Concentrations Of 15-HETE Research Articles

12-lipoxygenase in opioid-induced delayed cardioprotection: gene array, mass spectrometric, and pharmacological analyses.

12-lipoxygenase (12-LO) has been shown to be a factor in acute ischemic preconditioning (IPC) in the isolated rat heart; however, no studies have been reported in delayed PC. We characterized the role of 12-LO in an intact rat model of delayed PC induced by a delta-opioid agonist SNC-121 (SNC). Rats were pretreated with SNC and allowed to recover for 24 hours. They were then treated with either baicalein or phenidone, 2 selective 12-LO inhibitors. In addition, SNC-pretreated rats had plasma samples isolated at different times after ischemia-reperfusion for liquid chromatographic-mass spectrometric analysis of the major metabolic product of 12-LO, 12-HETE. Similar studies were conducted with inhibitors. Gene array data showed a significant induction of 12-LO message (P<0.05) after opioid pretreatment. This induction in 12-LO mRNA was confirmed by real-time polymerase chain reaction, and 12-LO protein expression was enhanced by SNC pretreatment at 24 hours relative to vehicle treatment. Both baicalein and phenidone attenuated the protective effects of SNC pretreatment on infarct size (50+/-4% and 42+/-3% versus 29+/-2%, P<0.05, respectively). No significant differences were observed in 12-HETE concentrations between baseline control and SNC-treated rats. However, 12-HETE concentrations were increased significantly at both 15 minutes during ischemia and at 1 hour of reperfusion in the SNC-treated rats compared with controls. Baicalein and phenidone attenuated the increase in 12-HETE at 1 hour of reperfusion. These data suggest that SNC-121 appears to enhance message and subsequently the activity and expression of 12-LO protein during times of stress, resulting in delayed cardioprotection.

Failure of frusemide to increase production of prostaglandin E2 in human nasal mucosa in vivo.

It has been suggested that inhaled frusemide protects subjects with asthma against bronchoconstriction by enhancing the synthesis of prostaglandin E2 (PGE2). To evaluate this hypothesis the effect of frusemide on PGE2 production from nasal mucosa was studied. Two main arachidonic acid metabolites produced by epithelial cells, PGE2 and 15-hydroxy 5,8,11,13-eicosatetraenoic acid (15-HETE), were measured by radioimmunoassay in nasal secretions obtained by nasal lavages with saline. Eleven healthy volunteers were randomly assigned to two study days, one week apart, in a double blind crossover study. Nasal instillation with three increasing doses of frusemide (5, 10, and 20 mg) or placebo was carried out at intervals of 15 minutes. Nasal lavages were performed immediately before nasal instillations and 15, 30, and 60 minutes after the last instillation. Baseline concentrations of 15-HETE were at least six times higher than PGE2. No differences between frusemide and placebo were detected either on PGE2 or 15-HETE release. The findings do not support the hypothesis that the antiasthmatic effect of frusemide may be due to increased synthesis of PGE2 or release in the respiratory mucosa.

Human tracheal epithelial cells selectively incorporate 15-hydroxyeicosatetraenoic acid into phosphatidylinositol.

15-hydroxyeicosatetraenoic acid (15-HETE) is the major lipoxygenase metabolite of arachidonic acid produced by human airway epithelial cells. Because HETEs have been shown to be rapidly metabolized and/or incorporated into cellular lipids in other cell types, we investigated the uptake, metabolism, and intracellular distribution of exogenous 15-HETE by primary monolayer cultures of human tracheal epithelial (HTE) cells. At concentrations of 0.1 microM, [3H]15-HETE was rapidly incorporated by HTE cells and also metabolized primarily by beta-oxidation to several more polar products that were released extracellularly. The majority of cell-associated [3H]15-HETE radiolabel was distributed into phospholipids, with phosphatidylinositol (PI) accounting for approximately 75% of phospholipid radiolabel. Exogenous 5- and 12-HETE were also metabolized by HTE cells but were less extensively incorporated into phospholipids and were distributed primarily into phosphatidylcholine and phosphatidylethanolamine. Phospholipase A2 hydrolysis indicated selective esterification of unmodified 15-HETE to the sn-2 position of phospholipids. 15-HETE incorporation into total phospholipids and into PI was saturable (half maximal incorporation at 0.82 and 0.68 microM, respectively), while incorporation into neutral lipids continued to increase at concentrations of 15-HETE up to 5 microM. The incorporation of 15-HETE into PI was metabolically stable, with an intracellular half-life of 12 h, and was not subject to mobilization in response to 5 microM calcium ionophore A23187. HTE cells can incorporate and metabolize HETEs that the cells themselves produce as well as those that might be released by inflammatory cells recruited into the airway.(ABSTRACT TRUNCATED AT 250 WORDS)

Langerhans cells of the human skin possess high-affinity 12(S)-hydroxyeicosa tetraenoic acid receptors.

The arachidonic acid metabolite 12-hydroxyeicosatetraenoic acid (12-HETE) is the main eicosanoid formed by epidermal cells and is assumed to play an important role in skin physiology and pathophysiology. Our aim was to find out whether epidermal Langerhans cells possess specific receptors for 12-HETE which would mediate the effects of this eicosanoid in their skin microenvironment. By radioligand binding studies on isolated human Langerhans cells, we could identify specific binding sites for 12(S)-HETE. The analysis of binding data revealed a single class of binding sites with a Kd of 3.32 +/- 0.45 nM and a Bmax of 691,000 +/- 58,000 receptors per cell. The binding was saturable, readily reversible, and specific for 12(S)-HETE. The receptor is likely to mediate the potent chemotactic response of human Langerhans cells towards 12(S)-HETE, which we previously described. Our results strongly suggest that extremely low concentrations of 12-HETE which is formed by epidermal keratinocytes may dramatically influence the biology of Langerhans cells by receptor-mediated effects.

Saturability of esterification pathways of major monohydroxyeicosatetraenoic acids in rat basophilic leukemia cells

The principal monohydroxyeicosatetraenoic acids (HETEs), 5-, 12-, and 15-HETE, which can be produced by rat basophilic leukemia (RBL-1) cells, are also esterified by these cells. Exogenously added 5-, 12-, and 15-HETE were rapidly incorporated as esters in RBL cells, reaching plateau levels within 25 min. In incubations in culture medium with protein added, all three HETEs were essentially completely metabolized within 24 h. 5-HETE was esterified more rapidly and to a greater extent than 12-HETE or 15-HETE when these were incubated together with RBL cells, indicating some degree of selectivity in the esterification pathways. When arachidonic acid (AA) was incubated in increasing concentrations with constant concentrations of 15-HETE and RBL cells, the free 15-HETE concentration increased and esterified 15-HETE concentration decreased markedly at AA: 15-HETE molar ratios above 9. 15-HETE esterification in RBL cells was also markedly inhibited by the polyunsaturated fatty acids, eicosatetraynoic and eicosapentanoic acids, but not by oleic or linoleic acids. In separate experiments with unlabeled and radiolabeled substrates, the extent of incorporation of esterified HETE in RBL cells decreased at higher concentrations of 15-HETE and AA, which showed that the pathway was saturable. The shapes of the curves for these fatty acid inhibitors suggest a concentration-dependent two-compartment pathway of esterification. These data indicate that the HETEs and other 20 carbon fatty acid substrates probably compete for activity of a specific arachidonyl-CoA synthetase, which is the first and rate-limiting step for esterification of arachidonic acid by many human cells. Esterified 15-HETE was found to be predominantly in the phosphatidylethanolamine fraction of RBL cell lipids.

Release of 15-hydroxy-5,8,11,13-eicosatetraenoic acid and cysteinyl-leukotrienes in carrageenin-induced inflammation: Effect of non-steroidal anti-inflammatory drugs

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.

12-HETE inhibits the binding of PGH 2/TxA 2 receptor ligands in human platelets

12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE), the end-lipoxygenase product of arachidonic acid in platelets has been previously shown to prevent PGH 2/TXA 2-induced aggregation. From the present study, we show that 12-HETE inhibits the binding of [ 125I]-PTA-OH, a thromboxane antagonist, to platelet membranes with an IC 50 of 8 μM. This value is in accordance with previously reported 12-HETE concentrations required to prevent the aggregation induced by TxA 2 mimetics, the methano analogues of PGH 2, U44069 and U46619. When [ 3H]-U44069 was used as a thromboxane agonist to label intact platelets, 12-HETE also inhibited its binding. We conclude that part of the inhibitory effect of 12-HETE on PGH 2/TxA 2-induced aggregation might be the result of interacting with PGH 2/TxA 2 receptor sites.

12-Hydroxyeicosatetraenoic acid (12-HETE) is a chemotactic stimulus for epidermal cells

Several observations point to an important role of 12HETE and LTB4 in cutaneous physiology and in the pathophysiology of inflammatory skin diseases, in particular psoriasis [13]. Normal epidermal cells possess a high capacity for the transformation of arachidonic acid (AA) into 12-HETE by 12-1ipoxygenase. It is the main eicosanoid which is generated in epidermal homogenates besides PGE2 and P G D 2 [9, 14]. In contrast, the 5lipoxygenase product LTB4 is formed mainly by leukocytes [4] which infiltrate skin during inflammation. In psoriasis, highly elevated concentrations of 12-HETE are found in lesional skin [2, 8]. In addition, 12-HETE and LTB4 are chemoattractants for PMNLs which could contribute to the infiltration of PMNLs in psoriatic skin and other cutaneous inflammatory reactions [3, 6]. Although the metabolic processes which lead to the formation of 12-HETE in skin are well understood, the exact function of this AA-metabolite in cutaneous biology is not clear. Growth stimulation of keratinocytes by 12-HETE was reported by Kragballe and Fallon [11], but has not been confirmed by other groups [7, 12]. Here we report a novel aspect of 12-HETE and LTB 4 in cutaneous biology. We detected a chemotactic response of epidermal cells towards 12-Sand 12-R-HETE as well as LTB4. The response of the cells towards 12-HETE was in the range of the KD of a high affinity 12-HETE receptor described recently by our group [7]. Eicosanoids (12-S-HETE, 12-R-HETE, LTB4) were supplied by Paesel (Frankfurt/Main, FRG) as free acids in ethanol. The ethanol was evaporated under nitrogen immediately before use, and the eicosanoids were dissolved in serum-free DMEM to the required concentration. EGF was purchased from Sebak (Aidenbach, FRG). Fibroblast-conditioned medium (FCM) was pre-

12-Hydroxyeicosatetraenoic acid is metabolized by beta-oxidation in mouse peritoneal macrophages. Identification of products and proposed pathway.

The products derived from the metabolism of 12-hydroxyeicosatetraenoic acid (12-HETE) by mouse peritoneal macrophages were characterized by high performance liquid chromatography (HPLC) and GC-mass spectrometry. HPLC analysis demonstrated two predominant polar products and several minor ones. The proportion and amounts of these products were dependent on the concentration of 12-HETE, the number of macrophages incubated with the monohydroxy fatty acid, and the time of incubation. The products identified by GC-mass spectrometry suggested that 12-HETE had undergone beta-oxidation. The intermediates identified were: 3,12-dihydroxy-5,8,10,14, 20:4; 10-hydroxy-3,6,8,12, 18:4; 3,10-dihydroxy-6,8,12, 18:3; 8-hydroxy-4,6,10, 16:3; 6-hydroxy-4,8, 14:2; and 4-hydroxy, 12:1. The major products, as identified by HPLC and GC-mass spectrometry, were 8-hydroxy-4,6,10, 16:3 and 4-hydroxy, 12:1. A minor product, 10-hydroxy-6,8,12, 18:3 was postulated to arise from either the isomerization and reduction of 10-hydroxy-3,6,8,12, 18:4 or from chain elongation of 8-hydroxy-4,6,10, 16:3. Inhibiting cyclooxygenase and lipoxygenase activities by ibuprofen and nordihydroguaiaretic acid, respectively, did not inhibit the formation of these products. 82% to 98% of 12-HETE was converted and released into the medium as products of beta-oxidation. The remainder was taken up into cellular lipids. beta-Oxidation of 12-HETE was decreased by only 12 and 21% after inhibiting mitochondrial fatty acid oxidation by 89 and 93% by 5 and 100 microM concentrations of the mitochondrial fatty acid oxidation inhibitor, methyl palmoxirate, respectively. It is thus postulated that the beta-oxidation of 12-HETE by mouse peritoneal macrophages occurs in peroxisomes.

Amniotic fluid arachidonate lipoxygenase metabolites in preterm labor

This study was conducted to determine if preterm labor with intact membranes is associated with changes in the amniotic fluid concentrations of arachidonate lipoxygenase metabolites. Amniotic fluid was obtained by transabdominal amniocentesis from 68 women with preterm labor. The patients were classified into three groups according to their response to tocolysis and their amniotic fluid culture results: Group 1 — women with a negative amniotic fluid culture who responded to tocolysis (n = 32); Group 2 — women with a negative culture, but who failed to respond to tocolysis (n = 22); and Group 3 — women with intraamniotic infection (n = 14). The following arachidonate lipoxygenase products were measured by radioimmunoassay: leukotriene B 4 (LTB 4); leukotriene C 4 (LTC 4); 12-hydroxyeicosatetraenoic acid (12-HETE); and 15-hydroxyeicosatetraenoic acid (15-HETE). The median concentrations of LTB 4 were significantly different among the three study groups (26 pg/ml, 67 pg/ml and 885 pg/ml, respectively, p > 0.05). Amniotic fluid concentrations of 12-HETE and LTC 4 did not vary among the three study groups. On the other hand, a significant difference in the distribution of amniotic fluid concentrations of 15-HETE was noted only between women with intraamniotic infection (Group 3) and women responding to tocolysis (Group 1). These results indicate that the arachidonate lipoxygenase pathway is activated during the course of preterm labor. Selective changes in the concentrations of the assayed metabolites were noted. Amniotic fluid LTB 4 concentrations may be a marker for the patient with preterm labor who is unresponsive to tocolysis.

Prostaglandins and Other Arachidonic Acid Metabolites in the Middle Ear Fluids

Prostaglandins (PGs) and other arachidonic acid (AA) metabolites have been recognized as mediators of inflammation and bone resorption, chemotactic agent to polymorphonuclear leukocytes (PMN), and regulators of inflammatory response. Although assays of some PGs in the middle ear fluid (MEF) were reported, there has been no report on comprehensive assay of PGs and other AA metabolites in MEF. Concentrations of PGs were measured by radioimmunoassay in human MEF. The data on human MEF were analyzed according to age group. In addition, levels of lipoxygenase-products, 5- and 15-hydroxyeicosatetraenoic acid (HETE) and leukotriene (LT)C4 were measured in human MEF. Levels of PGE2 in MEF from human serous otitis media (SOM) were highest in younger age group (below the age of 10) then the levels drop down to lower level in older age group. In human mucoid otitis media (MOM) the levels of PGE2 are lower than SOM in younger age group then become higher than SOM in older age group. Levels of 6-keto-PGF1 alpha were higher in SOM than in MOM overall. The age breakdown of data showed progressive increase in the 6-keto-PGF1 alpha levels with older age group. Levels of TxB2 in SOM were higher than in MOM overall. The age breakdown of SOM data remained almost the same in all the age groups but the MOM data showed lowest levels in 6-10 year-old group and highest in 11-15 year-old group. Concentrations of 15-HETE in human MOM were over two times higher than in SOM. LTC4 levels in MOM were higher than SOM.(ABSTRACT TRUNCATED AT 250 WORDS)