Metabolism of anandamide into eoxamides by 15-lipoxygenase-1 and glutathione transferases.

Human 15-lipoxygenase-1 (15-LO-1) can metabolize arachidonic acid (ARA) into pro-inflammatory mediators such as the eoxins, 15-hydroperoxyeicosatetraenoic acid (HPETE), and 15-hydroxyeicosatetraenoyl-phosphatidylethanolamine. We have in this study investigated the formation of various lipid hydroperoxide by either purified 15-LO-1 or in the Hodgkin lymphoma cell line L1236, which contain abundant amount of 15-LO-1. Both purified 15-LO-1 and L1236 cells produced lipid hydroperoxides more efficiently when anandamide (AEA) or 2-arachidonoyl-glycerol ester was used as substrate than with ARA. Furthermore, L1236 cells converted AEA to a novel class of cysteinyl-containing metabolites. Based on RP-HPLC, mass spectrometry and comparison to synthetic products, these metabolites were identified as the ethanolamide of the eoxin (EX) C(4) and EXD(4). By using the epoxide EXA(4)-ethanol amide, it was also found that platelets have the capacity to produce the ethanolamide of EXC(4) and EXD(4). We suggest that the ethanolamides of the eoxins should be referred to as eoxamides, in analogy to the ethanolamides of prostaglandins which are named prostamides. The metabolism of AEA into eoxamides might engender molecules with novel biological effects. Alternatively, it might represent a new mechanism for the termination of AEA signalling.

On the biosynthesis and biological role of eoxins and 15-lipoxygenase-1 in airway inflammation and Hodgkin lymphoma.

This mini-review is focused on the enzyme 15-lipoxygenase-1 (15-LO-1) and eoxins in airway inflammatory diseases and Hodgkin lymphoma. Several studies have demonstrated increased expression and activity of 15-LO-1 in the respiratory tissue from asthma patients , indicating a pathophysiological role of this enzyme in airway inflammation. Eoxins were recently identified as pro-inflammatory metabolites of arachidonic acid, formed through the 15-LO-1 pathway, in human eosinophils, mast cells, airway epithelial cells and Hodgkin lymphoma. Mice deficient of 12/15-LO, the ortholog to human 15-LO-1, had an attenuated allergic airway inflammation compared to wild type controls, also indicating a pathophysiological role of this enzyme in respiratory inflammation. The putative therapeutic implications of 15-LO-1 inhibitors in the treatment of asthma, chronic obstructive pulmonary disorder and Hodgkin lymphoma are discussed.

Hodgkin Reed-Sternberg cells express 15-lipoxygenase-1 and are putative producers of eoxins in vivo: novel insight into the inflammatory features of classical Hodgkin lymphoma.

Classical Hodgkin lymphoma has unique clinical and pathological features and tumour tissue is characterized by a minority of malignant Hodgkin Reed-Sternberg cells surrounded by inflammatory cells. In the present study, we report that the Hodgkin lymphoma-derived cell line L1236 has high expression of 15-lipoxygenase-1 and that these cells readily convert arachidonic acid to eoxin C(4), eoxin D(4) and eoxin E(4). These mediators were only recently discovered in human eosinophils and mast cells and found to be potent proinflammatory mediators. Western blot and immunocytochemistry analyses of L1236 cells demonstrated that 15-lipoxygenase-1 was present mainly in the cytosol and that the enzyme translocated to the membrane upon calcium challenge. By immunohistochemistry of Hodgkin lymphoma tumour tissue, 15-lipoxygenase-1 was found to be expressed in primary Hodgkin Reed-Sternberg cells in 17 of 20 (85%) investigated biopsies. The enzyme 15-lipoxygenase-1, however, was not expressed in any of 10 biopsies representing nine different subtypes of non-Hodgkin lymphoma. In essence, the expression of 15-lipoxygenase-1 and the putative formation of eoxins by Hodgkin Reed-Sternberg cells in vivo are likely to contribute to the inflammatory features of Hodgkin lymphoma. These findings may have important diagnostic and therapeutic implications in Hodgkin lymphoma. Furthermore, the discovery of the high 15-lipoxygenase-1 activity in L1236 cells demonstrates that this cell line comprises a useful model system to study the chemical and biological roles of 15-lipoxygenase-1.

Characterization of specific binding sites for cysteinyl leukotrienes in sheep lung.

Specific binding sites for [3H]leukotriene (LT)D4 and [3H]LTC4 have been identified in sheep lung parenchymal membranes. [3H] LTD4 specific binding was of high affinity (KD = 0.56 nM), saturable (Bmax = 43 fmol/mg of protein), stimulated by divalent cations and inhibited by nonhydrolyzable GTP analogs. LTs and LTD4-receptor antagonists competed for [3H]LTD4 specific binding with the rank order of potency predicted for the LTD4 receptor: LTD4 > ONO-1078 > ICI 204,219 > MK-571 > LTE4 > LTC4 > BAY u9773 >> LTB4. In contrast, [3H]LTC4 specific binding was of lower affinity (KD = 27 nM), abundant (Bmax = 87 pmol/mg of protein) and although stimulated by divalent cations was unaffected by GTP analogs. LTs and LTC4 analogs competed for [3H]LTC4 specific binding with the following rank order of potency: LTC2 > LTC3 > LTC4 > LTC5 >> N-methyl-LTC4 >> LTD4 approximately LTB4 approximately LTB4. [3H]LTD4 specific binding to sheep lung membranes has, therefore, the characteristics of being to a G-protein-coupled LTD4 receptor, whereas the profile of [3H]LTC4 specific binding strongly suggests that these sites are not LT-receptor related. Photolabeling of sheep lung membranes using [125I]azido-LTC4, a photoactivable LTC4 analog, resulted in the selective photolabeling of two polypeptides migrating at 30 kDa and 19 kDa. The selective photolabeling of the 19 kDa polypeptide could be modulated in an identical manner to [3H]LTC4 specific binding. This protein is, therefore, a candidate for being the principal [3H]LTC4 specific site in sheep lung membranes and has a comparable molecular mass to microsomal glutathione S-transferase, recently shown to be the predominant LTC4 binding protein in cellular membranes.

Photoaffinity labeling of the leukotriene D4 receptor in guinea pig lung.

The leukotriene (LT)D4 receptor has been defined as a G-protein-coupled receptor. In order to characterize this receptor, an iodinated, photoactivatable azido derivative of LTD4 (125I-azido-LTD4) has been synthesized for use as a photoaffinity probe. The characteristics of 125I-azido-LTD4 specific binding to guinea pig lung membranes were directly comparable to those of [3H]LTD4 specific binding to this tissue. 125I-Azido-LTD4 specific binding was saturable and of high affinity, enhanced by divalent cations and inhibited by sodium ions, but not potassium ions. 125I-Azido-LTD4 specific binding was also strongly inhibited by the nonhydrolyzable GTP analog, GTP gamma S, with ATP gamma S being 100-fold less potent, suggesting this inhibition was due to selective interaction with a G-protein. The cysteinyl leukotrienes competed for 125I-azido-LTD4 specific binding to guinea pig lung membranes with the following rank order of potency: LTD4 > LTE4 > LTC4, while the non-cysteinyl LTB4 was virtually inactive. Two structurally different LTD4 receptor antagonists, MK-571 and ICI 204,219, also competed for 125I-azido-LTD4 specific binding with nanomolar potency, whereas the leukotriene synthesis inhibitor, MK-886, was 10,000-fold less active. These data are in agreement with 125I-azido-LTD4 binding specifically to a G-protein-coupled LTD4 receptor. Photolysis of 125I-azido-LTD4 under equilibrium binding conditions resulted in the selective radiolabeling of a 45-kDa guinea pig lung membrane protein. The photolabeling of this 45-kDa protein was saturable, modulated by cations and inhibited by nucleotide analogs in an analogous way to 125I-azido-LTD4 specific binding. In addition, the photolabeling of this protein was inhibited in a concentration-dependent manner by all competing ligands, with the same rank order of potency and IC50 values as determined in the 125I-azido-LTD4 binding assay. It is proposed, therefore, that this novel 45-kDa protein is the guinea pig lung LTD4 receptor.