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W S Powell

Publications and source records attributed to W S Powell.

At least 37 records · Page 2Linked to original sources
PubMed1998

Calcium/calmodulin-dependent conversion of 5-oxoeicosanoids to 6, 7-dihydro metabolites by a cytosolic olefin reductase in human neutrophils.

We previously showed that 6-trans isomers of leukotriene B4 but not leukotriene B4 itself are converted to dihydro metabolites by human neutrophils. The first step in the formation of these metabolites is oxidation of the 5-hydroxyl group by 5-hydroxyeicosanoid dehydrogenase. The objective of the present investigation was to characterize the second step in the formation of the dihydro metabolites, reduction of an olefinic double bond. We found that the olefin reductase reduces the 6,7-double bond of 5-oxoeicosanoids, is localized in the cytosolic fraction of neutrophils, and requires NADPH as a cofactor. Neutrophil cytosol converts a variety of both 5-oxo- and 15-oxoeicosanoids to dihydro products. However, conversion of 5-oxoeicosanoids to their 6,7-dihydro metabolites is inhibited by EGTA and a calmodulin antagonist and stimulated by the addition of calcium and calmodulin, whereas the reduction of 15-oxoeicosanoids to their 13,14-dihydro metabolites is slightly inhibited by calcium. Furthermore, eicosanoid Delta6- and Delta13-reductases could be separated by chromatography on DEAE-Sepharose. 5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is converted by the Delta6-reductase to 6,7-dihydro-5-oxo-ETE, which is 1000 times less potent than 5-oxo-ETE in mobilizing calcium in neutrophils. We conclude that neutrophils contain both 5-oxoeicosanoid Delta6-reductase and prostaglandin Delta13-reductase. Metabolism of 5-oxo-ETE by the Delta6-reductase results in loss of its biological activity.

Calcium↗
PubMed1997

Effects of 5-oxo-6,8,11,14-eicosatetraenoic acid on expression of CD11b, actin polymerization, and adherence in human neutrophils.

Human neutrophils contain a highly specific dehydrogenase that converts 5-hydroxy-6,8,11,14-eicosatetraenoic acid to 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE). 5-Oxo-ETE is a potent stimulator of calcium mobilization, chemotaxis, and aggregation in these cells and has similar effects on eosinophils. The primary objectives of the current study were to determine whether this compound could increase the surface expression of integrins and stimulate actin polymerization in neutrophils. 5-Oxo-ETE stimulated the expression of CD11b and, to a lesser extent, CD11c, on neutrophils, but had no significant effects on the expression of CD11a, CD16 (Fc gammaRIII), or CD32 (Fc gammaRII). Surface expression of CD11b in response to 5-oxo-ETE was maximal after 12 min and remained constant thereafter. The EC50 for this response (50 nM) was lowered to 20 nM by preincubation of neutrophils with PMA. 5-Oxo-ETE (EC50, 10 nM) also rapidly stimulated actin polymerization in neutrophils, with a maximal response at 20 s. This response was blocked by pretreatment of neutrophils with the Gi protein inhibitor, pertussis toxin, and by homologous desensitization due to preincubation with 5-oxo-ETE. However, preincubation with leukotriene B4 or platelet-activating factor had no effect on the response of neutrophils to subsequent addition of 5-oxo-ETE. The adherence of neutrophils to plasma-coated plastic was also stimulated by 5-oxo-ETE with a time course similar to that for the surface expression of CD11b. Low concentrations of PMA (0.3 nM) enhanced this response. These results raise the possibility that 5-oxo-ETE could contribute to the infiltration of neutrophils into inflammatory sites.

Actins↗
PubMed1997

High-pressure liquid chromatography of oxo-eicosanoids derived from arachidonic acid.

Eicosanoids are a large group of biologically active metabolites of arachidonic acid and related C20 fatty acids. Many of these compounds contain hydroxyl groups which can be converted to oxo groups by a variety of substrate-specific dehydrogenases. In many cases, this results in a reduction in potency, but in others, such as the oxidation of 5-hydroxyeicosatetraenoic acid to its oxo metabolite 5-oxo-eicosatetraenoic acid, there is a dramatic increase in biological activity. Thus, it is often very important to analyze the relative amounts of oxo- and hydroxy-eicosanoids formed by various cells and tissues. The present study was designed to compare the chromatographic behavior of oxo-eicosanoids and their hydroxy counterparts in commonly used mobile phases for reversed-phase and normal-phase HPLC. We examined three groups of eicosanoids: prostaglandins, leukotriene B4 and some of its metabolites, and monohydroxy-eicosanoids and their oxo metabolites. We found that in reversed-phase HPLC, the retention times of oxo-eicosanoids were longer than those of the corresponding hydroxy-eicosanoids in mobile phases containing acetonitrile as the major organic component, whereas the reverse was true for mobile phases containing methanol. Normal-phase HPLC using mobile phases containing hexane, isopropanol, and acetic acid gave excellent separation of oxo- and hydroxy-eicosanoids. Increasing the concentration of acetic acid in the mobile phase selectively reduced the retention times of oxo-eicosatetraenoic acids compared to monohydroxy-eicosatetraenoic acids, whereas the reverse was true for isopropanol. Differences in the chromatographic behavior of oxo- and hydroxy-eicosanoids can be useful clues in the structural characterization of these compounds, as illustrated by the chromatographic properties of a complex series of LTB4 metabolites formed by rat neutrophils.

Animals↗
PubMed1997

Hyperpnea-induced bronchoconstriction is dependent on tachykinin-induced cysteinyl leukotriene synthesis.

The purpose of the study was to test the hypothesis that tachykinins mediate hyperpnea-induced induced bronchoconstriction indirectly by triggering cysteinyl leukotriene (LT) synthesis in the airways. Guinea pigs (350-600 g) were anesthetized with xylazine and pentobarbital sodium and received hyperpnea challenge (tidal volume 3.5-4.0 ml, frequency 150 breaths/min) with either humidified isocapnic gas (n = 6) or dry gas (n = 7). Dry gas challenge was performed on animals that received MK-571 (LTD4 antagonist; 2 mg/kg i.v.; n = 5), capsaicin (n = 4), neurokinin (NK) antagonists [NK1 (CP-99994) + NK2 (SR-48968) (1 mg/kg i.v.); n = 6], or the H1 antihistamine pyrilamine (2 mg/kg i.v.; n = 5). We measured the tracheal pressure and collected bile for 1 h before and 2 h after hyperpnea challenge. We examined the biliary excretion of cysteinyl LTs; the recovery of radioactivity in bile after instillation of 1 microCi [3H]LTC4 intratracheally averaged 24% within 4 h (n = 2). The major cysteinyl LT identified was LTD4 (32% recovery of radioactivity). Cysteinyl LTs were purified from bile of animals undergoing hyperpnea challenge by using reverse-phase high-pressure liquid chromatography and quantified by radioimmunoassay. There was a significant increase in the peak value of tracheal pressure after challenge, indicating bronchoconstriction in dry gas-challenged animals but not after humidified gas challenge. MK-571, capsaicin, and NK antagonists prevented the bronchoconstriction; pyrilamine did not. Cysteinyl LT levels in the bile after challenge were significantly increased from baseline in dry gas-challenged animals (P < 0.05) and were higher than in the animals challenged with humidified gas or dry gas-challenged animals treated with capsaicin or NK antagonists (P < 0.01). The results indicate that isocapnic dry gas hyperpnea-induced bronchoconstriction is LT mediated and the role of tachykinins in the response is indirect through release of LTs. Endogenous histamine does not contribute to the response.

Animals↗
PubMed1996

Metabolism and biologic effects of 5-oxoeicosanoids on human neutrophils.

5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is a recently discovered metabolite of arachidonic acid that activates human neutrophils by a mechanism independent of the receptor for leukotriene B4 (LTB4). The objectives of this study were to identify the major metabolites of 5-oxo-ETE in neutrophils and to compare the biologic activities of 5-oxo-ETE with those of its metabolites and other 5-oxoeicosanoids. Neutrophils rapidly converted 5-oxo-ETE to its omega-oxidation product, 5-oxo-20-hydroxy-6E,8Z,11Z,14Z- eicosatetraenoic acid. This compound was nearly 100 times less potent than 5-oxo-ETE in elevating cytosolic calcium levels in neutrophils. Methylation of the carboxyl group of 5-oxo-ETE resulted in a 20-fold loss of potency, whereas replacement of the 8,9-cis double bond by a trans double bond reduced potency by about sixfold. Similar results were obtained for the effects of the above compounds on neutrophil migration. 5-Oxo-20-hydroxy-6E,8Z,11Z,14Z- eicosatetraenoic acid, 5-oxo-8-trans-ETE, and 5-oxo-ETE methyl ester desensitized neutrophils to 5-oxo-ETE. 5-Oxo-ETE-induced calcium mobilization was inhibited by pretreatment of the cells with pertussis toxin. 5-Oxo metabolites of 6-trans-LTB4 and 12-epi-6-trans-LTB4 had weak stimulatory effects on calcium levels and migration that appeared to be mediated primarily by stimulation of LTB4 receptors. These studies indicate that the 5-oxo group, the omega-end of the molecule, and the carboxyl group are all important for the biologic activity of 5-oxo-ETE, which may be mediated by a G protein-linked receptor. The biologic activity of 5-oxo-ETE can be terminated by omega-oxidation.

Arachidonic Acids↗
PubMed1996

Synthesis of 5-oxo-6,8,11,14-eicosatetraenoic acid by human monocytes and lymphocytes.

We recently demonstrated that the arachidonate metabolite 5(S)-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) is converted by a highly specific dehydrogenase in human neutrophils to 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE), which is a potent stimulator of these cells. The objective of this study was to determine whether 5-oxo-ETE is also formed by monocytes and lymphocytes. Human monocytes (74 +/- 2% pure) and lymphocytes (86 +/- 1% pure) were prepared by successive centrifugations of leukocytes over Ficoll-Paque and Percoll. Both cell types converted 5-HETE to a single major product, which was identified as 5-oxo-ETE. The formation of 5-oxo-ETE was stimulated about twofold by phorbol myristate acetate (PMA; 30 nM). Dehydrogenase activity in monocyte fractions did not appear to be due to platelet contamination, since depletion of platelets did not reduce enzyme activity. The dehydrogenase was localized in membrane fractions from monocytes and required NADP+ as a cofactor. It was specific for eicosanoids containing a 5S-hydroxyl group followed by a 6-trans double bond. We also investigated the formation of 5-oxo-ETE from endogenous arachidonic acid by monocytes. 5-Oxo-ETE, 5-HETE, and leukotriene B4 (LTB4) were present in comparable amounts after incubation of these cells with A23187. PMA (EC50 approximately 4 nM) stimulated the formation of 5-oxo-ETE and 5-HETE and, to a lesser extent, LTB4. Although monocytes released considerably less 5-HETE and LTB4 than neutrophils, they released comparable amounts of 5-oxo-ETE. Unlike neutrophils, monocytes did not convert any of these substances to detectable amounts of omega-oxidation products. Although lymphocytes were capable of converting 5-HETE to 5-oxo-ETE, they released little or no 5-lipoxygenase products in response to A23187. We conclude that monocytes have a high capacity to synthesize 5-oxo-ETE and that its formation is stimulated by activation of protein kinase C.

Alcohol Oxidoreductases↗
PubMed1996

The role of endogenous corticosterone in the late-phase response to allergen challenge in the brown Norway rat.

The aim of this study was to assess the role of endogenous corticosterone (CCST) concentrations on the late airway response (LAR) to ovalbumin (OA). Thirty-two Brown Norway (BN) rats were sensitized to OA on Day 0, then divided into three groups. Group 1 (n = 11) received saline (1 ml, subcutaneously) at time (T) = -24, -12, and 0 h prior to a 5% OA challenge on Day 14. Group 2 (n = 11) received metyrapone (MTP), an 11 beta-hydroxylase inhibitor, (10 mg/100 g in 1 ml saline, subcutaneously) at time (T) = -24, -12, and 0 h. Group 3 (n = 10) received MTP on the same schedule as Group 2, plus CCST in the drinking water (16 micrograms/ml) from T = -24 to T = 0 h. Pulmonary resistance (RL) was measured for 8 h following OA challenge to determine early (EAR) and LAR. Blood samples were taken at T = 0 and T = 8 h to determine serum CCST levels. Bronchoalveolar lavage (BAL) fluid was collected at T = 8 h. Serum CCST levels were significantly reduced in the MTP group (235 ng/ml +/- 14 SEM) compared with control (564 +/- 38, p < 0.0001) and MTP+CCST animals (349 +/- 19, p < 0.0005). There were no differences in either baseline RL or EAR between the groups. However, the MTP group had a smaller LAR (6 ml/cm H2O/s*min +/- 2 SEM) than the control group (19 +/- 5, p < 0.02). The effect of MTP on LAR was reversed by treatment with CCST (21 +/- 3, p < 0.005). Total cell counts (p < 0.05) and eosinophils (p < 0.01) were increased in the BAL fluid of MTP rats versus control and MTP+CCST animals. We conclude that depletion of endogenous CCST in the BN rat diminishes the LAR to allergen challenge. These results indicate that physiologic levels of CCST are not necessary for development of the EAR, but they play a permissive role in the LAR to inhaled allergen.

Airway Resistance↗
PubMed1996

Effects of metabolites of leukotriene B4 on human neutrophil migration and cytosolic calcium levels.

Leukotriene B4 (LTB4) is metabolized by beta-oxidation, omega-oxidation and the 12-hydroxyeicosanoid dehydrogenase/delta 10-reductase pathway. We have investigated the effects of metabolites formed by the latter pathway on calcium mobilization and migration in human neutrophils and have compared their potencies with those of other LTB4 derivatives. 12-Oxo-LTB4 and 10,11-dihydro-LTB4 were 60 to 100 times less potent than LTB4 in stimulating neutrophils, whereas 10,11-dihydro-12-oxo-LTB4 and 10,11-dihydro-12-epi-LTB4 exhibited still lower potencies. The 6-trans isomers of 12-oxo-LTB4 and 10,11-dihydro-12-oxo-LTB4 were much less potent than the 6-cis compounds. The EC50 values for biologically and chemically (6-cis) synthesized 12-oxo-LTB4 were similar, indicating that the 6,7-double bond is retained in the cis configuration in the biologically formed compound. Methylation of LTB4 markedly reduced its effect on cytosolic calcium levels, whereas addition of a 3-hydroxyl group had a much more modest effect. Modifications of the omega end of the molecule also resulted in lower potencies for calcium mobilization. Nearly all of the compounds tested desensitized neutrophils to LTB4-induced calcium mobilization, which suggests that their effects were mediated by receptors for the latter compound. However, modifications in the carboxyl end of the molecule had smaller effects on desensitization than on calcium mobilization, whereas the reverse was true for modifications in the omega end of the molecule. This suggests that the structural requirements for agonist-induced desensitization to LTB4 may differ to some extent from the requirements for calcium mobilization.

Calcium↗
PubMed1995

5-Oxo-6,8,11,14-eicosatetraenoic acid is a potent stimulator of human eosinophil migration.

Human neutrophils and monocytes contain a highly specific dehydrogenase which converts 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) to 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE). We have previously shown that 5-oxo-ETE is a potent stimulus of neutrophil calcium levels and migration and have now investigated its effects on human eosinophils. 5-Oxo-ETE is a potent stimulus of eosinophil migration, with significant effects being detected at concentrations as low as 1 nM and a maximal response at 1 microM. The responses elicited by 5-oxo-ETE were about two to three times greater than those to platelet-activating factor (PAF) and 5-oxo-15-hydroxy-6,8,11,13-eicosatetraenoic acid (5-oxo-15-hydroxy-ETE) at all concentrations tested between 10 nM and 1 microM. Leukotrienes B4 and D4 also significantly stimulated eosinophil migration, but the maximal responses to these agonists were only about 4% of the maximal response to 5-oxo-ETE. A low concentration of 5-oxo-ETE (1 nM) potentiated eosinophil migration in response to PAF. Eosinophils were capable of converting 5-HETE to 5-oxo-ETE, and this reaction was enhanced by phorbol myristate acetate. Stimulation of eosinophils with A23187 in the presence of low concentrations of arachidonic acid and phorbol 12-myristate 13-acetate led to the formation of 5-oxo-ETE and 5-oxo-15-hydroxy-ETE, but the amounts were considerably less than those of other eicosanoids such as leukotriene C4, cysteine-containing lipoxins, and 5,15-dihydroxy-6E,8Z,11Z,13E-eicosatetraenoic acid. In summary, of all the lipid mediators tested, 5-oxo-ETE was the most effective in stimulating migration of human eosinophils. Although eosinophils are capable of synthesizing 5-oxo-eicosanoids, the amounts detected were relatively small, and other leukocytes such as neutrophils, monocytes, or macrophages may be more important sites for the synthesis of this compound.

Arachidonic Acid↗
PubMed1995

Analysis of leukotrienes, lipoxins, and monooxygenated metabolites of arachidonic acid by reversed-phase high-pressure liquid chromatography.

Arachidonic acid can be converted to a large number of metabolites by various lipoxygenases, cyclooxygenase, and other enzymes. Because of the complex profiles of products formed by many types of cells, high-pressure liquid chromatography (HPLC) has proved to be an invaluable technique for their purification and analysis. In the present study we have developed improved methods for the analysis of complex mixtures of eicosanoids by HPLC using binary gradients containing trifluoroacetic acid (TFA), which allows considerable manipulation of the retention times of the cysteine-containing leukotrienes (LTs) LTC4, LTD4, and LTE4 relative to those of other eicosanoids. With a gradient between 0.003 and 0.005% TFA and a 4.6-mm-i.d. column of Spherisorb ODS-2, cysteinyl-LTs are very well resolved from one another and are separated as a group with retention times longer than those of all other major eicosanoids. These conditions can be used for the analysis of prostaglandin B2 (PGB2), LTB4, monohydroxyeicosatetraenoic acids (HETEs), and cysteinyl-LTs in only 30 min. Slightly longer analysis times must be used for the separation of more polar eicosanoids such as hydroxy metabolites of LTB4 and lipoxins. We have also developed methods for the analysis of eicosanoids using a midbore (3.2 mm i.d.) column containing Spherisorb ODS-2, which improves sensitivity and reduces solvent consumption. In this case higher concentrations (0.04 to 0.05%) of TFA have been used, resulting in retention times for cysteinyl-LTs between those of the cyclooxygenase product 12-hydroxy-5,8,10-heptadecatrienoic acid and the HETEs. This approach permits analysis of PGB2, LTB4, HETEs, and cysteinyl-LTs in only 20 min. Samples which also contain hydroxy-LTB4 and lipoxins can be analyzed in 40 min. The above techniques are highly reproducible and give baselines which are free of interfering peaks.

Animals↗
PubMed1995

Effects of oxo and dihydro metabolites of 12-hydroxy-5,8,10,14-eicosatetraenoic acid on chemotaxis and cytosolic calcium levels in human neutrophils.

One of the pathways of metabolism of leukotriene B4 (LTB4) and 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) in leukocytes is oxidation of the 12-hydroxyl group, followed by reduction of the 10,11-double bond. In the case of 12R-HETE and 12S-HETE, this results in the formation of 12-oxo-ETE, 10,11-dihydro-12-oxo-ETE, and the 12R and 12S isomers of 10,11-dihydro-12-HETE (i.e., 12R-HETrE and 12S-HETrE). We investigated the effects of metabolites of 12-HETE formed by this pathway on cytosolic calcium levels and chemotaxis in human neutrophils. Of the above series of metabolites, 12S-HETrE (which has the same absolute stereochemistry at C-12 as 12R-HETE) was the most potent in stimulating both cytosolic calcium levels and chemotaxis. It was slightly less potent than 12R-HETE, consistent with the concept that reduction of the 10,11-double bond results in a loss of biological activity on neutrophils. The effect of 12S-HETrE on calcium levels was blocked by preincubation of these cells with LTB4, suggesting that it acted by stimulating the LTB4 receptor. 12R-HETrE was about 20 times less potent than its 12S isomer in stimulating cytosolic calcium in neutrophils and was also less active as a chemotactic agent. Oxidation of the 12-hydroxyl group to an oxo group resulted in a further loss of biological activity. 12-Oxo-ETE, 8-trans-12-oxo-ETE, and 12-oxo-ETrE had only modest effects on cytosolic calcium levels at concentrations as high as 10 microM and did not display detectable chemotactic activity. However, 12-oxo-ETE and its 8-trans isomer inhibited calcium responses to LTB4 by about 40%. It is concluded that reduction of the 10,11-double bond of 12-HETE results in a slight loss of biological activity on neutrophils, whereas oxidation of the 12-hydroxyl group results in a considerably greater loss of activity.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1995

Effects of dexamethasone on leukotriene synthesis and airway responses to antigen and leukotriene D4 in rats.

Cysteinyl-leukotrienes (cysteinyl-LTs) are important mediators of both early and late allergen-induced airway responses in atopic asthmatics. We have investigated the effects of glucocorticoids on the in vivo synthesis of cysteinyl-LTs and airway responses to these mediators. Ovalbumin (OA)-sensitized Brown Norway rats, which exhibit early responses and, in most cases, also late responses to late antigen challenge, were treated with either one dose (1 h before antigen challenge) or two doses (2 and 14 h before challenge) of dexamethasone (dex; 300 micrograms/kg). A third group of sensitized rats which did not receive dex served as control animals. All rats were subjected to bile duct cannulation and tracheal intubation 2 h before intratracheal instillation of OA. All of the OA-treated control rats had pronounced early responses (5.5 +/- 1.0 times baseline pulmonary resistance [RL]), whereas 5 of 7 rats had late responses. Biliary levels of N-acetyl-LTE4 increased in all control rats following antigen challenge with a maximum occurring between 1 to 2 h, followed by a decline. There appeared to be a second maximum between 6 to 7 h after challenge, although this could not be demonstrated statistically because of the variability in the response among animals. Treatment with two doses of dex reduced the early response by 57%, completely eliminated the late response, and reduced the levels of N-acetyl-LTE4 in bile by about 80% at all time points.(ABSTRACT TRUNCATED AT 250 WORDS)

Airway Resistance↗
PubMed1995

Cellular infiltration and eicosanoid synthesis in brown Norway rat lungs after allergen challenge.

Allergen challenge of sensitized Brown-Norway (BN) rats results in increased excretion of cysteinyl-leukotrienes (cLTs) in bile. It is unclear whether this reflects an increased capacity of lung cells to synthesize 5-lipoxygenase products, and, if so, which cells are of primary importance. We have examined the effects of allergen challenge on the capacity of a mixture of isolated lung cells from ovalbumin (OA)-sensitized BN rats to synthesize LTs and other eicosanoids. Cells were isolated by enzymatic digestion of lung tissue before and either 6 or 24 h after challenge of sensitized rats with either OA or saline. A23187-induced synthesis of eicosanoids by these cells was measured using high-pressure liquid chromatography. OA challenge resulted in a significant influx of neutrophils into the lungs and a significant increase in the synthesis of 5-lipoxygenase products, in particular LTB4, by lung cells after 6 h. There was a positive correlation between the percentage of neutrophils in unfractionated lung cells and the amounts of LTB4 produced by these cells. OA challenge had little or no effect on the production of cLTs and the cyclooxygenase product 12-hydroxy-5,8,10-heptadecatrienoic acid. There was a significant increase in the infiltration of eosinophils into the lungs 24 h after OA challenge but no increase in the production of cLTs by lung cells at this time, suggesting that eosinophils from BN rats are unlikely to be the major site for the production of these substances. This was confirmed in experiments with partially purified eosinophils obtained from Sephadex-treated rats. In contrast, cLTs were major products of arachidonic acid metabolism by alveolar macrophages from BN rats. We conclude that allergen challenge results in an increased capacity of lung cells to synthesize 5-lipoxygenase products, in particular LTB4. Macrophages, rather than eosinophils, may be an important site for the synthesis of cLTs in BN rat lungs.

Allergens↗
PubMed1995

Formation of a 5-oxo metabolite of 5,8,11,14,17-eicosapentaenoic acid and its effects on human neutrophils and eosinophils.

We recently showed that human neutrophils convert arachidonic acid to its 5-oxo metabolite, 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE). 5-Oxo-ETE, which is synthesized by oxidation of 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) by a highly specific microsomal dehydrogenase, is a potent stimulator of human neutrophils and eosinophils. The objective of the current investigation was to determine whether neutrophils can convert 5,8,11,14,17-eicosapentaenoic acid (EPA) to its 5-oxo metabolite, 5-oxo-6,8,11,14,17-eicosapentaenoic acid (5-oxo-EPE) and, if so, to compare the biological activities of 5-oxo-EPE and 5-oxo-ETE. The two major eicosanoids formed by neutrophils incubated with EPA in the presence of A23187 were 5-hydroxy-6,8,11,14,17-eicosapentaenoic acid (5-HEPE) and 5-oxo-EPE. Smaller amounts of LTB5 and 20-hydroxy-LTB5 were also formed. Phorbol myristate acetate stimulated the formation of 5-oxo-EPE from both EPA and 5-HEPE. 5-HEPE and 5-HETE were equally good substrates for 5-hydroxyeicosanoid dehydrogenase (Km, ca. 0.85 microM; Vmax, ca. 1.4 pmol/min per microgram protein). 5-Oxo-EPE mobilized calcium in neutrophils with an EC50 of 36 nM, about 10 times higher than that of 5-oxo-ETE. 5-Oxo-EPE was also about one-tenth as active as 5-oxo-ETE in stimulating the migration of both human neutrophils and human eosinophils. It is concluded that 5-oxo-EPE is readily formed from EPA via 5-HEPE. However, it is only about one-tenth as potent as 5-oxo-ETE in stimulating human neutrophils and eosinophils. These results support the contention that EPA can alleviate certain inflammatory diseases by reducing the contribution of arachidonate-derived eicosanoids.

Alcohol Oxidoreductases↗
PubMed1994

Phorbol myristate acetate stimulates the formation of 5-oxo-6,8,11,14-eicosatetraenoic acid by human neutrophils by activating NADPH oxidase.

We have shown previously that human neutrophil microsomes contain a highly specific dehydrogenase which, in the presence of NADP+, converts 5S-hydroxy-6,8,11,14-eicosatetraenoic acid (5S-HETE) to its 5-oxo metabolite, 5-oxo-ETE, a potent agonist of these cells. However, intact neutrophils convert 5S-HETE principally to its omega-oxidation product, 5,20-diHETE, and to only small amounts of 5-oxo-ETE. Phorbol myristate acetate (PMA) dramatically shifts the metabolism of 5S-HETE by intact cells so that 5-oxo-ETE is the major metabolite. The objective of this investigation was to determine the mechanism for the stimulatory effect of PMA on 5-oxo-ETE formation. The possibility that oxidants released in response to PMA nonenzymatically oxidized 5S-HETE was ruled out, since PMA did not appreciably stimulate the formation of 5-oxo-ETE from 5R-HETE. On the other hand, inhibition of NADPH oxidase either by diphenylene iodonium or by mild heating nearly completely prevented the stimulatory effect of PMA on the formation of 5-oxo-ETE. The possibility that this effect was mediated by superoxide seems unlikely, since it was still observed, although somewhat attenuated, in the presence of superoxide dismutase. Moreover, superoxide generated by another mechanism (xanthine/xanthine oxidase) did not appreciably affect the formation of 5-oxo-ETE by neutrophils. However, phenazine methosulfate, which can nonenzymatically convert NADPH to NADP+, mimicked the effect of PMA on 5-oxo-ETE formation by intact neutrophils. It is concluded that PMA acts by activating NADPH oxidase, resulting in conversion of NADPH to NADP+, which enhances the formation of 5-oxo-ETE and reduces the formation of 5,20-diHETE. Serum-treated zymosan has an effect on the metabolism of 5S-HETE similar to that of PMA in that it also stimulates the formation of 5-oxo-ETE and inhibits that of 5,20-diHETE.

Arachidonic Acids↗
PubMed1994

Effects of phorbol myristate acetate on the synthesis of 5-oxo-6,8,11,14-eicosatetraenoic acid by human polymorphonuclear leukocytes.

5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is a newly discovered chemotactic agent for human polymorphonuclear leukocytes (PMNL) which has potent stimulatory effects on cytosolic calcium levels in these cells. Although we have shown that it is synthesized from 5(S)-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) by a highly specific microsomal dehydrogenase, little is known about the synthesis of this substance by intact PMNL. In the present study we found that in contrast to PMNL microsomes, intact, unstimulated PMNL produced relatively small amounts of 5-oxo-ETE from 5-HETE, but instead converted 5-HETE primarily to its omega-oxidation product, 5,20-diHETE. However, preincubation of PMNL with phorbol myristate acetate (PMA; EC50, ca. 4 nM) dramatically increased the ratio of 5-oxo-ETE to 5,20-diHETE from 0.07 in its absence to 1.85 in the presence of 100 nM PMA. Both effects were completely reversed by staurosporine, indicated that they were mediated by a protein kinase. PMA also stimulated the formation of 5-oxo-ETE, 5-HETE, and leukotriene B4 (LTB4) from exogenous arachidonic acid. The greatest enhancement was observed for 5-oxo-ETE, which, under all conditions, was produced in greater quantities than LTB4. PMA stimulated the formation of 5-oxo-ETE by PMNL stimulated with either A23187 or zymosan. A23187-stimulated PMNL initially produced more LTB4 than 5-oxo-ETE, but at longer time points, 5-oxo-ETE predominated. These results demonstrate that PMA-activated human PMNL can synthesize substantial amounts of 5-oxo-ETE and raise the possibility that this substance may be an important inflammatory mediator.

Arachidonic Acid↗
PubMed1994

Effects of a fish-oil and vegetable-oil formula on aggregation and ethanolamine-containing lysophospholipid generation in activated human platelets and on leukotriene production in stimulated neutrophils.

The effects of consuming a liquid formula containing either fish oil enriched in omega-3 fatty acids or vegetable oil enriched in oleic acid was evaluated in 20 male subjects randomly allocated into two groups over a 42-d period. A decrease in collagen-induced aggregation by using washed platelet suspensions was found in both groups after nutritional supplementation. A considerable rise in omega-3 and a decrease in omega-6 fatty acids occurred in the platelet phospholipid with fish-oil consumption. The degree of eicosapentaenoic acid (EPA, 20:5n-3) enrichment (fish-oil group) was dramatically greater in the ether-containing plasmenylethanolamine (13.5 mol% of fatty acids; mol% of fatty acids = moles per 100 moles of total fatty acids) than in phosphatidylethanolamine (2.8 mol%) or phosphatidylcholine (2.9 mol%). Neither treatment significantly influenced the agonist-induced accumulation of lysoplasmenylethanolamine as derived via phospholipase A2 hydrolysis of plasmenylethanolamine. HPLC measurements of eicosanoid production in A23187-stimulated neutrophils revealed a considerable decrease in the formation of arachidonic acid-derived leukotriene B4 (LTB4), by 41%, and 5-HETE (5-hydroxyeicosatetraenoic acid), by 30%, in the fish-oil group along with the appearance of the corresponding EPA-derived products [LTB5 and 5-HEPE (5-hydroxyeicosapentaenoic acid)]. No such alterations in the formation of lipoxygenase products were found with the vegetable oil treatment.

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