Microsomal glutathione transferase 2 modulates LTC4 synthesis and ROS production in Apostichopus japonicus.

Microsomal glutathione transferase 2 (mGST2) is an integral membrane protein involved in detoxication of xenobiotics, and has also been suggested to catalyze the biosynthesis of pro-inflammatory mediator leukotriene C4 (LTC4) as homologous to LTC4 synthase (LTC4S) in mammals. In the present study, a novel mGST2 homology was identified from Apostichopus japonicus (designated as AjmGST2) by RACE approaches. The full-length cDNA of AjmGST2 was of 1917bp encoding a polypeptide of 161 amino acids residues. Multiple sequences alignment and phylogenetic analysis together supported that AjmGST2 belonged to a new member in invertebrate mGSTs family and close to mammalian LTC4S. Spatial expression analysis revealed that AjmGST2 was ubiquitously expressed in all examined tissues with the larger magnitude in intestine. AjmGST2 transcripts in coelomocytes were slightly induced post 6h challenge of pathogenic Vibrio splendidus and reached the peak expression at 48h. The increased expression profiles of AjmGST2 were also detected in lipopolysaccharide (LPS) exposed primary coelomocytes. Consistently, LTC4 contents were also induced by a 1.56-fold increase in the same condition. Functional assay further revealed that AjmGST2 might be functioned as LTC4S to promote LTC4 synthesis. AjmGST2 knock-down by specific siRNA significantly depressed LTC4 contents with 27.0% decrease at 24h. Meantime, ROS levels were elevated by 40.1% in vitro. All of these results indicated that AjmGST2 performed dual functions roles as LTC4S and ROS eliminator in sea cucumber immune response.

Pulmonary epithelial cancer cells and their exosomes metabolize myeloid cell-derived leukotriene C4 to leukotriene D4.

Leukotrienes (LTs) play major roles in lung immune responses, and LTD4 is the most potent agonist for cysteinyl LT1, leading to bronchoconstriction and tissue remodeling. Here, we studied LT crosstalk between myeloid cells and pulmonary epithelial cells. Monocytic cells (Mono Mac 6 cell line, primary dendritic cells) and eosinophils produced primarily LTC4 In coincubations of these myeloid cells and epithelial cells, LTD4 became a prominent product. LTC4 released from the myeloid cells was further transformed by the epithelial cells in a transcellular manner. Formation of LTD4 was rapid when catalyzed by γ-glutamyl transpeptidase (GGT)1 in the A549 epithelial lung cancer cell line, but considerably slower when catalyzed by GGT5 in primary bronchial epithelial cells. When A549 cells were cultured in the presence of IL-1ß, GGT1 expression increased about 2-fold. Also exosomes from A549 cells contained GGT1 and augmented LTD4 formation. Serine-borate complex (SBC), an inhibitor of GGT, inhibited conversion of LTC4 to LTD4 Unexpectedly, SBC also upregulated translocation of 5-lipoxygenase (LO) to the nucleus in Mono Mac 6 cells, and 5-LO activity. Our results demonstrate an active role for epithelial cells in biosynthesis of LTD4, which may be of particular relevance in the lung.

Leukotriene C4 is the major trigger of stress-induced oxidative DNA damage.

Endoplasmic reticulum (ER) stress and major chemotherapeutic agents damage DNA by generating reactive oxygen species (ROS). Here we show that ER stress and chemotherapy induce leukotriene C4 (LTC4) biosynthesis by transcriptionally upregulating and activating the enzyme microsomal glutathione-S-transferase 2 (MGST2) in cells of non-haematopoietic lineage. ER stress and chemotherapy also trigger nuclear translocation of the two LTC4 receptors. Acting in an intracrine manner, LTC4 then elicits nuclear translocation of NADPH oxidase 4 (NOX4), ROS accumulation and oxidative DNA damage. Mgst2 deficiency, RNAi and LTC4 receptor antagonists abolish ER stress- and chemotherapy-induced ROS and oxidative DNA damage in vitro and in mouse kidneys. Cell death and mouse morbidity are also significantly attenuated. Hence, MGST2-generated LTC4 is a major mediator of ER stress- and chemotherapy-triggered oxidative stress and oxidative DNA damage. LTC4 inhibitors, commonly used for asthma, could find broad clinical use in major human pathologies associated with ER stress-activated NOX4.

Cinnamaldehyde is the main mediator of cinnamon extract in mast cell inhibition.

PURPOSE: In terms of their involvement in allergic and inflammatory conditions, mast cells (MC) can be promising targets for medical agents in therapy. Because of their good compliance and effectiveness, phytochemicals are of great interest as new therapeutic tools in form of nutraceuticals. We found recently that cinnamon extract (CE) inhibits mast cell activation. Here, we analysed the effects of a major compound of CE, cinnamaldehyde (CA), on mast cell activation. METHODS: Release of prestored and de novo synthesised mediators as well as expression of pro-inflammatory cytokines and mast cell-specific proteases were analysed in RBL-2H3 cells or in human mast cells isolated from intestinal tissue (hiMC) treated with CA prior to stimulation by FcεRI crosslinking or IONO/PMA. The results were compared with the corresponding effects of CE. RESULTS: Following treatment with CA, release of ß-hexosaminidase in IgE-dependent or IgE-independent activated RBL-2H3 cells was down-regulated in a dose-dependent manner to about 10%. In hiMC, release of ß-hexosaminidase was also significantly reduced, and release of LTC4 and CXCL8 was almost completely inhibited by CA. Moreover, IgE-mediated expression of CXCL8, CCL2, CCL3 and CCL4 in hiMC was significantly down-regulated by CA. With the exception of the expression of the mast cell proteases tryptase and chymase, the inhibitory effects of CA were very similar to the effects shown for CE treatment. The reducing effect of CA on mast cell mediators-seen for long- and for short-term incubations-could be related to particular signalling pathways as CA caused a down-regulation in ERK as well as PLCγ1 phosphorylation. CONCLUSIONS: CA decreases release and expression of pro-inflammatory mast cell mediators. This inhibitory action is similar to the effects observed for CE indicating CA as the main active compound in CE leading to its anti-allergic properties.

Identification of GPR99 protein as a potential third cysteinyl leukotriene receptor with a preference for leukotriene E4 ligand.

The cysteinyl leukotrienes (cys-LTs), leukotriene C4 (LTC4), a conjugation product of glutathione and eicosatetraenoic acid, and its metabolites, LTD4 and LTE4, are lipid mediators of smooth muscle constriction and inflammation in asthma. LTD4 is the most potent ligand for the type 1 cys-LT receptor (CysLT1R), and LTC4 and LTD4 have similar lesser potency for CysLT2R, whereas LTE4 has little potency for either receptor. Cysltr1/Cysltr2(-/-) mice, lacking the two defined receptors, exhibited a comparable dose-dependent vascular leak to intradermal injection of LTC4 or LTD4 and an augmented response to LTE4 as compared with WT mice. As LTE4 retains a cysteine residue and might provide recognition via a dicarboxylic acid structure, we screened cDNAs within the P2Y nucleotide receptor family containing CysLTRs and dicarboxylic acid receptors with trans-activator reporter gene assays. GPR99, previously described as an oxoglutarate receptor (Oxgr1), showed both a functional and a binding response to LTE4 in these transfectants. We generated Gpr99(-/-) and Gpr99/Cysltr1/Cysltr2(-/-) mice for comparison with WT and Cysltr1/Cysltr2(-/-) mice. Strikingly, GPR99 deficiency in the Cysltr1/Cysltr2(-/-) mice virtually eliminated the vascular leak in response to the cys-LT ligands, indicating GPR99 as a potential CysLT3R active in the Cysltr1/Cysltr2(-/-) mice. Importantly, the Gpr99(-/-) mice showed a dose-dependent loss of LTE4-mediated vascular permeability, but not to LTC4 or LTD4, revealing a preference of GPR99 for LTE4 even when CysLT1R is present. As LTE4 is the predominant cys-LT species in inflamed tissues, GPR99 may provide a new therapeutic target.

Saucerneol D inhibits eicosanoid generation and degranulation through suppression of Syk kinase in mast cells.

Previously we reported that saucerneol D (SD), a naturally occurring sesquilignan isolated from Saururus chinensis (S. chinensis) suppressed lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 cells. The aim of this study was to elucidate whether SD modulates the generation of other inflammatory mediators in activated mast cells. We investigated the effects of SD on cyclooxygenase-2 (COX-2)-dependent prostaglandin D(2) (PGD(2)) and 5-lipoxygenase (5-LO)-dependent leukotriene C(4) (LTC(4)) generations as well as degranulation in cytokine-stimulated mouse bone marrow-derived mast cells (BMMCs). Biochemical analyses of the cytokine-mediated signaling pathways showed that SD suppressed the phosphorylation of Syk kinase and multiple downstream signaling processes including phospholipase Cγ1 (PLCγ1)-mediated intracellular Ca(2+) influx and activation of mitogen-activated protein kinases (MAPKs; including extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun NH(2)-terminal kinase (JNK) and p38) and the nuclear factor-κB (NF-κB) pathway. Taken together, the present study suggests that SD suppresses eicosanoid generation and degranulation through Syk-dependent pathway in BMMCs.

Lipoxin A4 inhibits 5-lipoxygenase translocation and leukotrienes biosynthesis to exert a neuroprotective effect in cerebral ischemia/reperfusion injury.

Lipoxin A(4) (LXA(4)), a biologically active eicosanoid with anti-inflammatory and pro-resolution properties, was recently found to have neuroprotective effects in brain ischemia. As 5-lipoxygenase (5-LOX) and leukotrienes are generally considered to aggravate cerebral ischemia/reperfusion (I/R) injury, we investigated their effects on LXA(4)-mediated neuroprotection by studying middle cerebral artery occlusion (MCAO)/reperfusion in rats and oxygen-glucose deprivation (OGD)/recovery in neonatal rat astrocyte primary cultures. LXA(4) effectively reduced infarct volumes and brain edema, and improved neurological scores in the MCAO/reperfusion experiments; this effect was partially blocked by butoxycarbonyl-Phe-Leu-Phe-Leu-Phe (Boc2), a specific antagonist of the LXA(4) receptor (ALXR). Total 5-LOX expression did not change, regardless of treatment, but LXA(4) could inhibit nuclear translocation induced by MCAO or OGD. We also found that LXA(4) inhibits the upregulation of both leukotriene B(4) (LTB(4)) and leukotriene C(4) (LTC(4)) and the phosphorylation of extracellular signal-regulated kinase (ERK) induced by MCAO or OGD. The phosphorylation of the 38-kDa protein kinase (p38) and c-Jun N-terminal kinase (JNK) was not altered throughout the experiment. These results suggest that the neuroprotective effects of LXA(4) are probably achieved by anti-inflammatory mechanisms that are partly mediated by ALXR and through an ERK signal transduction pathway.

Effect of multiple cysteine substitutions on the functionality of human multidrug resistance protein 1 expressed in human embryonic kidney 293 cells: identification of residues essential for function.

Multidrug resistance protein 1 (MRP1) is a broad-specificity membrane transporter belonging to the C branch of the ATP binding cassette (ABC) superfamily. MRP1 confers resistance to various chemotherapeutic drugs and transports a wide range of conjugated organic anions. Several ABCC proteins, including MRP1, are unusual among ABC transporters in having a third membrane-spanning domain (MSD), MSD0, at their N termini. MRP1 lacking this additional MSD (ΔMRP1) is able to traffic to the plasma membrane of mammalian cells and to transport a number of well characterized substrates. A cysteineless (cysless) ΔMRP1 has been expressed in yeast and reported to be functional. However, we found that trafficking of such a construct in human cells was severely compromised, and, even when expressed in insect Sf21 cells, the protein had extremely low transport activity. Therefore, we have systematically examined the effects of substituting cysteines in the four domains of ΔMRP1, initially with alanine. These studies allowed us to identify five cysteines that cannot be replaced with alanine without inactivating the protein. Substitution of two of these residues with alternative amino acids has allowed us to produce an almost cysless form of ΔMRP1 that traffics to the plasma membrane and transports leukotriene C(4), 17ß-estradiol 17-ß-D-glucuronide, and estrone-3-sulfate with kinetic characteristics similar to those of the wild-type protein. The distribution of the remaining Cys residues is such that the protein will provide a useful template for a variety of cysteine based mutagenesis studies.

The catalytic architecture of leukotriene C4 synthase with two arginine residues.

Leukotriene (LT) C(4) and its metabolites, LTD(4) and LTE(4), are involved in the pathobiology of bronchial asthma. LTC(4) synthase is the nuclear membrane-embedded enzyme responsible for LTC(4) biosynthesis, catalyzing the conjugation of two substrates that have considerably different water solubility; that amphipathic LTA(4) as a derivative of arachidonic acid and a water-soluble glutathione (GSH). A previous crystal structure revealed important details of GSH binding and implied a GSH activating function for Arg-104. In addition, Arg-31 was also proposed to participate in the catalysis based on the putative LTA(4) binding model. In this study enzymatic assay with mutant enzymes demonstrates that Arg-104 is required for the binding and activation of GSH and that Arg-31 is needed for catalysis probably by activating the epoxide group of LTA(4).

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.

Mast cell activation syndrome: Proposed diagnostic criteria.

The term mast cell activation syndrome (MCAS) is finding increasing use as a diagnosis for subjects who present with signs and symptoms involving the dermis, gastrointestinal track, and cardiovascular system frequently accompanied by neurologic complaints. Such patients often have undergone multiple extensive medical evaluations by different physicians in varied disciplines without a definitive medical diagnosis until the diagnosis of MCAS is applied. However, MCAS as a distinct clinical entity has not been generally accepted, nor do there exist definitive criteria for diagnosis. Based on current understanding of this disease "syndrome" and on what we do know about mast cell activation and resulting pathology, we will explore and propose criteria for its diagnosis. The proposed criteria will be discussed in the context of other disorders involving mast cells or with similar presentations and as a basis for further scientific study and validation.

Leukotrienes modulate secretion of progesterone and prostaglandins during the estrous cycle and early pregnancy in cattle: an in vivo study.

Recently, we showed that leukotrienes (LTs) regulate ovarian cell function in vitro. The aim of this study was to examine the role of LTs in corpus luteum (CL) function during both the estrous cycle and early pregnancy in vivo. mRNA expression of LT receptors (BLT for LTB(4) and CYSLT for LTC(4)), and 5-lipoxygenase (5-LO) in CL tissue and their localization in the ovary were studied during the estrous cycle and early pregnancy. Moreover, concentrations of LTs (LTB(4) and C(4)) in the CL tissue and blood were measured. 5-LO and BLT mRNA expression increased on days 16-18 of the cycle, whereas CYSLT mRNA expression increased on days 16-18 of the pregnancy. The level of LTB(4) was evaluated during pregnancy compared with the level of LTC(4), which increased during CL regression. LT antagonists influenced the duration of the estrous cycle: the LTC(4) antagonist (azelastine) prolonged the luteal phase, whereas the LTB(4) antagonist (dapsone) caused earlier luteolysis in vivo. Dapsone decreased progesterone (P(4)) secretion and azelastine increased P(4) secretion during the estrous cycle. In summary, LT action in the bovine reproductive tract is dependent on LT type: LTB(4) is luteotropic during the estrous cycle and supports early pregnancy, whereas LTC(4) is luteolytic, regarded as undesirable in early pregnancy. LTs are produced/secreted in the CL tissue, influence prostaglandin function, and serve as important factors during the estrous cycle and early pregnancy in cattle.

The phosphoinositide 3-kinase-dependent activation of Btk is required for optimal eicosanoid production and generation of reactive oxygen species in antigen-stimulated mast cells.

Activated mast cells are a major source of the eicosanoids PGD(2) and leukotriene C(4) (LTC(4)), which contribute to allergic responses. These eicosanoids are produced following the ERK1/2-dependent activation of cytosolic phospholipase A(2), thus liberating arachidonic acid, which is subsequently metabolized by the actions of 5-lipoxygenase and cyclooxygenase to form LTC(4) and PGD(2), respectively. These pathways also generate reactive oxygen species (ROS), which have been proposed to contribute to FcepsilonRI-mediated signaling in mast cells. In this study, we demonstrate that, in addition to ERK1/2-dependent pathways, ERK1/2-independent pathways also regulate FcepsilonRI-mediated eicosanoid and ROS production in mast cells. A role for the Tec kinase Btk in the ERK1/2-independent regulatory pathway was revealed by the significantly attenuated FcepsilonRI-dependent PGD(2), LTC(4), and ROS production in bone marrow-derived mast cells of Btk(-/-) mice. The FcepsilonRI-dependent activation of Btk and eicosanoid and ROS generation in bone marrow-derived mast cells and human mast cells were similarly blocked by the PI3K inhibitors, Wortmannin and LY294002, indicating that Btk-regulated eicosanoid and ROS production occurs downstream of PI3K. In contrast to ERK1/2, the PI3K/Btk pathway does not regulate cytosolic phospholipase A(2) phosphorylation but rather appears to regulate the generation of ROS, LTC(4), and PGD(2) by contributing to the necessary Ca(2+) signal for the production of these molecules. These data demonstrate that strategies to decrease mast cell production of ROS and eicosanoids would have to target both ERK1/2- and PI3K/Btk-dependent pathways.

Analysis of 927T > C CYSLTR1 and –444A > C LTC4S polymorphisms in children with asthma

Introduction: The cysteinyl leukotrienes (Cys-LTs) are potent inflammatory mediators in asthma. It has been suggested that the different response of patients to Cys-LTs inhibitors could be due to the presence of polymorphisms in the genes implicated in this pathway. Methods: In this study, polymorphisms 927T > CCYSLTR1 and –444A > C LTC4S were analysed in a Spanish population of 188 individuals (109 asthmatic children and 79 controls). Standardised history, skinprick tests and lung function measurements were performed in all patients. Genotypes were determined by sequencing after PCR amplification. Results: Differences were observed in 927T > CCYSLTR1, regarding the severity of asthma in males. A greater presence of allele C in the population with persistent asthma versus the control group (Fisher’sp-value = 0.001; Monte Carlo p-value = 0.003; OR:12.35; 95 %CI: 2.18-70.00) was observed. Differences were also detected in the combined study of both polymorphisms, among controls and asthmatic patients (Monte Carlo p-value = 0.0002). In the group of males with asthma, an increase of AC variant (–444A LTC4S and 927C CYSLTR1) and a reduction in the AT genetic combination were detected. Conclusions: The combined study of polymorphisms in genes of the leukotriene pathway could explain the differences observed in the studies reported on polymorphism –444A < C LTC4S individually analysed

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Interaction between the bound Mg.ATP and the Walker A serine residue in NBD2 of multidrug resistance-associated protein MRP1 plays a crucial role for the ATP-dependent leukotriene C4 transport.

Structural analysis of human MRP1-NBD1 revealed that the Walker A S685 forms a hydrogen bond with the Walker B D792 and interacts with the Mg (2+) cofactor and the beta-phosphate of the bound Mg.ATP. We have found that substitution of the S685 with an amino acid that potentially prevents the formation of the hydrogen bond resulted in misfolding of the protein and significantly affect the ATP-dependent leukotriene C4 (LTC4) transport. In this report we tested whether the corresponding substitution in NBD2 would also result in misfolding of the protein. In contrast to the NBD1 mutations, none of the mutations in NBD2, including S1334A, S1334C, S1334D, S1334H, S1334N, and S1334T, caused misfolding of the protein. However, elimination of the hydroxyl group at S1334 in mutations including S1334A, S1334C, S1334D, S1334H, and S1334N drastically reduced the ATP binding and the ATP-enhanced ADP trapping at the mutated NBD2. Due to this low efficient ATP binding at the mutated NBD2, the inhibitory effect of ATP on the LTC4 binding is significantly decreased. Furthermore, ATP bound to the mutated NBD2 cannot be efficiently hydrolyzed, leading to almost completely abolishing the ATP-dependent LTC4 transport. In contrast, S1334T mutation, which retained the hydroxyl group at this position, exerts higher LTC4 transport activity than the wild-type MRP1, indicating that the hydroxyl group at this position plays a crucial role for ATP binding/hydrolysis and ATP-dependent solute transport.

The effects of isoimperatorin isolated from Angelicae dahuricae on cyclooxygenase-2 and 5-lipoxygenase in mouse bone marrow-derived mast cells.

Isoimperatorin (4-[(3-Methyl-2-butenyl)oxy]-7H-furo[3,2-g][1]benzopyran-7-one) is a medicinal herbal product that is isolated from the dried roots of Angelicae dahuricae. Isoimperatorin inhibits the cyclooxygenase-2 (COX-2) and COX-1-dependent phases of prostaglandin D2 (PGD2) generation in bone marrow-derived mast cells (BMMC) in a concentration-dependent manner, with IC50 values of 10.7 microM and 24 microM, respectively. However, this compound was not able to inhibit COX-1 and 2 protein expression in BMMC that were treated with concentrations of up to 50 microM, which indicates that isoimperatorin directly inhibits COX-2 activity. Furthermore, this compound consistently inhibited the production of leukotriene C4 (LTC4), as well as the degranulation reaction in BMMC, with an IC50 value of 5.7 microM and 9 microM, respectively, and these effects occurred in a dose dependent fashion. These results demonstrate that isoimperatorin has a dual cyclooxygenase-2 selective/5-lipoxygenase inhibitory activity, and therefore may provide the basis for novel anti-inflammatory drugs.

Ginkgetin, a Biflavone from Ginko biloba leaves, inhibits cyclooxygenases-2 and 5-lipoxygenase in mouse bone marrow-derived mast cells.

Ginkgetin, a biflavone from Ginkgo biloba leaves, was previously reported to be a phospholipase A(2) inhibitor and this compound showed the potent antiarthritic activity in rat adjuvant-induced arthritis as well as analgesic activity. This investigation was carried out to find effects on cyclooxygenase-2 (COX-2) in vitro effect. Ginkgetin inhibits COX-2 dependent phases of prostaglandin D(2) (PGD(2)) generation in bone marrow-derived mast cells (BMMC) in a concentration-dependent manner with IC(50) values of 0.75 microM. Western blotting probed with specific anti-COX-2 antibodies showed that the decrease in quantity of the PGD(2) product was accompanied by a decrease in the COX-2 protein level. In addition, this compound consistently inhibited the production of leukotriene C(4) (LTC(4)) in a dose dependent manner, with an IC(50) value of 0.33 microM. These results demonstrate that ginkgetin has a dual cyclooxygenase-2/5-lipoxygenase inhibitory activity. Furthermore, this compound also inhibited degranulation reaction in a dose dependent manner, with an IC(50) value of 6.52 microM. Therefore, this compound might provide a basis for novel anti-inflammatory agents.

Post-transcriptional regulation of LTC4 synthase activity by retinoic acid in rat basophilic leukemia cells.

Calcium ionophore-stimulated production of leukotriene (LT) C4 was enhanced by 16- to 26-h incubation with retinoic acid (RA) in rat basophilic leukemia-1 cells. Production of LTC4 by enzyme assay using cell lysates as the enzyme source and LTA4 as the substrate was also enhanced by RA-treatment. Production of LTB4 was not enhanced under these two experimental conditions, suggesting the preferential activation of LTC4 synthase activity. The RA-induced enhancement of LTC4 synthesis by the cells was suppressed by co-incubation with dexamethasone (DEX) or cyclosporine A (CSA). However, the expression of mRNA for LTC4 synthase was not affected by the exposure to RA, DEX or CSA. These results indicate that RA-induced enhancement of LTC4 production and its inhibition by DEX and CSA was determined by post-transcriptional regulation of LTC4 synthase.

Consequences of transcellular biosynthesis of leukotriene C4 on organ function.

Formation of eicosanoids is a special mode of cell communication whereby production of eicosanoids by mixed cell populations differs from that expected from each individual cell. Transcellular biosynthesis of leukotriene C4 occurs via transfer of the reactive intermediate leukotriene A4 from neutrophils to vicinal acceptor cells devoid of 5-lipoxygenase activity such as platelets or vascular cells. Evidence for the in vivo relevance of transcellular eicosanoid metabolism results from experiments using the isolated beating rabbit heart perfused with activated neutrophils. The resultant leukotriene C4 synthesis is timely related to the pressor response of the coronary arteries and inflammatory damage of the heart by edema formation and neutrophil infiltration into the organ. Blockade of leukotriene C4 synthesis by 5-lipoxygenase inhibitors or leukotriene C4 actions by respective receptor antagonists facilitated significant protective effects. Further confirmation of the potential role of LTC4 in myocardial ischemia comes from in vivo studies in the rabbit.