETE) and 11-dehydrothromboxane B2 (11-dehydro-TXB2) were measured by radioimmunoassay in each sample. HFOV (stroke volume: 6 ml/kg, f: 10 Hz, bias flow: 5 l/min) and CMV (stroke volume: 12 ml/kg, f: 15/min) were performed in random sequence and achieved comparable gas exchange. The concentration of 15-HETE in tracheal fluid during HFOV (87 +/- 67 pg/ml) was decreased to less than half of that during CMV (286 +/- 184 pg/ml, P less than 0.05), while there was no significant change of 11-dehydro-TXB2 either in tracheal fluid or in plasma. This reduction of 15-HETE was tended to be enhanced by vagotomy (HFOV: 42 +/- 14, CMV: 120 +/- 103 pg/ml) with the concentration ratio of CMV/HFOV remaining unchanged. HFOV may provide hitherto unrecognized advantage over CMV by reducing airway secretion of 15-HETE, a potent inflammatory mediator.

Topics: Animals; Catheters, Indwelling; Dogs; High-Frequency Ventilation; Hydroxyeicosatetraenoic Acids; Radioimmunoassay; Thromboxane B2; Trachea; Vagotomy

Prostaglandins (PGs) and leukotrienes (LTs) are known to play an important role in allergic inflammatory reactions. The triad of aspirin sensitivity, nasal polyposis, and asthma led us to suspect that PGs, LTs and other arachidonic acid metabolites may be involved in the pathogenesis of nasal polyps. The purpose of this study was to determine arachidonic acid metabolites and to measure concentrations of PGs and LTs in nasal polyps and nasal mucosa. Samples of nasal polyps and nasal mucosa were obtained at the time of polypectomies and nasal procedures. Metabolites of arachidonic acid in tissue were determined by incubation of tissue-homogenates with 14C-arachidonic acid and analyses with thin-layer chromatography and high performance liquid chromatography (HPLC). Levels of PGE2, 6-keto-PGF1 alpha, thromboxane (Tx)B2, 15-hydroxyeicosatetraenoic acid (HETE), LTC4, LTB4 were measured by radioimmunoassay. The predominant arachidonic acid metabolite in both nasal polyps and mucosa with 15-HETE. The HPLC analysis showed that the predominant metabolite in nasal polyp was 15-HETE, especially in polyps from aspirin sensitive patients. Levels of 15-HETE and PGE2 were higher in polyps from patients with a history of allergy than from nonallergic patients. Levels of LTC4 and LTB4 in nasal polyps were determined. The findings of this study will help to explain biochemical basis of the pathogenesis of aspirin-sensitive nasal polyps and to develop better medical treatment for them.

Topics: 6-Ketoprostaglandin F1 alpha; Arachidonic Acid; Arachidonic Acids; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Dinoprostone; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Nasal Mucosa; Nasal Polyps; Prostaglandins; Prostaglandins E; Radioimmunoassay; SRS-A; Thromboxane B2

Topics: Arachidonic Acids; Blood Platelets; Collagen; Dinoprost; Gas Chromatography-Mass Spectrometry; Humans; Hydroxyeicosatetraenoic Acids; Platelet Aggregation; Prostaglandins F; Thrombin; Thromboxane B2

Interactions between vascular endothelial cells and blood platelets have been investigated using a model microcirculation consisting of microcarrier beads colonized with human umbilical vein endothelial cells (HUVECs) and perfused with washed platelet suspensions. To simulate the effects of endothelial desquamation and exposure of subendothelium, fibrillar collagen in suspension was coinjected with the platelets. In this model, neither the passage of platelets alone nor collagen alone stimulated prostacyclin (PGI2) production by the HUVECs. Platelets activated by coinjection with collagen released thromboxane A2 (TXA2), and this was associated with the simultaneous production of PGI2 by the HUVECs. By means of double-isotope experiments with [3H]arachidonic acid (AA) incorporated into platelets and [14C]-AA into HUVECs, it was shown that all the PGI2 generated was derived from platelet AA and/or endoperoxides. This interpretation was strengthened by the finding that PGI2 production was not prevented by treatment of HUVECs with indomethacin followed by perfusion with collagen-stimulated platelets. AA metabolites in double-isotope label experiments were further characterized by reverse-phase chromatography, and it was shown that both cyclooxygenase and lipoxygenase products of the HUVECs were derived from platelet membrane lipid. Thrombin regularly produced transient PGI2 release, but showed rapid tachyphylaxis. Platelet-derived compounds including ADP, ATP, and platelet-activating factor (PAF) did not produce PGI2 release by HUVECs in this system. Thus, the transfer of AA and metabolites from collagen-stimulated platelets is likely to be the mechanism for PGI2 production in the context of minor degrees of endothelial desquamation.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonic Acid; Arachidonic Acids; Blood Platelets; Collagen; Endothelium; Epoprostenol; Female; Humans; Hydroxyeicosatetraenoic Acids; Pregnancy; Thromboxane B2; Time Factors; Umbilical Cord

The purpose of this study was to investigate the cellular composition of peritoneal fluid during post-surgical re-epithelialization and to determine the metabolism of arachidonic acid by these cells. Rabbits underwent a midline laparotomy followed by abrasion of the broad ligament. The presence of adhesions was graded and the peritoneal exudative cells collected up to 14 days thereafter. Ascitic fluid and cells were separated by centrifugation and the cellular percipitate incubated with [14C]arachidonic acid. The aliquot was separated by silica Gel G thin-layer chromatography and the specific radioactivity of each strip determined. To evaluate the reformation of adhesions 14 days after the first abrasion, rabbits underwent reabrasion of the same area and the pattern of arachidonic acid metabolism by the ascites cells was similarly evaluated. Six hours after the abrasion, PMNs comprised 87.5% of the peritoneal exudative cells (total 0.03 X 10(7) cells/rabbit). On Day 3, the total cell number increased to 2.92 X 10(7), 97.6% of which were large mononuclear cells. No significant change in the type of distribution of adhesions was evident from 6 hr through Day 2. After Day 7, the total number of adhesions was minimal; however, those that were present were primarily severe. The second-look evaluation of adhesion formation was not found to be consistent prior to the 7th postoperative day, since many filmy bands present prior to that time were not present later. The in vitro formation of 5-HETE by these cells increased from 6 hr through Day 11. Production of di-HETE increased beginning Day 3 and maintained high steady state levels thereafter.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Arachidonic Acids; Ascitic Fluid; Dinoprostone; Female; Hydroxyeicosatetraenoic Acids; Kinetics; Leukocytes; Peritoneum; Prostaglandins E; Rabbits; Thromboxane B2; Wound Healing

The effects of 5-, 5-lactone, 12- and 15-hydroxyeicosatetraenoic acids (HETEs) on the synthesis of leukotriene C4 (LTC4), thromboxane B2 (TXB2) and prostaglandin E2 (PGE2) by mouse resident peritoneal macrophages incubated with zymosan particles (100 micrograms/ml) were investigated. Zymosan phagocytosis stimulated a 110-, 16-, and 16-fold increase in LTC4, TXB2 and PGE2 synthesis, respectively. 15-HETE inhibited zymosan-induced LTC4 (IC50 = 1.1 microM) and TXB2 (IC50 = 38.9 microM) synthesis; in contrast, 15-HETE induced a consistent but variable enhancement of PGE2 synthesis. 5-HETE (IC50 = 15 microM), 5-lactone HETE (IC50 = 10.4 microM) and 12-HETE (IC50 = 13 microM) also inhibited LTC4 synthesis but they were approximately an order of magnitude less potent than 15-HETE. Furthermore, 5-HETE, 5-lactone HETE and 12-HETE inhibited TXB2 (IC50 = 20.4, 16.9 and 11.8 microM, respectively) and PGE2 (IC50 = 38.6, 2.3 and 11.6 microM, respectively) synthesis. Thus, monoHETEs exert modulatory actions on arachidonic acid metabolism and the different isomers of HETE differ quantitatively and qualitatively in their actions.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acid; Arachidonic Acids; Dinoprostone; Female; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Macrophages; Mice; Prostaglandins E; SRS-A; Thromboxane B2; Zymosan

A procedure using high performance liquid chromatography (HPLC) is described for the separation of major primary cyclooxygenase metabolites (prostacyclin metabolite-6ketoPGF1 alpha, thromboxane B2, and prostaglandins F2 alpha, E2, and D2), leukotrienes (C4, B4, and D4), monohydroxyeicosatetraenoic acids (15-, 11-, 12-, and 5HETEs), and free arachidonic acid. It is therefore possible to quantitate major arachidonic acid metabolites by a single chromatographic procedure. Using this technique we have determined that a major arachidonic acid metabolite of human lung macrophages co-elutes with leukotriene B4.

Topics: Arachidonic Acids; Chromatography, High Pressure Liquid; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Lung; Macrophages; Prostaglandins; Prostaglandins F; SRS-A; Thromboxane B2

Mono-hydroxy-eicosatetraenoic acids (HETE's) are frequently the principal lipoxygenase-derived products in a number of cell types. This paper describes the development of a selective and sensitive radioimmunoassay procedure for 15-HETE, a metabolite which has previously been shown to be both an activator and inhibitor of leukotriene formation in various cells. Initially, rabbits were immunized with 15-HETE conjugated to bovine serum albumin. After seven months, the anti-plasma showed significant binding of tritiated 15-HETE (40-45% binding with a 1:600 dilution of the anti-plasma) which was displaceable by cold 15-HETE. The sensitivity of the assay was approximately 20 pg. of 15-HETE. The anti-plasma exhibited very little (less than 1%) cross-reactivity with arachidonic acid, 5-, 8-, 9-, 11- and 12-HETE's, HHT, TXB2, PGE2 and 6-Keto-PGF1 alpha. Significant cross-reactivity was observed with 5,15-diHETE (53%), 8, 15-diHETE (6.6%), and several other 15-hydroxy-eicosanoids. Rabbit reticulocytes have a very active 15S-lipoxygenase and converted arachidonic acid (final concentration 7 microM) principally to 15-HETE. Unstimulated reticulocytes were found to release negligible amounts of 15-HETE as determined by radioimmunoassay. Treatment of these cells with the calcium ionophore A23187 (0.16 to 4.0 micrograms/ml) elicited a level of 15-HETE release (8 - 14 ng/ml) that was twenty to forty times less than that obtained with exogenous arachidonic acid (2.5 micrograms/ml). The radioimmunoassay reported here may be useful for identifying factors which stimulate cellular release of 15-HETE and other 15-hydroxy-eicosanoids from endogenous arachidonic acid.

Topics: Animals; Arachidonic Acids; Calcimycin; Cross Reactions; Hydroxyeicosatetraenoic Acids; Rabbits; Radioimmunoassay; Reticulocytes; Thromboxane B2; Time Factors

Within 5 min, resting macrophages metabolize microM quantities of exogenous arachidonic acid (20:4) to cyclooxygenase and lipoxygenase products. Mono-HETEs represent a major class of metabolites recovered from the medium. However, the quantity of mono-Hetes progressively decreases over a 60-min incubation period, with a concomitant increase in more polar lipoxygenase products, suggesting additional metabolic fates for these hydroxy acids. This was directly confirmed by exposing resident macrophage cultures to radiolabeled 15-, 12-, and 5-HETEs (1 microM). 12-30% of the recovered HETEs were cell-associated and predominantly esterified into phospholipid. High pressure liquid chromatography analyses of medium extracts indicated that 50% of each HETE was also converted to 10 or more metabolites over a 60-min time-course, a rate slower than for 20:4. The major metabolite generated from each mono-HETE had the elution characteristics of a di-HETE. The 5-HETE product has a triene spectrum similar to that of 5(S), 12(S)-di-HETE, whereas the 15- and 12-HETE products exhibited single ultraviolet absorption maxima, indicating a metabolic pathway for 5-HETE distinct from the other mono-HETEs. None of the stable cyclooxygenase products of 20:4 (6-keto PGF1 alpha, PGF2 alpha, PGE2, TXB2) nor polar metabolites of mono-HETEs are either incorporated or metabolized. The results indicate that macrophages have the capacity to specifically metabolize 20:4 and mono-HETEs to polar oxygenated products in the absence of a discernible trigger.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acids; Dinoprost; Dinoprostone; Female; Humans; Hydroxyeicosatetraenoic Acids; Macrophage Activation; Macrophages; Male; Mice; Mice, Inbred ICR; Phosphatidylcholines; Phospholipids; Prostaglandins E; Prostaglandins F; Thromboxane B2; Time Factors

The functional interrelationships between the cyclooxygenase and lipoxygenase systems for arachidonic acid metabolism in platelets have not yet been clarified. Although a number of specific inhibitors of the cyclooxygenase, such as aspirin and indomethacin, have been described, few inhibitors of the lipoxygenase have been found. Several hydroxy and hydroperoxy derivatives of arachidonic acid were prepared and purified by high performance liquid chromatography, and their structures were confirmed by gas chromatography-mass spectrometry. The effect of these compounds on oxygenation of [1-14C]arachidonic acid by platelet cyclooxygenase and lipoxygenase enzymes was monitored both with an oxygen electrode and by analysis of radioactive products formed. It was found that 15-hydroxy-5,8,11,13-eicosatetraenoic acid selectively inhibited platelet lipoxygenase activity at micromolar concentrations (I50 = 8 microM) without inhibiting the cyclooxygenase. The 15-hydroperoxy analog, although more potent, was less selective, whereas neither 12-hydroxy-5,8,10,14-eicosatetraenoic acid nor 12-hydroxy-9-octadecenoic acid (ricinoleic acid) appeared to affect either enzyme. The high degree of selectivity of the 15-hydroxy derivative of eicosatetraenoic acid makes it the most suitable inhibitor so far discovered for studying the functions of the platelet lipoxygenase system.

Topics: Arachidonic Acids; Blood Platelets; Cell-Free System; Humans; Hydroxyeicosatetraenoic Acids; Lipoxygenase; Lipoxygenase Inhibitors; Oxygen Consumption; Prostaglandin-Endoperoxide Synthases; Thromboxane B2