The eosinophil chemoattractant 5-oxo-ETE and the OXE receptor

5-Oxo-ETE (5-oxo-6,8,11,14-eicosatetraenoic acid) is formed from the 5-lipoxygenase product 5-HETE (5S-hydroxy-6,8,11,14-eicosatetraenoic acid) by 5-hydroxyeicosanoid dehydrogenase (5-HEDH). The cofactor NADP⁺ is a limiting factor in the synthesis of 5-oxo-ETE because of its low concentrations in unperturbed cells. Activation of the respiratory burst in phagocytic cells, oxidative stress, and cell death all dramatically elevate both intracellular NADP⁺ levels and 5-oxo-ETE synthesis. 5-HEDH is widely expressed in inflammatory, structural, and tumor cells. Cells devoid of 5-lipoxygenase can synthesize 5-oxo-ETE by transcellular biosynthesis using inflammatory cell-derived 5-HETE. 5-Oxo-ETE is a chemoattractant for neutrophils, monocytes, and basophils and promotes the proliferation of tumor cells. However, its primary target appears to be the eosinophil, for which it is a highly potent chemoattractant. The actions of 5-oxo-ETE are mediated by the highly selective OXE receptor, which signals by activating various second messenger pathways through the release of the βγ-dimer from G_i/o proteins to which it is coupled. Because of its potent effects on eosinophils, 5-oxo-ETE may be an important mediator in asthma, and, because of its proliferative effects, may also contribute to tumor progression. Selective OXE receptor antagonists, which are currently under development, could be useful therapeutic agents in asthma and other allergic diseases.     

Oxidative stress stimulates the synthesis of the eosinophil chemoattractant 5-oxo-6,8,11,14-eicosatetraenoic acid by inflammatory cells

5-Oxo-ETE (5-oxo-6,8,11,14-eicosatetraenoic acid) is a highly potent granulocyte chemoattractant that acts through a selective G-protein coupled receptor. It is formed by oxidation of the 5-lipoxygenase product 5-HETE (5S-hydroxy-6,8,11,14-eicosatetraenoic acid) by 5-hydroxyeicosanoid dehydrogenase (5-HEDH). Although leukocytes and platelets display high microsomal 5-HEDH activity, unstimulated intact cells do not convert 5-HETE to appreciable amounts of 5-oxo-ETE. To attempt to resolve this dilemma we explored the possibility that 5-oxo-ETE synthesis could be enhanced by oxidative stress. We found that hydrogen peroxide and t-butyl hydroperoxide strongly stimulate 5-oxo-ETE formation by U937 monocytic cells. This was dependent on the GSH redox cycle, as it was blocked by depletion of GSH or inhibition of glutathione reductase and mimicked by oxidation of GSH to GSSG by diamide. Glucose inhibited the response to H2O2 through its metabolism by the pentose phosphate pathway, as its effect was reversed by the glucose-6-phosphate dehydrogenase inhibitor dehydroepiandrosterone. 5-Oxo-ETE synthesis was also strongly stimulated by hydroperoxides in blood monocytes, lymphocytes, and platelets, but not neutrophils. Unlike monocytic cells, lymphocytes and platelets were resistant to the inhibitory effects of glucose. 5-Oxo-ETE synthesis following incubation of peripheral blood mononuclear cells with arachidonic acid and calcium ionophore was also strongly enhanced by t-butyl hydroperoxide. Oxidative stress could act by depleting NADPH, resulting in the formation NADP+, the cofactor for 5-HEDH. This is opposed by the pentose phosphate pathway, which converts NADP+ back to NADPH. Oxidative stress could be an important mechanism for stimulating 5-oxo-ETE production in inflammation, promoting further infiltration of granulocytes into inflammatory sites.     

Biochemistry, biology and chemistry of the 5-lipoxygenase product 5-oxo-ETE

5-Oxo-ETE (5-oxo-6,8,11,14-eicosatetraenoic acid) is an arachidonic acid metabolite formed by the oxidation of 5S-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) by 5-hydroxyeicosanoid dehydrogenase (5-HEDH), a microsomal enzyme found in leukocytes and platelets. 5-HEDH is highly selective for 5S-HETE, and displays little activity for other monohydroxy metabolites of arachidonic acid. The synthesis of 5-oxo-ETE requires NADP(+) and can be stimulated by activation of the respiratory burst and by oxidative stress. 5-Oxo-ETE is a chemoattractant for eosinophils and neutrophils, and elicits a variety of responses in these cells, including actin polymerization, calcium mobilization, integrin expression, and degranulation. Its primary target appears to be the eosinophil, and among lipid mediators it is the strongest chemoattractant for these cells. It is also a chemoattractant for monocytes and stimulates the proliferation of prostate tumor cells. Its actions are mediated by a G(i) protein-coupled receptor (OXE receptor) that is highly expressed by eosinophils>neutrophils>monocytes. When administered in vivo in both humans and rodents it elicits tissue eosinophilia, suggesting that it may be an important mediator in allergic diseases such as asthma, and that the development of drugs designed to prevent its formation or effects may be useful therapeutic agents in these diseases.     

Design and synthesis of affinity chromatography ligands for the purification of 5-hydroxyeicosanoid dehydrogenase

Arachidonic acid (AA) is converted to biologically active metabolites by different pathways, one of the most important of which is initiated by 5-lipoxygenase (5-LO). 5-Hydroxyeicosatetraenoic acid (5-HETE), although possessing only weak biological activity itself, is oxidized to 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE), a potent chemoattractant for eosinophils and neutrophils. Our main goal is to determine how the biosynthesis of 5-oxo-ETE is regulated and to determine its pathophysiological roles. To achieve this task, we designed and synthesized affinity chromatography ligands for the purification of 5-hydroxyeicosanoid dehydrogenase (5-HEDH), the enzyme responsible for the formation of 5-oxo-ETE.     

Airway epithelial cells synthesize the lipid mediator 5-oxo-ETE in response to oxidative stress

5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is a potent eosinophil chemoattractant that is synthesized from the 5-lipoxygenase product 5S-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) by the NADP+-dependent enzyme 5-hydroxyeicosanoid dehydrogenase (5-HEDH), previously reported only in inflammatory cells. Because of their critical location at the interface of the lung with the external environment, we sought to determine whether epithelial cells could also synthesize this substance. We found that HEp-2, T84, A549, and BEAS-2B cells all synthesize 5-oxo-ETE from 5-HETE in amounts comparable to leukocytes. The epithelial dehydrogenase is localized in the microsomal fraction, requires NADP+, and is selective for the S-isomer of 5-HETE, suggesting that it is identical to leukocyte 5-HEDH. Normal human bronchial epithelial cells have an even greater capacity to synthesize 5-oxo-ETE. H2O2 dramatically stimulates its synthesis in association with increased levels of intracellular GSSG and NADP+. These responses were all blocked by removal of GSH/GSSG with N-ethylmaleimide, suggesting that H2O2 stimulates 5-oxo-ETE synthesis by raising NADP+ levels through activation of the GSH redox cycle. Airway smooth muscle cells can also synthesize 5-oxo-ETE, but to a lesser extent. These results suggest that epithelial cells may be a major source of 5-oxo-ETE under conditions of oxidative stress, which may contribute to eosinophil infiltration in allergic diseases.     

Structure-activity relationship study of β-oxidation resistant indole-based 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) receptor antagonists

5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is formed from 5S-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) by the 5-lipoxygenase (5-LO) pathway under conditions associated with oxidative stress. 5-Oxo-ETE is an important pro-inflammatory mediator, which stimulates the migration of eosinophils via a selective G-protein coupled receptor, known as the OXE receptor (OXE-R). Previously, we designed and synthesized structural mimics of 5-oxo-ETE such as 1 using an indole scaffold. In the present work, we added various substituents at C-3 of this moiety to block potential β-oxidation of the 5-oxo-valerate side chain, and investigated the structure-activity relationships of the resulting novel β-oxidation-resistant antagonists. Cyclopropyl and cyclobutyl substituents were well tolerated in this position, but were less potent as the highly active 3S-methyl compound. It seems likely that 3-alkyl substituents can affect the conformation of the 5-oxovalerate side chain containing the critical keto and carboxyl groups, thereby affecting interaction with the OXE-receptor.     

LTA4-derived 5-oxo-eicosatetraenoic acid: pH-dependent formation and interaction with the LTB4 receptor of human polymorphonuclear leukocytes

5-Oxo-(7E,9E,11Z,14Z)-eicosatetraenoic acid (5-oxo-ETE) has been identified as a non-enzymatic hydrolysis product of leukotriene A₄ (LTA₄) in addition to 5,12-dihydroxy-(6E,8E,10E,14Z)-eicosatetraenoic acids (5,12-diHETEs) and 5,6-dihydroxy-(7E,9E,11Z,14Z)-eicosatetraenoic acids (5,6-diHETEs). The amount of 5-oxo-ETE detected in the mixture of the hydrolysis products of LTA₄ was found to be pH-dependent. After incubation of LTA₄ in aqueous medium, the ratio of 5-oxo-ETE to 5,12-diHETE was 1:6 at pH 7.5, and 1:1 at pH 9.5. 5-Oxo-ETE was isolated from the alkaline hydrolysis products of LTA₄ in order to evaluate its effects on human polymorphonuclear (PMN) leukocytes. 5-Oxo-ETE induced a rapid and dose-dependent mobilization of calcium in PMN leukocytes with an EC₅₀ of 250 nM, as compared to values of 3.5 nM for leukotriene B₄ (LTB₄) and >500 nM for 5(S)-hydroxy-(6E,8Z,11Z,14Z)-eicosatetraenoic acid (5-HETE). Pretreatment of the cells with LTB₄ totally abolished the calcium response induced by 5-oxo-ETE. In contrast, the preincubation with 5-oxo-ETE did not affect the calcium mobilization induced by LTB₄. The calcium response induced by 5-oxo-ETE was totally inhibited by the specific LTB₄ receptor antagonist LY223982. These data demonstrate that 5-oxo-ETE can induce calcium mobilization in PMN leukocyte via the LTB₄ receptor in contrast to the closely related analog 5-oxo-(6E,8Z,11Z,14Z)-eicosatetraenoic acid which is known to activate human neutrophils by a mechanism independent of the receptor for LTB₄.     

LTA(4)-derived 5-oxo-eicosatetraenoic acid: pH-dependent formation and interaction with the LTB(4) receptor of human polymorphonuclear leukocytes

5-oxo-(7E,9E,11Z,14Z)-eicosatetraenoic acid (5-oxo-ETE) has been identified as a non-enzymatic hydrolysis product of leukotriene A(4) (LTA(4)) in addition to 5,12-dihydroxy-(6E,8E,10E, 14Z)-eicosatetraenoic acids (5,12-diHETEs) and 5,6-dihydroxy-(7E,9E, 11Z,14Z)-eicosatetraenoic acids (5,6-diHETEs). The amount of 5-oxo-ETE detected in the mixture of the hydrolysis products of LTA(4) was found to be pH-dependent. After incubation of LTA(4) in aqueous medium, the ratio of 5-oxo-ETE to 5,12-diHETE was 1:6 at pH 7.5, and 1:1 at pH 9.5. 5-Oxo-ETE was isolated from the alkaline hydrolysis products of LTA(4) in order to evaluate its effects on human polymorphonuclear (PMN) leukocytes. 5-Oxo-ETE induced a rapid and dose-dependent mobilization of calcium in PMN leukocytes with an EC(50) of 250 nM, as compared to values of 3.5 nM for leukotriene B(4) (LTB(4)500 nM for 5(S)-hydroxy-(6E,8Z,11Z,14Z)-eicosatetraenoic acid (5-HETE). Pretreatment of the cells with LTB(4) totally abolished the calcium response induced by 5-oxo-ETE. In contrast, the preincubation with 5-oxo-ETE did not affect the calcium mobilization induced by LTB(4). The calcium response induced by 5-oxo-ETE was totally inhibited by the specific LTB(4) receptor antagonist LY223982. These data demonstrate that 5-oxo-ETE can induce calcium mobilization in PMN leukocyte via the LTB(4) receptor in contrast to the closely related analog 5-oxo-(6E,8Z,11Z, 14Z)-eicosatetraenoic acid which is known to activate human neutrophils by a mechanism independent of the receptor for LTB(4).     

Regulation of 5-oxo-ETE synthesis by nitric oxide in human polymorphonuclear leucocytes upon their interaction with zymosan and Salmonella typhimurium

In the present study we have presented data on the regulation of LT (leukotriene) and 5-oxo-ETE (5-oxo-6,8,11,14-eicosatetraenoic acid) syntheses in human neutrophils upon interaction with OZ (opsonized zymosan) or Salmonella typhimurium. Priming of neutrophils with PMA (phorbol 12-myristate 13-acetate) and LPS (lipopolysaccharide) elicits 5-oxo-ETE formation in neutrophils exposed to OZ, and the addition of AA (arachidonic acid) significantly increases 5-oxo-ETE synthesis. We found that NO (nitric oxide)-releasing compounds induce 5-oxo-ETE synthesis in neutrophils treated with OZ or S. typhimurium. Exposure of neutrophils to zymosan or bacteria in the presence of the NO donor DEA NONOate (1,1-diethyl-2-hydroxy-2-nitroso-hydrazine sodium) considerably increased the conversion of endogenously formed 5-HETE (5S-hydroxy-6,8,11,14-eicosatetraenoic acid) to 5-oxo-ETE. To our knowledge, this study is the first to demonstrate that NO is a potent regulator of 5-oxo-ETE synthesis in human polymorphonuclear leucocytes exposed to Salmonella typhimurium and zymosan.     

5-oxo-ETE is a major oxidative stress-induced arachidonate metabolite in B lymphocytes

B lymphocytes convert arachidonic acid (AA) to the 5-lipoxygenase products leukotriene B4 (LTB4) and 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) when subjected to oxidative stress. 5-HETE has little biological activity, but can be oxidized by a selective dehydrogenase in some cells to 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE), a potent eosinophil chemoattractant. We found that CESS cells, a B lymphocyte cell line, convert AA to 5-oxo-ETE and this is selectively stimulated by oxidative stress. In the presence of H2O2, 5-oxo-ETE is a major AA metabolite in these cells (5-oxo-ETE≈5-HETE>LTB4). The cyclooxygenase product 12-hydroxy-5,8,10-heptadecatrienoic acid is also formed, but is not affected by H2O2. Diamide had effects similar to those of H2O2 and both substances had similar effects on human tonsillar B cells. H2O2 also stimulated 5-oxo-ETE formation from its direct precursor 5-HETE in tonsillar B and CESS cells, and this was inhibited by the glutathione reductase inhibitor carmustine. H2O2 concomitantly induced rapid increases in GSSG and NADP+ and reductions in GSH and NADPH. We conclude that oxidative stress stimulates 5-oxo-ETE synthesis in B lymphocytes by two mechanisms: activation of 5-lipoxygenase and increased oxidation of 5-HETE by NADP+-dependent 5-hydroxyeicosanoid dehydrogenase. B lymphocyte-derived 5-oxo-ETE could contribute to eosinophilic inflammation in asthma and other allergic diseases.     

NADP-Utilizing Enzymes in the Matrix of Plant Mitochondria

Purified potato tuber (Solanum tuberosum L. cv Bintie) mitochondria contain soluble, highly latent NAD⁺- and NADP⁺-isocitrate dehydrogenases, NAD⁺- and NADP⁺-malate dehydrogenases, as well as an NADPH-specific glutathione reductase (160, 25, 7200, 160, and 16 nanomoles NAD(P)H per minute and milligram protein, respectively). The two isocitrate dehydrogenase activities, but not the two malate dehydrogenase activities, could be separated by ammonium sulfate precipitation. Thus, the NADP⁺-isocitrate dehydrogenase activity is due to a separate matrix enzyme, whereas the NADP⁺-malate dehydrogenase activity is probably due to unspecificity of the NAD⁺-malate dehydrogenase. NADP⁺-specific isocitrate dehydrogenase had much lower K_ms for NADP⁺ and isocitrate (5.1 and 10.7 micromolar, respectively) than the NAD⁺-specific enzyme (101 micromolar for NAD⁺ and 184 micromolar for isocitrate). A broad activity optimum at pH 7.4 to 9.0 was found for the NADP⁺-specific isocitrate dehydrogenase whereas the NAD⁺-specific enzyme had a sharp optimum at pH 7.8. Externally added NADP⁺ stimulated both isocitrate and malate oxidation by intact mitochondria under conditions where external NADPH oxidation was inhibited. This shows that (a) NADP⁺ is taken up by the mitochondria across the inner membrane and into the matrix, and (b) NADP⁺-reducing activities of malate dehydrogenase and the NADP⁺-specific isocitrate dehydrogenase in the matrix can contribute to electron transport in intact plant mitochondria. The physiological relevance of mitochondrial NADP(H) and soluble NADP(H)-consuming enzymes is discussed in relation to other known mitochondrial NADP(H)-utilizing enzymes.     

Effect of NADP+ on light-induced cytochrome changes in membrane fragments from a blue-green alga

The effect of NADP⁺ on light-induced steady-state redox changes of membrane-bound cytochromes was investigated in membrane fragments prepared from the blue-green algae Nostoc muscorum (Strain 7119) that had high rates of electron transport from water to NADP⁺ and from an artificial electron donor, reduced dichlorophenolindophenol (DCIPH₂) to NADP⁺. The membrane fragments contained very little phycocyanin and had excellent optical properties for spectrophotometric assays. With DCIPH₂ as the electron donor, NADP⁺ had no effect on the light-induced redox changes of cytochromes: with or without NADP⁺, 715- or 664-nm illumination resulted mainly in the oxidation of cytochrome f and of other component(s) which may include a c-type cytochrome with an α peak at 549 nm. With 664 nm illumination and water as the electron donor, NADP⁺ had a pronounced effect on the redox state of cytochromes, causing a shift toward oxidation of a component with a peak at 549 nm (possibly a c-type cytochrome), cytochrome f, and particularly cytochrome b₅₅₉. Cytochrome b₅₅₉ appeared to be a component of the main noncyclic electron transport chain and was photooxidized at physiological temperatures by Photosystem II. This photooxidation was apparent only in the presence of a terminal acceptor (NADP⁺) for the electron flow from water.     

Formation of leukotrienes and other hydroxy acids during platelet-neutrophil interactions in vitro

Interactions of human platelets with neutrophils were studied in suspensions of [³H]arachidonate-labeled platelets and unlabeled neutrophils stimulated with ionophore A23187. Several radioactive arachidonate metabolites, not produced by platelets alone, were detected, including [³H]-labeled leukotriene B₄ (LTB₄), dihydroxyeicosatetraenoic acid (DHETE) and 5-hydroxyeicosatetraenoic acid (5-HETE). When [³H]12-HETE, a platelet product, was added to stimulated neutrophils, DHETE was formed. Similarly, when [³H]5-HETE, a neutrophil product, was added to stimulated platelets, DHETE was the major product. These results suggest that upon stimulation: 1) platelet-derived arachidonate may serve as precursor for the neutrophil-derived eicosanoids LTB₄ and 5-HETE, and 2) that platelet-derived 12-HETE can be converted to DHETE by human neutrophils. The present investigation documents cell-cell interactions via the lipoxygenase pathway, which may be important in hemostasis, thrombosis and inflammation.     

12-L-hydroxy-5,8,10,14-eicosatetraenoic acid, a chemotactic fatty acid, is incorporated into neutrophil phospholipids and triglyceride

Platelets contain a lipoxygenase which converts arachidonic acid to 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) which has been shown to be chemotactic for human neutrophils and eosinophils. [¹⁴C]-12-HETE was biosynthesized, purified and incubated at a concentration of 1 μM with human neutrophils. Lipids were extracted from the neutrophils and the media, and the radiolabeled products identified. 26 percent of the radiolabel was found in the cells after 30 min incubation, essentially all of it esterified into phospholipid and triglyceride. The radiolabeled phospholipids and triglycerides were transesterified and the liberated fatty acid was identified as [¹⁴C]-12-HETE.This is the first demonstration of direct alteration of membrane components by a chemotactic agent and may be an example of a more generalized mechanism for altering membrane characteristics.     

Glutamate Dehydrogenases: The Why and How of Coenzyme Specificity

NAD⁺ and NADP⁺, chemically similar and with almost identical standard oxidation–reduction potentials, nevertheless have distinct roles, NAD⁺ serving catabolism and ATP generation whereas NADPH is the biosynthetic reductant. Separating these roles requires strict specificity for one or the other coenzyme for most dehydrogenases. In many organisms this holds also for glutamate dehydrogenases (GDH), NAD⁺-dependent for glutamate oxidation, NADP⁺-dependent for fixing ammonia. In higher animals, however, GDH has dual specificity. It has been suggested that GDH in mitochondria reacts only with NADP(H), the NAD⁺ reaction being an in vitro artefact. However, contrary evidence suggests mitochondrial GDH not only reacts with NAD⁺ but maintains equilibrium using the same pool as accessed by β-hydroxybutyrate dehydrogenase. Another complication is the presence of an energy-linked dehydrogenase driving NADP⁺ reduction by NADH, maintaining the coenzyme pools at different oxidation–reduction potentials. Its coexistence with GDH makes possible a futile cycle, control of which is not yet properly explained. Structural studies show NAD⁺-dependent, NADP⁺-dependent and dual-specificity GDHs are closely related and a few site-directed mutations can reverse specificity. Specificity for NAD⁺ or for NADP⁺ has probably emerged repeatedly during evolution, using different structural solutions on different occasions. In various GDHs the P7 position in the coenzyme-binding domain plays a key role. However, whereas in other dehydrogenases an acidic P7 residue usually hydrogen bonds to the 2′- and 3′-hydroxyls, dictating NAD⁺ specificity, among GDHs, depending on detailed conformation of surrounding residues, an acidic P7 may permit binding of NAD⁺ only, NADP⁺ only, or in higher animals both.     

5-oxo-6,8,11,14-eicosatetraenoic acid stimulates the release of the eosinophil survival factor granulocyte/macrophage colony-stimulating factor from monocytes

Allergic diseases such as asthma are characterized by tissue eosinophilia induced by the combined effects of chemoattractants and cytokines. Lipid mediators are a major class of endogenous chemoattractants, among which 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is the most potent for human eosinophils. In this study, we investigated the effects of 5-oxo-ETE on eosinophil survival by flow cytometry. We found that this compound could promote eosinophil survival in the presence of small numbers of contaminating monocytes, but not in their absence. The conditioned medium from monocytes treated for 24 h with 5-oxo-ETE also strongly promoted eosinophil survival, whereas the medium from vehicle-treated monocytes had no effect. An antibody against the granulocyte/macrophage colony-stimulating factor (GM-CSF) completely blocked the response of eosinophils to the conditioned medium from 5-oxo-ETE-treated monocytes, whereas an antibody against interleukin-5 had no effect. Furthermore, 5-oxo-ETE stimulated the release of GM-CSF from cultured monocytes in amounts compatible with eosinophil survival activity, with a maximal effect being observed after 24 h. This effect was concentration-dependent and could be observed at concentrations in the picomolar range. 5-Oxo-ETE and leukotriene B(4) had similar effects on GM-CSF release at low concentrations, but 5-oxo-ETE induced a much stronger response at concentrations of 10 nm or higher. This is the first report that 5-oxo-ETE can induce the release of any cytokine, suggesting that it could be an important mediator in allergic and other inflammatory diseases due both to its chemoattractant properties and to its potent effects on the synthesis of the survival factor GM-CSF.     

Metabolism of 5(S)-hydroxy-6,8,11,14-eicosatetraenoic acid and other 5(S)-hydroxyeicosanoids by a specific dehydrogenase in human polymorphonuclear leukocytes.

Human polymorphonuclear leukocytes (PMNL) convert 6-trans isomers of leukotriene B4 (LTB4) to dihydro metabolites (Powell, W.S., and Gravelle, F. (1988) J. Biol. Chem. 263, 2170-2177). In the present study we investigated the mechanism for the initial step in the formation of these products. We found that the 1,500 x g supernatant fraction from human PMNL converts 12-epi-6-trans-LTB4 to its 5-oxo metabolite which was identified by mass spectrometry and UV spectrophotometry. The latter compound was subsequently converted to the corresponding dihydro-oxo product, which was further metabolized to 6,11-dihydro-12-epi-6-trans-LTB4, which was the major product after longer incubation times. The 5-hydroxyeicosanoid dehydrogenase activity is localized in the microsomal fraction and requires NADP+ as a cofactor. These experiments therefore suggest that the initial step in the formation of dihydro metabolites of 6-trans isomers of LTB4 is oxidation of the 5-hydroxyl group by a microsomal dehydrogenase. Studies with a variety of substrates revealed that the microsomal dehydrogenase in human PMNL oxidizes the hydroxyl groups of a number of other eicosanoids which contain a 5(S)-hydroxyl group followed by a 6-trans double bond. There is little or no oxidation of hydroxyl groups in the 8-, 9-, 11-, 12-, or 15-positions of eicosanoids, or of the 5-hydroxyl group of LTB4, which has a 6-cis rather than a 6-trans double bond. The preferred substrate for this enzyme is 5(S)-hydroxy-6,8,11,14-eicosatetraenoic acid (5(S)-HETE) (Km, 0.2 microM), which is converted to 5-oxo-6,8,11,14-eicosatetraenoic acid. Unlike 5(S)-HETE, 5(R)-HETE is a poor substrate for the 5(S)-hydroxyeicosanoid dehydrogenase, indicating that in addition to exhibiting a high degree of positional specificity, this enzyme is also highly stereospecific. In addition to 5(S)-HETE and 6-trans isomers of LTB4, 5,15-diHETE is also a good substrate for this enzyme, being converted to 5-oxo-15-hydroxy-6,8,11,13-eicosatetraenoic acid (5-oxo-15-hydroxy-ETE). The oxidation of 5(S)-HETE to 5-oxo-ETE is reversible since human PMNL microsomes stereospecifically reduce 5-oxo-ETE to the 5(S)-hydroxy compound in the presence of NADPH. 5-Oxo-ETE is formed rapidly from 5(S)-HETE by intact human PMNL, but because of the reversibility of the reaction, its concentration only reaches about 25% that of 5(S)-HETE.     

C20-trifluoro-5-oxo-ETE: a metabolically stable 5-oxo-ETE derivative

The total synthesis of C₂₀-trifluoro-6(E),8(Z),11(Z),14(Z) 5-oxo-ETE is reported. This compound was designed as an ω-oxidation-resistant analog of 5-oxo-ETE that would be resistant to metabolism. The trifluoro derivative of 5-oxo-ETE stimulated calcium mobilization in neutrophils and desensitized these cells to subsequent exposure to 5-oxo-ETE.     

Detection of 15-hydroxyprostaglandin dehydrogenase in bovine mesenteric blood vessels

Two types of 15-hydroxyprostaglandin dehydrogenase (NAD⁺ and NADP⁺ dependent) were demonstrated in bovine mesentric arteries and veins. The 15-hydroxyprostaglandin dehydrogenase activity was found in the high-speed supernatant, suggesting that these enzymes are associated with the cytoplasmic fraction of the blood vessels. The levels of activities of both NAD⁺- and NADP⁺-dependent dehydrogenases were similar in mesentric blood vessels. Prostaglandin F_2α was preferred to the prostaglandin E₂ as subtrate by both NAD⁺ and NADP⁺ dependent enzymes. The presence of 15-hydroxyprostaglandin dehydrogenase in blood vessels may play a siginificant role in the regulation of intracellular levels of prostaglandins of the E and F series in blood vessels.     

Glyceraldehyde 3-Phosphate:NADP Reductase of Spinach Leaves : Steady State Kinetics and Effect of Inhibitors

The steady state kinetics of glyceraldehyde 3-phosphate:NADP⁺ oxidoreductase (GNR) (EC 1.2.1.9) have been investigated. The enzyme exhibits hyperbolic behavior over a wide range of substrate concentrations. Double-reciprocal plots are nearly parallel or distantly convergent with limiting K_m values of 2 to 5 micromolar for NADP⁺ and 20 to 40 micromolar for D-glyceraldehyde 3-phosphate (G3P). The velocity response to NADP⁺ as the varied substrate is however sigmoidal if G3P concentration exceeds 10 micromolar, whereas the response to G3P may show inhibition above this concentration. This `G3P-inhibited state' is alleviated by saturating amounts of NADP⁺ or NADPH. Product inhibition patterns indicate NADPH as a potent competitive inhibitor to NADP⁺ (K_i 30 micromolar) and mixed inhibitor towards G3P, and 3-phosphoglycerate (3PGA) as mixed inhibitor to both NADP⁺ and G3P (K_i 10 millimolar). The data, and those obtained with dead-end inhibitors, are consistent with a nonrapid equilibrium random mechanism with two alternative kinetic pathways. Of these, a rapid kinetic sequence (probably ordered with NADP⁺ binding first and G3P binding as second substrate) is dominant in the range of hyperbolic responses. A reverse reaction with 3PGA and NADPH as substrates is unlikely, and was not detected. Of a number of compounds tested, erythrose 4-phosphate (K_i 7 micromolar) and Pi (K_i 2.4 millimolar) act as competitive inhibitors to G3P (uncompetitive towards NADP⁺) and are likely to affect the in vivo activity. Ribose 5-phosphate, phosphoenolpyruvate, ATP, and ADP are also somewhat inhibitory. Full GNR activity in the leaf seems to be allowed only under high photosynthesis conditions, when levels of several inhibitors are low and substrate is high. We suggest that a main function of leaf GNR is to supply NADPH required for photorespiration, the reaction product 3PGA being cycled back to chloroplasts.