Comparison of leukotriene A4 metabolism into leukotriene C4 by human platelets and endothelial cells.

Leukotriene (LT) A4 metabolism was studied in human platelets and endothelial cells, since both cells could be involved in transcellular formation of LTC4. Upon addition of exogenous LTA4, both cells produced LTC4 as a major metabolite at various incubation times, and no LTB4, LTD4, or LTE4 was detected. Kinetic studies revealed a higher apparent Km for LTA4 in endothelial cells as compared to platelets (5.8 microM for human umbilical vein endothelial cells (HUVEC) versus 1.3 microM for platelets); platelets were more efficient in this reaction with a higher Vmax (174 pmol/mg protein/min) versus 15 pmol/mg protein/min in HUVEC. The formation of LTC4 and corresponding kinetic parameters were not modified when platelets or endothelial cells were stimulated by thrombin prior to or simultaneously with the addition of LTA4. In both cells LTC4 synthase activity was not modified by repeated addition of LTA4 showing that it is not a suicide-inactivated enzyme. Furthermore, in platelets and endothelial cells, the enzyme activity was localized in the membrane fraction and was distinct from cytosolic glutathione-S-transferases. Platelet membrane fractions showed apparent Km values of 31 microM and 1.2 mM for LTA4 and GSH, respectively. Inhibition of LTC4 formation from platelets and endothelial cells preparations by S-substituted glutathione derivatives was correlated to the length of the S-alkyl chain. The same substances inhibited cytosolic glutathione-S-transferases with significantly lower IC50, confirming the distinct nature of the two enzymes. These results show that platelets and HUVEC possess similar enzymes for the production of LTC4 from LTA4; however, platelets seem to have a higher efficiency than HUVEC in performing this reaction.     

Endothelial cell leukotriene C4 synthesis results from intercellular transfer of leukotriene A4 synthesized by polymorphonuclear leukocytes

Leukotriene (LT) synthesis and metabolism were studied in porcine aortic endothelial cells. Leukotrienes were identified by combinations of guinea pig lung parenchymal strip bioassay, radioimmunoassay, and UV spectrophotometry with high performance liquid chromatography. Endothelial cells stimulated with the calcium ionophore, A23187, were unable to convert arachidonic acid to detectable levels of LTA4-derived products including the biologically active metabolites, LTB4 or LTC4. However, these cells readily converted exogenous LTA4 to the potent slow-reacting substance, LTC4. Smaller quantities of 11-trans-LTC4 and LTD4 were also observed. LTB4 was not detectable in these incubations nor was LTB4 metabolism observed. The possible intercellular transfer of LTA4 between polymorphonuclear leukocytes (PMNL) and endothelial cells was tested since PMNL release LTA4 when stimulated and have significant contact with endothelium. When A23187-stimulated neutrophils were coincubated with endothelial cells, a significant increase in LTC4 levels was detected over PMNL alone. LTC4 is formed by the enzymatic conjugation of glutathione (GSH) with LTA4. Therefore in some experiments, endothelial cells were prelabeled with [35S]cysteine to allow intracellular synthesis of [35S]GSH. When unlabeled PMNL were added, as a source of LTA4 to the prelabeled endothelial cells, substantial levels of [35S] LTC4 were recovered. The data indicate that endothelial cells synthesize LTC4 from LTA4. They also demonstrate a specific PMNL-endothelial cell interaction in which endothelial cell LTC4 synthesis results from the intercellular transfer of LTA4 produced by PMNL.     

Identification of regions of leukotriene C4 synthase which direct the enzyme to its nuclear envelope localization

Leukotrienes (LTs) are fatty acid derivatives formed by oxygenation of arachidonic acid via the 5-lipoxygenase (5-LO) pathway. Upon activation of inflammatory cells 5-LO is translocated to the nuclear envelope (NE) where it converts arachidonic acid to the unstable epoxide LTA4. LTA4 is further converted to LTC4 by conjugation with glutathione, a reaction catalyzed by the integral membrane protein LTC4 synthase (LTC4S), which is localized on the NE and endoplasmic reticulum (ER). We now report the mapping of regions of LTC4S that are important for its subcellular localization. Multiple constructs encoding fusion proteins of green fluorescent protein (GFP) as the N-terminal part and various truncated variants of human LTC4S as C-terminal part were prepared and transfected into HEK 293/T or COS-7 cells. Constructs encoding hydrophobic region 1 of LTC4S (amino acids 6-27) did not give distinct membrane localized fluorescence. In contrast hydrophobic region 2 (amino acids 60-89) gave a localization pattern similar to that of full length LTC4S. Hydrophobic region 3 (amino acids 114-135) directed GFP to a localization indistinguishable from that of full length LTC4S. A minimal directing sequence, amino acids 117-132, was identified by further truncation. The involvement of the hydrophobic regions in the homo-oligomerization of LTC4S was investigated using bioluminescence resonance energy transfer (BRET) analysis in living cells. BRET data showed that hydrophobic regions 1 and 3 each allowed oligomerization to occur. These regions most likely form transmembrane helices, suggesting that homo-oligomerization of LTC4S is due to helix-helix interactions in the membrane.     

Synthesis and metabolism of leukotrienes by human endothelial cells: influence on prostacyclin release

The synthesis and metabolism of leukotrienes (LTs) by endothelial cells was investigated using reverse-phase high-performance liquid chromatography. Cells were incubated with [14C]arachidonic acid. LTA4 or [3H]LTA4 and stimulated with ionophore A23187. The cells did not synthesize leukotrienes from [14C]arachidonic acid. LTA4 and [3H]LTA4 were converted to LTC4, LTD4, LTE4 and 5,12-diHETE. Endothelial cells metabolized [3H]LTC4 to [3H]LTD4 and [3H]LTE4. The metabolism of [3H]LTC4 was inhibited by L-serine-borate complex, phenobarbital and acivicin in a concentration-related manner, with maximal inhibition occurring at a concentration of 0.1 M, 0.01 M and 0.01 M, respectively. LTC4, LTB4 and LTD4 stimulated the synthesis of prostacyclin, measured by radioimmunoassays as 6-keto-PGF1 alpha. The stimulation by LTC4 was greater than that by LTD4 or LTB4. LTE4, 14,15-LTC4 and 14,15-LTD4 failed to stimulate the synthesis of prostacyclin. LTD4 and LTB4 also stimulated the release of PGE2, whereas LTC4 did not. Serine-borate and phenobarbital inhibited LTC4-stimulated synthesis of prostacyclin in a concentration-related manner. They also inhibited the release of prostacyclin by histamine, A23187 and arachidonic acid. Acivicin had no effect on the release of prostacyclin by LTC4, histamine or A23187. Furthermore, FPL-55712, an LT receptor antagonist, inhibited LTC4-stimulated prostacyclin synthesis but had no effect on histamine-stimulated release of prostacyclin or PGE2. Indomethacin inhibited both LTC4- and histamine-stimulated release. The results show that (a) endothelial cells metabolize LTA4, LTC4 and LTD4 but do not synthesize LTs from arachidonic acid; (b) LTC4 act directly at the leukotriene receptor to stimulation prostacyclin synthesis; (c) the presence of the glutathione moiety at the C-6 position of the eicosatetraenoic acid skeleton is necessary for leukotriene stimulation of prostacyclin release; and (d) the metabolism of LTC4 to LTD4 and LTE4 does not appear to alter the ability of LTC4 to stimulate the synthesis of PGI2.     

Characterization of leukotriene C4 synthetase in mouse peritoneal exudate cells

Cell lysates of mouse peritoneal macrophages, in the presence of reduced glutathione, converted leukotriene LTA4 to LTC4, and neither LTD4 nor LTE4 was detected. Therefore, like cultured rat basophilic leukemia cells (RBL cells), the peritoneal macrophage contains LTC4 synthetase and appears to contain little, if any, gamma-glutamyl transpeptidase. When LTA4 was added to subcellular fractions of mouse macrophage lysate, the highest specific activity of LTC4 synthetase (nmol LTC4/mg protein per 10 min) was associated with the particulate or membrane fractions (i.e., 10(4) and 10(5) X g pellets). The 10(5) X g supernatant contains approx. 1% of the specific activity and 6% of the total LTC4 synthetase activity compared with that of the 10(5) X g pellet. Conversely, the 10(5) X g supernatant had four-times more specific activity and 19-times more total GSH S-transferase activity than did the 10(5) X g pellet when evaluated using 1-chloro-2,4-dinitrobenzene (DNCB) as the substrate. LTA4 was converted to LTC4 by the membrane enzyme LTC4 synthetase in a dose-dependent manner at low LTA4 concentrations (3-50 microM) and reached a plateau of approx. 30 microM LTA4 using the macrophage 10(5) X g pellet as an enzyme source. The apparent Km value of LTC4 synthetase for LTA4 was estimated to be 5 microM based on Lineweaver-Burk plots. Enzyme in the 10(5) X g supernatant produced negligible quantities of LTC4 (1% or less of the particulate fractions) over a wide range of LTA4 concentrations. However, an enzyme in the 10(5) X g supernatant fraction presumed to be GSH S-transferase effectively catalyzes the conjugation of glutathione (GSH) with the aromatic compound DNCB. The apparent Km value of GSH S-transferase for DNCB was estimated to be 1.0-1.5 mM. On the other hand, enzyme from the membrane fraction (i.e., 10(5) X g pellet) catalyzed this reaction at a negligible rate over a wide range of DNCB concentrations. The apparent Km value of LTC4 synthetase for GSH was estimated to be 0.36 mM and the corresponding Km value estimated for the glutathione S-transferase was 0.25-0.76 mM. These values indicate similar kinetics for GSH utilization by both enzymes. These Km values are also significantly lower than the intracellular GSH levels of 2 to 5 mM. Therefore, it is suggested that the substrate limiting LTC4 synthetase activity is LTA4 and not GSH. Our results indicate that LTC4 synthetase from mouse peritoneal macrophages is a particulate or membrane-bound enzyme, as was reported by Bach et al.(ABSTRACT TRUNCATED AT 400 WORDS)     

Transcellular metabolism of neutrophil-derived leukotriene A4 by human platelets. A potential cellular source of leukotriene C4

Transformation of leukotriene (LT) A4 into leukotriene C4 has been found to be carried out by human platelets in a rather efficient manner. LTC4 was characterized by a combination of high performance liquid chromatography, UV spectrophotometry, use of labeled precursor, guinea pig ileum bioassay, and enzyme immunoassay. LTA4 metabolism was found to be substrate-dependent, time-dependent, and proportional to platelet concentration even at sub- or supraphysiological levels (0.0019-1 X 10(9) platelets/ml). Neither plasma alone nor the supernatant of resting or activated platelets was found to catalyze the production of LTC4 in the presence or in the absence of reduced glutathione. These data suggest that platelets contain a glutathione S-transferase specific for LTC4 biosynthesis. The formation of LTC4 was greatly enhanced when LTA4 was incubated with platelets in the presence of albumin. Low concentrations of albumin (2-4 g/liter) stabilized LTA4 to an extent that conversion into LTC4 by the platelets could be detected after 1 h of incubation. The possible intercellular transfer of LTA4 between neutrophils and platelets was tested. The production of LTC4 by neutrophils was greatly enhanced in the presence of platelets. Furthermore, the supernatant of neutrophils stimulated with the calcium ionophore contained a short-lived acid-labile substance which was converted by the platelets into LTC4. When platelets were prelabeled with [35S]cysteine to allow intracellular synthesis of [35S]glutathione, the coincubation of both cell types challenged with the calcium ionophore resulted in the production of [35S] LTC4. These data indicate that platelets can produce large amounts of LTC4 from neutrophil-derived LTA4. They also suggest that such interactions may occur in vivo and that platelets could be an important contribution to the generation of the biologically active LTC4.     

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.     

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.     

Human leukotriene C4 synthase expression in dimethyl sulfoxide-differentiated U937 cells.

Leukotriene C4 (LTC4) synthase was highly expressed in the human U937 monoblast leukemia cell line when differentiated into monocyte/macrophage-like cells by growth in the presence of dimethyl sulfoxide. The specific activity of LTC4 synthase in differentiated cells (399.0 +/- 84.1 pmol of LTC4 formed.min-1.mg-1) was markedly higher (10-fold; p less than 0.001) than in undifferentiated U937 cells (39.9 +/- 16.7 pmol of LTC4 formed.min-1.mg-1) or freshly isolated blood monocytes (21.5 +/- 4.8 pmol of LTC4 formed.min-1.mg-1). The increase in LTC4 synthase activity following dimethyl sulfoxide-induced differentiation was substantially higher than the increase observed for other proteins involved in leukotriene biosynthesis. LTC4 synthase activity was unaffected in U937 cells differentiated by growth in the presence of phorbol 12-myristate 13-acetate. The HL-60 myeloblast leukemia cell line expressed higher LTC4 synthase levels when differentiated into either neutrophil-like or macrophage-like cells by growth in the presence of dimethyl sulfoxide or phorbol 12-myristate 13-acetate (respectively), but reached a specific activity comparable only to undifferentiated U937 cells. Human LTC4 synthase was found to be a unique membrane-bound enzymatic activity completely distinct from alpha, mu, pi, theta, and microsomal glutathione S-transferases, as determined by differential detergent solubilization, chromatographic separation, substrate specificity, and Western blot analysis. An 18-kDa polypeptide was specifically labeled in membranes from dimethyl sulfoxide-differentiated U937 cells using azido 125I-LTC4, a photoaffinity probe based on the product of the LTC4 synthase-catalyzed reaction. Photolabeling of the 18-kDa polypeptide was specifically competed for by LTC4 (greater than 50% at 0.1 microM) but not by 100,000-fold higher concentrations of reduced glutathione (10 mM). Elevation of both the level of the specifically photolabeled 18-kDa polypeptide and of LTC4 synthase specific activity occurred concomitantly with dimethyl sulfoxide differentiation of U937 cells. We conclude that differentiation of U937 cells into monocyte/macrophage-like cells by growth in the presence of dimethyl sulfoxide results in high levels of expression of LTC4 synthase activity. Human LTC4 synthase is a unique enzyme with a high degree of specificity for LTA4 and may therefore be dedicated exclusively to the formation of LTC4 in vivo. An 18-kDa membrane polypeptide, specifically labeled by a photoaffinity derivative of LTC4, is a candidate for being either LTC4 synthase or a subunit thereof.     

Molecular cloning and functional expression of rat leukotriene A4 hydrolase using the polymerase chain reaction.

We isolated a cDNA encoding rat leukotriene A4 (LTA4) hydrolase from mesangial cells by the polymerase chain reaction according to the human amino acid sequence. The deduced amino acid sequence shows that rat LTA4 hydrolase is a 609 amino acid protein with an Mr 69 kDa. Comparison of human LTA4 hydrolase revealed 93% homology, and include zinc-binding motifs of aminopeptidases. COS-7 cells transfected with the cDNA revealed substantial LTA4 hydrolase activity, and their activities were abolished by preincubation with captopril, representing the first reported cDNA expression of recombinant enzyme in mammalian cells. RNA blot analysis indicated that LTA4 hydrolase was expressed in glomerular endothelial, epithelial and mesangial cells.     

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.     

Activation of sphingosine kinase by tumor necrosis factor-alpha inhibits apoptosis in human endothelial cells

Human umbilical vein endothelial cells (HUVEC), like most normal cells, are resistant to tumor necrosis factor-alpha (TNF)-induced apoptosis in spite of TNF activating sphingomyelinase and generating ceramide, a known inducer of apoptosis. Here we report that TNF activates another key enzyme, sphingosine kinase (SphK), in the sphingomyelin metabolic pathway resulting in production of sphingosine-1-phosphate (S1P) and that S1P is a potent antagonist of TNF-mediated apoptosis. The TNF-induced SphK activation is independent of sphingomyelinase and ceramidase activities, suggesting that TNF affects this enzyme directly other than through a mass effect on sphingomyelin degradation. In contrast to normal HUVEC, in a spontaneously transformed endothelial cell line (C11) TNF stimulation failed to activate SphK and induced apoptosis as characterized by morphological and biochemical criteria. Addition of exogenous S1P or increasing endogenous S1P by phorbol ester markedly protected C11 cell line from TNF-induced apoptosis. Conversely, N, N-dimethylsphingosine, an inhibitor of SphK, profoundly sensitized normal HUVEC to killing by TNF. Thus, we demonstrate that the activation of SphK by TNF is an important signaling for protection from the apoptotic effect of TNF in endothelial cells.     

Differential metabolism of exogenous and endogenous arachidonic acid in human neutrophils.

Leukotrienes can be produced by cooperative interactions between cells in which, for example, arachidonate derived from one cell is oxidized to leukotriene A(4) (LTA(4)) by another and this can then be exported for conversion to LTB(4) or cysteinyl leukotrienes (cys-LTs) by yet another. Neutrophils do not contain LTC(4) synthase but are known to cooperate with endothelial cells or platelets (which do have this enzyme) to generate cys-LTs. Stimulation of human neutrophils perfusing isolated rabbit hearts resulted in production of cys-LTs, whereas these were not seen with perfused hearts alone or isolated neutrophils. In addition, the stimulated, neutrophil-perfused hearts generated much greater amounts of total LTA(4) products, suggesting that the hearts were supplying arachidonate to the neutrophils and, in addition, that this externally derived arachidonate was preferentially used for exported LTA(4) that could be metabolized to cys-LTs by the coronary endothelium. Stable isotope-labeled arachidonate and electrospray tandem mass spectrometry were used to differentially follow metabolism of exogenous and endogenous arachidonate. Isolated, adherent neutrophils at low concentrations (to minimize transcellular metabolism between them) were shown to generate higher proportions of nonenzymatic LTA(4) products from exogenous arachidonate (deuterium-labeled) than from endogenous (unlabeled) sources. The endogenous arachidonate, on the other hand, was preferentially used for conversion to LTB(4) by the LTA(4) hydrolase. This result was not because of saturation of the LTA(4) hydrolase, because it occurred at widely differing concentrations of exogenous arachidonate. Finally, in the presence of platelets (which contain LTC(4) synthase), the LTA(4) synthesized from exogenous deuterium-labeled arachidonate was converted to cys-LTs to a greater degree than that from endogenous sources. These experiments suggest that exogenous arachidonate is preferentially converted to LTA(4) for export (not intracellular conversion) and raises the likelihood that there are different intracellular pathways for arachidonate metabolism.     

The innate defense antimicrobial peptides hBD3 and RNase7 are induced in human umbilical vein endothelial cells by classical inflammatory cytokines but not Th17 cytokines

Antimicrobial peptides are multifunctional effector molecules of innate immunity. In this study we investigated whether endothelial cells actively contribute to innate defense mechanisms by expression of antimicrobial peptides. We therefore stimulated human umbilical vein endothelial cells (HUVEC) with inflammatory cytokines, Th17 cytokines, heat-inactivated bacteria, bacterial conditioned medium (BCM) of Staphylococcus aureus and Streptococcus sanguinis, and lipoteichoic acid (LTA). Stimulation with single cytokines induced discrete expression of human β-defensin 3 (hBD3) by IFN-γ or IL-1β and of ribonuclease 7 (RNase7) by TNF-α without any effects on LL-37 gene expression. Stronger hBD3 and RNase7 induction was observed after combined stimulation with IL-1β, TNF-α and IFN-γ and was confirmed by high hBD3 and RNase7 peptide levels in cell culture supernatants. In contrast, Th17 cytokines or stimulation with LTA did not result in AMP production. Moreover, only BCM of an invasive S. aureus bacteremia isolate induced hBD3 in HUVEC. We conclude that endothelial cells actively contribute to prevent dissemination of pathogens at the blood-tissue-barrier by production of AMPs that exhibit microbicidal and immunomodulatory functions. Further investigations should focus on tissue-specific AMP induction in different endothelial cell types, on pathogen-specific induction patterns and potentially involved pattern-recognition receptors of endothelial cells.     

Bioinformatic and enzymatic characterization of the MAPEG superfamily

The membrane associated proteins in eicosanoid and glutathione metabolism (MAPEG) superfamily includes structurally related membrane proteins with diverse functions of widespread origin. A total of 136 proteins belonging to the MAPEG superfamily were found in database and genome screenings. The members were found in prokaryotes and eukaryotes, but not in any archaeal organism. Multiple sequence alignments and calculations of evolutionary trees revealed a clear subdivision of the eukaryotic MAPEG members, corresponding to the six families of microsomal glutathione transferases (MGST) 1, 2 and 3, leukotriene C4 synthase (LTC4), 5-lipoxygenase activating protein (FLAP), and prostaglandin E synthase. Prokaryotes contain at least two distinct potential ancestral subfamilies, of which one is unique, whereas the other most closely resembles enzymes that belong to the MGST2/FLAP/LTC4 synthase families. The insect members are most similar to MGST1/prostaglandin E synthase. With the new data available, we observe that fish enzymes are present in all six families, showing an early origin for MAPEG family differentiation. Thus, the evolutionary origins and relationships of the MAPEG superfamily can be defined, including distinct sequence patterns characteristic for each of the subfamilies. We have further investigated and functionally characterized representative gene products from Escherichia coli, Synechocystis sp., Arabidopsis thaliana and Drosophila melanogaster, and the fish liver enzyme, purified from pike (Esox lucius). Protein overexpression and enzyme activity analysis demonstrated that all proteins catalyzed the conjugation of 1-chloro-2,4-dinitrobenzene with reduced glutathione. The E. coli protein displayed glutathione transferase activity of 0.11 micromol.min(-1).mg(-1) in the membrane fraction from bacteria overexpressing the protein. Partial purification of the Synechocystis sp. protein yielded an enzyme of the expected molecular mass and an N-terminal amino acid sequence that was at least 50% pure, with a specific activity towards 1-chloro-2,4-dinitrobenzene of 11 micromol.min(-1).mg(-1). Yeast microsomes expressing the Arabidopsis enzyme showed an activity of 0.02 micromol.min(-1).mg(-1), whereas the Drosophila enzyme expressed in E. coli was highly active at 3.6 micromol.min(-1).mg(-1). The purified pike enzyme is the most active MGST described so far with a specific activity of 285 micromol.min(-1).mg(-1). Drosophila and pike enzymes also displayed glutathione peroxidase activity towards cumene hydroperoxide (0.4 and 2.2 micromol.min(-1).mg(-1), respectively). Glutathione transferase activity can thus be regarded as a common denominator for a majority of MAPEG members throughout the kingdoms of life whereas glutathione peroxidase activity occurs in representatives from the MGST1, 2 and 3 and PGES subfamilies.     

Peritoneal macrophages of guinea pig possibly lack LTC4 synthetase

Peritoneal cells and adherent cells of mice and rats synthesized LTC4 and LTB4 when stimulated with A23187 in vitro. On the other hand, neither peritoneal cells nor adherent cells of guinea pigs generated LTC4, D4, and E4, but did the lower amounts of LTB4. Only generation of LTB4 was potentiated by simultaneous addition of 10 microM A.A. in this species. Enzyme solutions which were extracted from peritoneal cells of these three species were capable of converting DNCB to a colored product in the presence of glutathione and then these potencies were in the following order; guinea pig greater than mouse greater than rat. On the other hand, the potencies of converting LTA4 to LTC4 in the presence of glutathione were in the following order; mouse greater than rat much greater than guinea pig approximately equal to 0. These results suggest that macrophages of guinea pigs lack "LTC4 synthetase" and also this enzyme is different from usual GSH S-transferases.     

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.     

Hydrogen sulfide attenuated tumor necrosis factor-α-induced inflammatory signaling and dysfunction in vascular endothelial cells

Background

Hydrogen sulfide (H₂S), the third physiologically relevant gaseous molecule, is recognized increasingly as an anti-inflammatory mediator in various inflammatory conditions. Herein, we explored the effects and mechanisms of sodium hydrosulfide (NaHS, a H₂S donor) on tumor necrosis factor (TNF)-α-induced human umbilical vein endothelial cells (HUVEC) dysfunction.

Methodology and Principal Findings

Application of NaHS concentration-dependently suppressed TNF-α-induced mRNA and proteins expressions of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), mRNA expression of P-selectin and E-selectin as well as U937 monocytes adhesion to HUVEC. Western blot analysis revealed that the expression of the cytoprotective enzyme, heme oxygenase-1 (HO-1), was induced and coincident with the anti-inflammatory action of NaHS. Furthermore, TNF-α-induced NF-κB activation assessed by IκBα degradation and p65 phosphorylation and nuclear translocation and ROS production were diminished in cells subjected to treatment with NaHS.

Significance

H₂S can exert an anti-inflammatory effect in endothelial cells through a mechanism that involves the up-regulation of HO-1.     

Effect of NADPH oxidase inhibition on endothelial cell ELAM-1 mRNA expression.

Neutrophil adherence to endothelium is partially mediated by the expression of endothelial leukocyte adhesion molecule-1 (ELAM-1) on endothelial cells activated by agents such as lipopolysaccharide (LPS) and phorbol myristate acetate (PMA). To elucidate molecular mechanisms involved in the induction of ELAM-1 on endothelial cells, we investigated the effect of the NADPH oxidase inhibitor, apocynin (4-hydroxy-3-methoxyacetophenone), on ELAM-1 mRNA expression in human umbilical vein endothelial cells (HUVEC) by Northern blot analysis. Apocynin downregulated both LPS- and PMA-induced ELAM-1 mRNA expression in a dose-dependent manner. Our results suggest NADPH oxidase might play a key role in ELAM-1 mRNA expression in HUVEC.