Leukotriene C4 synthase homo-oligomers detected in living cells by bioluminescence resonance energy transfer

Leukotrienes (LTs) are biologically active compounds derived from arachidonic acid which have important pathophysiological roles in asthma and inflammation. The cysteinyl leukotriene LTC(4) and its metabolites LTD(4) and LTE(4) stimulate bronchoconstriction, airway mucous formation and generalized edema formation. LTC(4) is formed by addition of glutathione to LTA(4), catalyzed by the integral membrane protein, LTC(4) synthase (LTCS). We now report the use of bioluminescence resonance energy transfer (BRET) to demonstrate that LTCS forms homo-oligomers in living cells. Fusion proteins of LTCS and Renilla luciferase (Rluc) and a variant of green fluorescent protein (GFP), respectively, were prepared. High BRET signals were recorded in transiently transfected human embryonic kidney (HEK 293) cells co-expressing Rluc/LTCS and GFP/LTCS. Homo-oligomer formation in living cells was verified by co-transfection of a plasmid expressing non-chimeric LTCS. This resulted in dose-dependent attenuation of the BRET signal. Additional evidence for oligomer formation was obtained in cell-free assays using glutathione S-transferase (GST) pull-down assay. To map interaction domains for oligomerization, GFP/LTCS fusion proteins were prepared with truncated variants of LTCS. The results obtained identified a C-terminal domain (amino acids 114-150) sufficient for oligomerization of LTCS. Another, centrally located, interaction domain appeared to exist between amino acids 57-88. The functional significance of LTCS homo-oligomer formation is currently being investigated.     

Leukotriene C4 Transport and Metabolism in the Central Nervous System

The transport and metabolism of radiolabeled leukotriene (LT) C4 in the CNS were investigated after intraventricular injection. Under thiopental (Pentothal) anesthesia, New Zealand white rabbits were injected intracerebroventricularly with 0.2 ml of artificial CSF containing 2.5 microCi of [3H]LTC4 (36 Ci/mmol), 0.3 microCi of [14C]mannitol, and, in some cases, 0.9 mg of probenecid, 1.8 mg of cysteine, 1.4 micrograms of unlabeled LTC4, or 2 mg of tolazoline HCl. After 2 h, the conscious rabbits were killed, and the quantity and nature of the 3H and 14C were determined in CSF, choroid plexus, and brain. The [3H]LTC4 recovered in CSF and brain was not extensively metabolized, as greater than 70% of the 3H remained [3H]LTC4, although some spontaneous conversion to 11-trans-[3H]LTC4 occurred. Oxidized forms of [3H]LTC4, [3H]LTD4, and [3H]LTE4 did not exceed 18% in CSF and brain. After intraventricular injection of [3H]LTC4, 3H was transferred from the CSF to blood by a probenecid-sensitive, but tolazoline-insensitive, transport system in the CNS much more rapidly than mannitol. Cysteine decreased the retention of [3H]LTC4 in brain. These results are consistent with previous in vitro observations that [3H]LTC4 is transferred from CSF into blood by an efficient transport system for LTC4 in choroid plexus.     

Leukotriene C4 synthase homo-oligomers detected in living cells by bioluminescence resonance energy transfer

Leukotrienes (LTs) are biologically active compounds derived from arachidonic acid which have important pathophysiological roles in asthma and inflammation. The cysteinyl leukotriene LTC₄ and its metabolites LTD₄ and LTE₄ stimulate bronchoconstriction, airway mucous formation and generalized edema formation. LTC₄ is formed by addition of glutathione to LTA₄, catalyzed by the integral membrane protein, LTC₄ synthase (LTCS). We now report the use of bioluminescence resonance energy transfer (BRET) to demonstrate that LTCS forms homo-oligomers in living cells. Fusion proteins of LTCS and Renilla luciferase (Rluc) and a variant of green fluorescent protein (GFP), respectively, were prepared. High BRET signals were recorded in transiently transfected human embryonic kidney (HEK 293) cells co-expressing Rluc/LTCS and GFP/LTCS. Homo-oligomer formation in living cells was verified by co-transfection of a plasmid expressing non-chimeric LTCS. This resulted in dose-dependent attenuation of the BRET signal. Additional evidence for oligomer formation was obtained in cell-free assays using glutathione S-transferase (GST) pull-down assay. To map interaction domains for oligomerization, GFP/LTCS fusion proteins were prepared with truncated variants of LTCS. The results obtained identified a C-terminal domain (amino acids 114–150) sufficient for oligomerization of LTCS. Another, centrally located, interaction domain appeared to exist between amino acids 57–88. The functional significance of LTCS homo-oligomer formation is currently being investigated.     

Characterization of leukotriene C4 binding sites in the brain of the American bullfrog, Rana catesbeiana.

In this study, specific binding sites for [3H]-LTC4 on membrane preparations from American bullfrog (Rana catesbeiana) brain were characterized. Binding assays were done in the presence of serine (5mM) borate (10 mM) for 30 min at 23 degrees C. Under these conditions, no metabolism of LTC4 to LTD4 occurred. Specific binding of [3H]-LTC4 reached steady state within 10 min, remained constant for 60 min, and was reversible with the addition of 1,000-fold excess unlabelled LTC4. Scatchard analysis of the binding data indicated a single class of binding sites with an estimated Kd of 89.83 nM and Bmax of 43.79 pmol/mg protein. Competition binding studies demonstrated that LTD4 and LTE4 were ineffective in displacing [3H]-LTC4 from its binding site. The Ki for LTC4 was 51 nM. S-decylglutathione, glutathione and hematin had Ki values of 44, 312,602, and 25,576 nM, respectively. The mammalian cysteinyl leukotriene antagonist L-660,711 inhibited specific binding of [3H]-LTC4, with a Ki of 87,149 nM. Guanosine-5'-0-3-thiotriphosphate (GTP gamma S) did not affect specific binding of [3H]-LTC4 indicating that, like mammalian LTC4 receptors, a Gi protein is not involved in the transduction mechanism. The LTC4 binding site in bullfrog brain demonstrates both similarities and differences from its mammalian counterpart.     

Human umbilical vein endothelial cells generate leukotriene C4 via microsomal glutathione S-transferase type 2 and express the CysLT(1) receptor.

Certain immunocompetent myeloid cells, such as eosinophils, basophils and mast cells, have a large capacity to synthesize the potent proinflammatory and spasmogenic mediator leukotriene (LT) C4 via a specific microsomal glutathione S-transferase (MGST) termed LTC4 synthase (LTC4S). Here, we report that MGST2, a distant homologue of LTC4S, is abundantly expressed in Human umbilical vein endothelial cells (HUVEC) and converts LTA4 into a single product, LTC4. Thus, using Northern blot, RT-PCR, Western blot, and enzyme activity assays, we show that MGST2 is the main, if not the only, enzyme that converts LTA4 into LTC4 in membrane preparations of HUVEC. In fact, we failed to detect any expression of LTC4S, MGST1 or MGST3 in these cells, indicating that MGST2 is a critical enzyme for transcellular LTC4 biosynthesis in the vascular wall. Unlike LTC4S, MGST2 prefers the naturally occurring free acid of LTA4 over the methyl ester as substrate and is also susceptible to product inhibition with an IC50 of about 1 microM for LTC4. Moreover, HUVEC were found to express the CysLT1 receptor in line with a paracrine and autocrine role for cysteinyl-leukotrienes in endothelial cell function.     

Oxidative stress suppresses cysteinyl leukotriene generation by mouse bone marrow-derived mast cells

Cysteinyl leukotrienes and oxidative stress have both been implicated in bronchial asthma; however, there is no previous study that focused on the ability of oxidative stress to alter cysteinyl leukotriene generation. In this study, treatment of bone marrow-derived mast cells with prostaglandin D(2) reduced their ability to generate leukotriene (LT) C(4) upon calcium ionophore stimulation but had little effect on LTB(4) generation. This effect could be reproduced by a selective agonist of the DP(2) receptor, 15R-methyl prostaglandin D(2) (15R-D(2)). 15R-D(2) dose-dependently inhibited LTC(4) generation with an IC(50) of 2 μM, and the effect was not altered by a DP(2)/thromboxane antagonist or by a peroxisome proliferator-activated receptor-γ antagonist. 15R-D(2) exerted its suppressive effect via a reduction in intracellular GSH, a mechanism that involved the conjugation of its non-enzymatic breakdown product to GSH. At 10 μM, 15R-D(2) reduced LTC(4) generation to 10%, intracellular GSH to 50%, and LTC(4) synthase (LTC(4)S) activity to 33.5% of untreated cells without altering immunoreactive LTC(4)S protein expression or 5-lipoxygenase activity. The effects of 15R-D(2) on LTC(4)S activity could be partially reversed by reducing reagent. The sulfhydryl-reactive oxidative agent diamide suppressed LTC(4)S activity and induced a reversible formation of covalent dimer LTC(4)S. LTC(4)S bearing a C56S mutation was resistant to the effect of diamide. Covalent dimer LTC(4)S was observed in nasal polyp biopsies, indicating that dimerization and inactivation of LTC(4)S can occur at the site of inflammation. These results suggest a cellular redox regulation of LTC(4)S function through a post-translational mechanism.     

Up-regulation of cysteinyl leukotriene 1 receptor by IL-13 enables human lung fibroblasts to respond to leukotriene C4 and produce eotaxin

Cysteinyl leukotrienes (CysLTs) play an important role in eosinophilic airway inflammation. In addition to their direct chemotactic effects on eosinophils, indirect effects have been reported. Eotaxin is a potent eosinophil-specific chemotactic factor produced mainly by fibroblasts. We investigated whether CysLTs augment eosinophilic inflammation via eotaxin production by fibroblasts. Leukotriene (LT)C(4) alone had no effect on eotaxin production by human fetal lung fibroblasts (HFL-1). However, LTC(4) stimulated eotaxin production by IL-13-treated fibroblasts, thereby indirectly inducing eosinophil sequestration. Unstimulated fibroblasts did not respond to LTC(4), but coincubation or preincubation of fibroblasts with IL-13 altered the response to LTC(4). To examine the mechanism(s) involved, the expression of CysLT1R in HFL-1 was investigated by quantitative real-time PCR and flow cytometry. Only low levels of CysLT1R mRNA and no CysLT1R protein were expressed in unstimulated HFL-1. In contrast, stimulation with IL-13 at a concentration of 10 ng/ml for 24 h significantly up-regulated both CysLT1R mRNA and protein expression in HFL-1. The synergistic effect of LTC(4) and IL-13 on eotaxin production was abolished by CysLT1R antagonists pranlukast and montelukast. These findings suggest that IL-13 up-regulates CysLT1R expression, which may contribute to the synergistic effect of LTC(4) and IL-13 on eotaxin production by lung fibroblasts. In the Th2 cytokine-rich milieu, such as that in bronchial asthma, CysLT1R expression on fibroblasts might be up-regulated, thereby allowing CysLTs to act effectively and increase eosinophilic inflammation.     

The identification of a distinct export step following the biosynthesis of leukotriene C4 by human eosinophils

We have examined the requirements for the export of leukotriene C4 (LTC4) from cultured human eosinophils. To define saturability and kinetics of LTC4 export, eosinophils were interacted with leukotriene A4 (LTA4) at 37 degrees C, and the methanolic extracts of the cell-associated and extracellular compartments were then analyzed for LTC4 content by reverse phase high performance liquid chromatography with on-line monitoring of absorbance at 280 nm. When LTA4 was added at concentrations from 0 to 100 microM for 10 min at 37 degrees C, the amount of LTC4 released extracellularly became constant at an LTA4 concentration of 7.5 microM or greater even though the amount of intracellular LTC4 continued to increase. When eosinophils were incubated with 50 microM LTA4 for 0-60 min at 37 degrees C and then held at 0 degrees C for the remainder of the 60-min interval, 54.2 and 77.3% (n = 3), respectively, of the total LTC4 was released extracellularly after 15 and 30 min of incubation at 37 degrees C. Eosinophils incubated with 50 microM LTA4 at 0 degrees C for 1 h synthesized 290 pmol of LTC4 (n = 3) which was approximately half-maximal, all of which was retained intracellularly. We utilized the time and temperature dependence of LTC4 export to preload eosinophils with both LTC4 and leukotriene C5 (LTC5) by sequentially supplying them with specific substrates. With increasing concentrations of intracellular LTC5, there was dose-dependent inhibition of the subsequent release of LTC4 at 37 degrees C, with the sum of the released glutathionyl leukotrienes remaining constant. In addition, only minimal competition for LTC4 release occurred when cells were preloaded with both LTC4 and the conjugate of 1-chloro-2,4-dinitrobenzene and reduced glutathione, S-(dinitrophenyl)glutathione. The criteria of saturability, time dependence of LTC4 release at 37 degrees C, competition of LTC4 with LTC5 for release, and the inhibition of LTC4 release at 0 degrees C establish the export of LTC4 from cells as a novel and specific biochemical step distinct from both LTA4 uptake and the conjugation of LTA4 with reduced glutathione by LTC4 synthase to form LTC4.     

Induction of leukotriene C4 synthase activity in differentiating human erythroleukemia cells.

Leukotriene (LT)C4 synthase is a membrane-bound, specific glutathione transferase which catalyzes the transformation of LTA4 to LTC4. It was originally shown to be present in rodent mastocytoma and basophilic leukemia cells as well as in macrophages. Recently, expression of human LTC4 synthase was demonstrated in platelets (S?derstr?m, M., et al. (1992) Arch. Biochem. Biophys. 294, 70-74). The present report describes the induction of LTC4 synthase activity during differentiation of human erythroleukemia (HEL) cells by the protein kinase C stimulator 12-O-tetradecanoyl phorbol 13-acetate (TPA), ligands of the steroid-thyroid hormone receptor superfamily: all-trans-retinoic acid (RA) and 1 alpha, 25-dihydroxy-vitamin D3 and in addition dimethylsulfoxide (DMSO). TPA was the most powerful inducer of enzyme activity followed by 1 alpha, 25-dihydroxy-vitamin D3 and DMSO. RA did not induce LTC4 synthase activity.     

Attenuated zymosan-induced peritoneal vascular permeability and IgE-dependent passive cutaneous anaphylaxis in mice lacking leukotriene C4 synthase

Leukotriene C(4) synthase (LTC(4)S), the terminal 5-lipoxygenase pathway enzyme that is responsible for the biosynthesis of cysteinyl leukotrienes, has been deleted by targeted gene disruption to define its tissue distribution and integrated pathway function in vitro and in vivo. The LTC(4)S (-/-) mice developed normally and were fertile. LTC(4)S activity, assessed by conjugation of leukotriene (LT) A(4) methyl ester with glutathione, was absent from tongue, spleen, and brain and > or = 90% reduced in lung, stomach, and colon of the LTC(4)S (-/-) mice. Bone marrow-derived mast cells (BMMC) from the LTC(4)S (-/-) mice provided no LTC(4) in response to IgE-dependent activation. Exocytosis and the generation of prostaglandin D(2), LTB(4), and 5-hydroxyeicosatetraenoic acid by BMMC from LTC(4)S (-/-) mice and LTC(4)S (+/+) mice were similar, whereas the degraded product of LTA(4), 6-trans-LTB(4), was doubled in BMMC from LTC(4)S (-/-) mice because of lack of utilization. The zymosan-elicited intraperitoneal extravasation of plasma protein and the IgE-mediated passive cutaneous anaphylaxis in the ear were significantly diminished in the LTC(4)S (-/-) mice. These observations indicate that LTC(4)S, but not microsomal or cytosolic glutathione S-transferases, is the major LTC(4)-producing enzyme in tissues and that its integrated function includes mediation of increased vascular permeability in either innate or adaptive immune host inflammatory responses.     

Leukotriene C4 synthase is a novel PPARγ target gene,and leukotriene C4 and D4 activate adipogenesis through cysteinyl LT1 receptors in adipocytes

Leukotriene (LT) C₄ synthase (LTC4S) catalyzes the conversion from LTA₄ to LTC_4, which is a proinflammatory lipid mediator in asthma and other inflammatory diseases. LTC₄ is metabolized to LTD₄ and LTE₄, all of which are known as cysteinyl (Cys) LTs and exert physiological functions through CysLT receptors. LTC4S is expressed in adipocytes. However, the function of CysLTs and the regulatory mechanism in adipocytes remain unclear. In this study, we investigated the expression of LTC4S and production of CysLTs in murine adipocyte 3T3-L1 cells and their underlying regulatory mechanisms. Expression of LTC4S and production of LTC₄ and CysLTs increased during adipogenesis, whereas siRNA-mediated suppression of LTC4S expression repressed adipogenesis by reducing adipogenic gene expression. The CysLT1 receptor, one of the two LTC₄ receptors, was expressed in adipocytes. LTC₄ and LTD₄ increased the intracellular triglyceride levels and adipogenic gene expression, and their enhancement was suppressed by co-treatment with pranlukast, a CysLT1 receptor antagonist. Moreover, the expression profiles of LTC4S gene/protein during adipogenesis resembled those of peroxisome proliferator-activated receptor (PPAR) γ. LTC4S expression was further upregulated by treatment with troglitazone, a PPARγ agonist. Promoter-luciferase and chromatin immunoprecipitation assays showed that PPARγ directly bound to the PPAR response element of the LTC4S gene promoter in adipocytes. These results indicate that the LTC4S gene expression was enhanced by PPARγ, and LTC₄ and LTD₄ activated adipogenesis through CysLT1 receptors in 3T3-L1 cells. Thus, LTC4S and CysLT1 receptors are novel potential targets for the treatment of obesity.     

Demonstration of cell-specific phosphorylation of LTC4 synthase

PMA-induced leukotriene C4 synthase (LTC4S) phosphorylation was investigated over a period of 8 h in a monocytic cell line (THP-1). The level of LTC4S phosphorylation was increased 3-5 fold over a 4 h period decreasing to basal levels after 8 h. This phosphorylation event was found to be specific to THP-1 cells as there was a lack of LTC4S phosphorylation in both COS-7 and K-562 cells, and was also found to be dependent on the cellular confluency. In the presence of specific protein kinase C (PKC) inhibitors, a dose-dependent inhibition of the phosphorylation of LTC4S became evident, an effect not seen with PKA and tyrosine kinase inhibitors. This represents the first direct demonstration of LTC4S phosphorylation in whole cells.     

Membrane localization and topology of leukotriene C4 synthase

Leukotriene C(4) (LTC(4)) synthase conjugates LTA(4) with GSH to form LTC(4). Determining the site of LTC(4) synthesis and the topology of LTC(4) synthase may uncover unappreciated intracellular roles for LTC(4), as well as how LTC(4) is transferred to its export carrier, the multidrug resistance protein-1. We have determined the membrane localization of LTC(4) synthase by immunoelectron microscopy. In contrast to the closely related five-lipoxygenase-activating protein, LTC(4) synthase is distributed in the outer nuclear membrane and peripheral endoplasmic reticulum but is excluded from the inner nuclear membrane. We have combined immunofluorescence with differential membrane permeabilization to determine the topology of LTC(4) synthase. The active site of LTC(4) synthase is localized in the lumen of the nuclear envelope and endoplasmic reticulum. These results indicate that the synthesis of LTB(4) and LTC(4) occurs in different subcellular locations and suggests that LTC(4) must be returned to the cytoplasmic side of the membrane for export by multidrug resistance protein-1. The differential localization of two very similar integral membrane proteins suggests that mechanisms other than size-dependent exclusion regulate their passage to the inner nuclear membrane.     

Characterization of Seizure-Induced Cysteinyl-Leukotriene Formation in Brain Tissue of Convulsion-Prone Gerbils

Tonic-clonic seizures elicited in convulsion-prone gerbils resulted in a large increase in immunoreactive prostaglandin (PG) F2 alpha and in a smaller increase in immunoreactive leukotriene (LT) C4-like material in brain tissue. Brain tissue contents of both eicosanoids were found to reach a maximum at 6 min after the onset of seizures and were still elevated at 54 min after the beginning of convulsions. By reversed phase HPLC the immunoreactive LTC4-like material was identified as LTC4 and LTD4 at 6 min after the onset of convulsions, whereas at 54 min after the onset, transformation of LTD4 to LTE4 could be detected as well. In gerbils showing only weak seizure activity a small increase in PGF2 alpha but no increase in immunoreactive LTC4-like material could be detected at 6 min after the onset of convulsions. Pretreatment with indomethacin abolished the formation of PGF2 alpha but significantly enhanced the biosynthesis of immunoreactive LTC4-like material at 18 min after the beginning of seizures. The results demonstrate formation of cysteinyl-LT following tonic-clonic convulsions in spontaneously convulsing gerbils which could be enhanced by inhibition of the cyclooxygenase pathway of arachidonic acid metabolism. Since cysteinyl-LT have potent biological actions in various organs this finding warrants further investigations on the potential role of cysteinyl-LT in the CNS.     

Cutting edge: Interleukin 4-dependent mast cell proliferation requires autocrine/intracrine cysteinyl leukotriene-induced signaling

Reactive mastocytosis (RM) in epithelial surfaces is a consistent Th2-associated feature of allergic disease. RM fails to develop in mice lacking leukotriene (LT) C4 synthase (LTC4S), which is required for cysteinyl leukotriene (cys-LT) production. We now report that IL-4, which induces LTC4S expression by mast cells (MCs), requires cys-LTs, the cys-LT type 1 receptor (CysLT1), and Gi proteins to promote MC proliferation. LTD4 (10-1000 nM) enhanced proliferation of human MCs in a CysLT1-dependent, pertussis toxin-sensitive manner. LTD4-induced phosphorylation of ERK required transactivation of c-kit. IL-4-driven comitogenesis was likewise sensitive to pertussis toxin or a CysLT1-selective antagonist and was attenuated by treatment with leukotriene synthesis inhibitors. Mouse MCs lacking LTC4S or CysLT1 showed substantially diminished IL-4-induced comitogenesis. Thus, IL-4 induces proliferation in part by inducing LTC4S and cys-LT generation, which causes CysLT1 to transactivate c-kit in RM.     

Cysteinyl leukotrienes regulate Th2 cell-dependent pulmonary inflammation

The Th2 cell-dependent inflammatory response is a central component of asthma, and the ways in which it is regulated is a critical question. The cysteinyl leukotrienes (cys-LTs) are 5-lipoxygenase pathway products implicated in asthma, in particular, by their function as smooth muscle constrictors of airways and microvasculature. To elucidate additional roles for cys-LTs in the pathobiology of pulmonary inflammation, we used an OVA sensitization and challenge protocol with mice lacking leukotriene C(4) synthase (LTC(4)S), the terminal enzyme for cys-LT generation. Ag-induced pulmonary inflammation, characterized by eosinophil infiltration, goblet cell hyperplasia with mucus hypersecretion, and accumulation and activation of intraepithelial mast cells was markedly reduced in LTC(4)S(null) mice. Furthermore, Ag-specific IgE and IgG1 in serum, Th2 cell cytokine mRNA expression in the lung, and airway hyperresponsiveness to methacholine were significantly reduced in LTC(4)S(null) mice compared with wild-type controls. Finally, the number of parabronchial lymph node cells from sensitized LTC(4)S(null) mice and their capacity to generate Th2 cell cytokines ex vivo after restimulation with Ag were also significantly reduced. In contrast, delayed-type cutaneous hypersensitivity, a prototypic Th1 cell-dependent response, was intact in LTC(4)S(null) mice. These findings provide direct evidence of a role for cys-LTs in regulating the initiation and/or amplification of Th2 cell-dependent pulmonary inflammation.     

Selective inhibition of leukotriene C4 synthesis and natural killer activity by ethacrynic acid

We have investigated the role of arachidonic acid (AA) metabolism in natural killer (NK) cell activity. Human nonadherent (NA) peripheral blood lymphocytes were used as effector cells against 51Cr-labeled K562 target cells. Synthesis of leukotriene C4 (LTC4) is dependent on glutathione S-transferase (GST). We have chosen to study three putative GST inhibitors, namely, ethacrynic acid (ET), caffeic acid (CA), and ferulic acid (FA), with regard to NK activity and with regard to their effect on AA metabolism. The GST inhibitors inhibited NK lysis when added directly to the NK assay. The GST inhibitors inhibited LTC4 synthesis as induced by calcium ionophore A23187 in a dose-dependent manner similar to their inhibition of NK activity. However, only ET was selective, for it had little effect on LTB4, 5-hydroxyeicosatetraenoic acid, and prostaglandin E2 synthesis. LTC4 synthesis was associated with the NK-enriched fractions obtained from discontinuous Percoll gradients. NK-specific anti-Leu-11b antibody and C treatment could abrogate NK activity and LTC4 synthesis. ET was also inhibitory when NA cells were cultured at 37 degrees C for 18 hr. In this case, LTC4 could reverse the inhibitory effect of ET. Our data suggest that LTC4 plays an important role in NK activity.     

Biosynthesis and metabolism of leukotrienes

Leukotrienes are metabolites of arachidonic acid derived from the action of 5-LO (5-lipoxygenase). The immediate product of 5-LO is LTA4 (leukotriene A4), which is enzymatically converted into either LTB4 (leukotriene B4) by LTA4 hydrolase or LTC4 (leukotriene C4) by LTC4 synthase. The regulation of leukotriene production occurs at various levels, including expression of 5-LO, translocation of 5-LO to the perinuclear region and phosphorylation to either enhance or inhibit the activity of 5-LO. Several other proteins, including cPLA2a (cytosolic phospholipase A2a) and FLAP (5-LO-activating protein) also assemble at the perinuclear region before production of LTA4. LTC4 synthase is an integral membrane protein that is present at the nuclear envelope; however, LTA4 hydrolase remains cytosolic. Biologically active LTB4 is metabolized by w-oxidation carried out by specific cytochrome P450s (CYP4F) followed by b-oxidation from the w-carboxy position and after CoA ester formation. Other specific pathways of leukotriene metabolism include the 12-hydroxydehydrogenase/15-oxo-prostaglandin-13-reductase that forms a series of conjugated diene metabolites that have been observed to be excreted into human urine. Metabolism of LTC4 occurs by sequential peptide cleavage reactions involving a g-glutamyl transpeptidase that forms LTD4 (leukotriene D4) and a membrane-bound dipeptidase that converts LTD4 into LTE4 (leukotriene E4) before w-oxidation. These metabolic transformations of the primary leukotrienes are critical for termination of their biological activity, and defects in expression of participating enzymes may be involved in specific genetic disease.