Purification of human leukotriene C4 synthase from dimethylsulfoxide-differentiated U937 cells.

Human leukotriene C4 (LTC4) synthase was purified > 10000-fold from dimethylsulfoxide-differentiated U937 cells. Steps included: (a) solubilization of membrane-bound LTC4 synthase from microsomal membranes by the anionic detergent taurocholate; (b) successive anion-exchange chromatography steps in the presence of taurocholate plus Triton X-100 (primary anion exchange) then taurocholate plus n-octyl glucoside (secondary anion exchange); and (c) LTC2-affinity chromatography on a matrix that was constructed by first biotinylating synthetic LTC2 then immobilizing the biotinylated LTC2 on streptavidin agarose. The purification of human LTC4 synthase was enabled by the finding that LTC4 synthase activity in preparations enriched > 500-fold was absolutely dependent on the presence in LTC4 synthase incubation mixtures of divalent cations (specifically Mg2+) and phospholipids (specifically phosphatidylcholine), and that reduced glutathione, which was required at 2-4 mM for stabilization of LTC4 synthase, irreversibly inactivated the enzyme when present at > or = 5 mM during freeze/thaw cycles. The > 10000-fold purified LTC4 synthase preparation was comprised of three polypeptides having molecular masses of 37.1, 24.5 and 18.0 kDa. An 18-kDa polypeptide in both microsomal membranes and in the LTC2-affinity purified fraction was specifically labelled by a radioiodinated LTC4 photoaffinity probe (azido 125I-LTC4). The Km values in the LTC2-affinity purified preparation for reduced glutathione and LTA4 were 1.83 mM and 19.6 microM (respectively), closely resembling the Km values in isolated human blood monocytes. The Vmax of LTC2-affinity purified LTC4 synthase was 2-4 mumol LTC4 formed .min-1 x mg-1.     

Isolation of leukotriene C4 from human seminal fluid

LTC₄ was isolated and characterized from seminal fluid of seven human volunteers. A compound with a similar retention time of that of synthetic LTC₄ was obtained using reverse-phase high performance liquid chromatography. The ultraviolet absorbance of the extracted substance was identical to synthetic LTC₄. Furthermore this compound contracted the guinea pig ileum and lung parenchymal strip. Its effects were antagonized by the leukotriene antagonist FPL55712. It was concluded that LTC₄ is present in human seminal fluid in very small amounts (about 100 ng/ejaculate). The possible physiological functions of LTC₄ in the reproductive tract area discussed.     

Leukotrienes C4 and D4 in psoriatic skin lesions

Chemoattractant arachidonate lipoxygenase products have been recovered from the skin lesions of psoriasis, and may play a role in eliciting the intra-epidermal neutrophil infiltrate that characterises this disease. In view of evidence for lipoxygenase activity in psoriasis, the characteristic vasodilation in psoriatic lesions, and the vasodilator properties of leukotriene (LT) C4 and D4 in human skin, the presence of these LTs in psoriatic lesions has been investigated. Skin chamber fluid from abraded psoriatic lesions contained significantly greater amounts of immunoreactive material than that from clinically normal skin, as determined by a double antibody radioimmunoassay (RIA) that uses antiserum cross-reacting with both LTC4 and LTD4. Purification of lesional chamber fluid and scale extracts by high performance liquid chromatography (HPLC) and RIA of fractions showed immunoreactivity which co-eluted with standard LTC4 and LTD4. These findings suggest that LTC4 and LTD4 may play a role in mediating the vasodilation and increased blood flow that characterise psoriatic skin lesions.     

Characterization of primate bronchoalveolar mast cells. I. IgE-dependent release of histamine, leukotrienes, and prostaglandins

Large numbers of functional mast cells were obtained by bronchoalveolar lavage (BAL) of Macaca arctoides monkeys that had been infected with the nematode Ascaris suum. These lavage cells, of which 21% were mast cells, released histamine, LTC4, and PGD2 in a concentration-dependent fashion when challenged with ascaris antigen or antibody to human IgE. However, there was no release of histamine when these cells were challenged with compound 48/80. The amount of mediator released was highly dependent on the sensitivity of the cells to immunologic challenge, but was generally in the range of 2 to 5 micrograms histamine (30 to 70% of total), 20 to 80 ng LTC4, and 100 to 300 ng PGD2 per 10(6) mast cells when maximally challenged. Other eicosanoids measured were released only in much smaller quantities. Maximal values were 4 ng LTB4, 2 ng PGE2, and approximately 10 to 20 ng PGF2 alpha per 10(6) mast cells. The amount of LTC4 and PGD2 released correlated with the release of histamine, the calculated regression line indicating that 18 ng LTC4 and 50 ng PGD2 were released per microgram of histamine released. This correlation suggests that the majority of the LTC4 and PGD2 released was probably mast cell-derived. Further support for this conclusion was given by the observation that when lavage cells were fractioned on continuous Percoll gradients, the ability to release LTC4 and PGD2 on immunologic challenge coincided with the peak of mast cells.     

Distribution and metabolism of leukotriene C4 after cisternal injection in guinea pigs

Following cisternal injection of [3H8]LTC4 into guinea pigs, leukotriene metabolites were identified in the brain, cerebellum, perilymph, blood, liver and kidneys. LTC4 was metabolized into LTD4 and LTE4 in the cerebrospinal fluid and LTE4 was transported into the blood for general circulation and uptake into the liver and kidneys. The excretion of LTE4 from CNS to blood seemed to be the rate-limiting step in the elimination of leukotrienes from the body. Leukotrienes were also transported into the perilymph. The conversion of LTC4 into LTD4 and LTE4 was lower in perilymph as compared to the cerebrospinal fluid, suggesting a rate limiting function of the cochlear aqueduct that can be defined as a cerebrospinal fluid-labyrinth barrier.     

Amniotic fluid embolism and leukotrienes--the role of amniotic fluid surfactant in leukotriene production.

Surfactant rich lipid (lipid) was extracted from cell free 10,000 x g pellets of amniotic fluid. White blood cells (WBC) were isolated from human donors. 36 x 10(7) WBC and 5 g rabbit lung were incubated with pretreated lipid or dipalmitoyl lecithin (lecithin). Leukotrienes (LTs) were identified by high performance liquid chromatography (HPLC) and bioassay, and quantified by radioimmunoassay. Peaks of LTC4 and LTD4 on HPLC and guinea-pig ileum contraction could be identified in lipid and lecithin groups, but not in the control group. LTC4 production by lipid and lecithin groups was significantly higher than that by the control group. An involvement of amniotic fluid surfactant in leukotriene production is suggested.     

Structural and physicochemical requirements of endotoxins for the activation of arachidonic acid metabolism in mouse peritoneal macrophages in vitro

Lipopolysaccharides of different wild-type and mutant gram-negative bacteria, as well as synthetic and bacterial free lipid A, were studied for their ability to activate arachidonic acid metabolism in mouse peritoneal macrophages in vitro. It was found that lipopolysaccharides of deep-rough mutants of Salmonella minnesota and Escherichia coli (Re to Rc chemotypes) stimulated macrophages to release significant amounts of leukotriene C4 (LTC4) and prostaglandin E2 (PGE2). Lipopolysaccharides of wild-type strains (S. abortus equi, S. friedenau) only induced PGE2 and not LTC4 formation. Unexpectedly, free bacterial and synthetic E. coli lipid A were only weak inducers of LTC4 and PGE2 production. Deacylated Re-mutant lipopolysaccharide preparations were inactive. However, co-incubation of macrophages with both deacylated lipopolysaccharide and lipid A lead to the release of significant amounts of LTC4 and PGE2, similar to those obtained with Re-mutant lipopolysaccharide. The significance of the lipid A portion of lipopolysaccharide for the induction of LTC4 was indicated by demonstrating that peritoneal macrophages of endotoxin-low-responder mice or of mice rendered tolerant to endotoxin did not respond with the release of arachidonic acid metabolites on stimulation with Re-mutant lipopolysaccharide and that polymyxin B prevented the Re-lipopolysaccharide-induced LTC4 and PGE2 release. Physical measurements showed that the phase-transition temperatures of both free lipid A and S-form lipopolysaccharide were above 37 degrees C while those of R-mutant lipopolysaccharides were significantly lower (30-35 degrees C). Thus, with the materials investigated, an inverse relationship between the phase-transition temperature and the capacity to elicit LTC4 production was revealed.     

Human leukotriene C(4) synthase at 4.5 A resolution in projection

Leukotriene (LT) C(4) synthase, an 18 kDa integral membrane enzyme, conjugates LTA(4) with reduced glutathione to form LTC(4), the parent compound of all cysteinyl leukotrienes that play a crucial role in the pathobiology of bronchial asthma. We have calculated a projection map of recombinant human LTC(4) synthase at a resolution of 4.5 A by electron crystallography, which shows that the enzyme is a trimer. A map truncated at 7.5 A visualizes four transmembrane alpha helices per protein monomer. The densities in projection indicate that most of the alpha helices run nearly perpendicular to the plane of the membrane. At this resolution, LTC(4) synthase is strikingly similar to microsomal glutathione S-transferase 1, which belongs to the same gene family but bears little sequence identity and no resemblance in substrate specificity to the LTC(4) synthase. These results provide new insight into the structure and function of membrane proteins involved in eicosanoid and glutathione metabolism.     

Characterization of sulfidopeptide leukotriene responses in sheep tracheal smooth muscle in vitro.

We have investigated the effects of leukotrienes (LTs) on isolated tracheal smooth muscle from sheep sensitive to Ascaris suum antigen. LTC4 and LTD4 produced dose-dependent contractions of sheep trachea, but LTE4 was virtually inactive. YM-17690, a non-analogous LT agonist, produced no contractile response up to 100 microM. Indomethacin (5 microM) had no effect on LTC4- and LTD4-induced contractions. L-Serine borate (45 mM), an inhibitor of gamma-glutamyl transpeptidase, shifted the dose-response curve of LTC4 to the left by 161-fold, and L-cysteine (6 mM), an inhibitor of aminopeptidase, shifted the dose-response curves of LTC4 and LTD4 to the left by 67- and 23-fold, respectively. YM-16638 (1 microM), an LT antagonist, shifted the dose-response curves of LTC4 and LTD4 to the right with pKB values of 6.57 and 7.13, respectively. YM-16638 did not affect LTC4-induced contractions of L-serine borate-treated tissues, indicating that the compound acts only on LTD4 receptors in sheep trachea, LTE4 (1 microM) shifted the dose-response curves of LTC4 and LTD4 to the right with pKB values of 6.87 and 7.31, respectively. YM-17690 (10 microM) showed effects similar to LTE4, suggesting that the compound acts as an LTE4 agonist in sheep trachea. These results suggest that in sheep tracheal smooth muscle (a) LTC4 and LTD4 produce contractions, (b) these LT-induced contractions are not mediated by cyclooxygenase products, (c) LTC4 is converted to LTD4 and then to LTE4, and (d) the potency of the LTC4- and LTD4-induced contractions is increased when their conversion to LTE4 is inhibited.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of a leukotriene C4 export mechanism in human platelets: possible involvement of multidrug resistance-associated protein 1

Platelets express leukotriene (LT) C4 synthase and can thus participate in the formation of bioactive LTC4. To further elucidate the relevance of this capability, we have now determined the capacity of human platelets to export LTC4. Endogenously formed LTC4 was efficiently released from human platelets after incubation with LTA4 at 37 degrees C, whereas only 15% of produced LTC4 was exported when the cells were incubated at 0 degrees C. The activation energy of the process was calculated to 49.9 +/- 7.7 kJ/mol, indicating carrier-mediated LTC4 export. This was also supported by the finding that the transport was saturable, reaching a maximal export rate of 470 +/- 147 pmol LTC4/min x 10(9) platelets. Furthermore, markedly suppressed LTC4 transport was induced by a combination of the metabolic inhibitors antimycin A and 2-deoxyglucose, suggesting energy-dependent export.The presence in platelets of multidrug resistance-associated protein 1 (MRP1), a protein described to be an energy-dependent LTC4 transporter in various cell types, was demonstrated at the mRNA and protein level. Additional support for a role of MRP1 in platelet LTC4 export was obtained by the findings that the process was inhibited by probenecid and the 5-lipoxygenase-activating protein (FLAP) inhibitor, MK-886. The present findings further support the physiological relevance of platelet LTC4 production.     

Measurement of peptidoleukotrienes in biological fluids

Samples of human bronchoalveolar lavage fluid (BALF) and urine were utilized to demonstrate methods for quantitation and validation of leukotrienes (LTs). These methods utilize an enzyme immunoassay (EIA) that uses commercially available reagents, the antibody recognizing LTC4, LTD4, LTE4, and N-acetyl LTE4. BALF containing epithelial lining fluid was collected from atopic asthmatics both before and 5 min after the subjects had been challenged with a local instillation of allergen into the airways. BALF samples collected without allergen challenge had low levels of immunoreactive LTs, whereas samples collected after allergen were markedly elevated. After high-performance liquid chromatography (HPLC) separation of LTs, EIA revealed the presence of LTC4. The identity was validated by incubating LTC4 with a bovine gamma-glutamyl transpeptidase with dipeptidase activity that converted added [3H]-LTC4 as well as LTC4 immunoreactivity to LTE4. Urine samples collected from six healthy volunteers, one patient with adult respiratory distress syndrome (ARDS), and three patients in status asthmaticus were also analyzed for LTs. After HPLC separation of LTs and quantitation by EIA, urine samples from healthy subjects were found to have low but measurable LTE4. In contrast, the urine samples from the patients in status asthmaticus and from the ARDS patient had large elevations of LTE4 levels compared with healthy subjects. When the HPLC fractions containing [3H]LTE4 and LT immunoreactivity in the ARDS sample were treated with acetic anhydride, HPLC analysis indicated that both radiolabel and immunoreactivity now eluted at the retention time of N-acetyl LTE4, the derivatized product of LTE4. The methods described are relatively easy and can be used to measure and validate the existence of peptidoleukotrienes in biological samples.     

Ovarian follicular fluid eicosanoid concentrations during the pre-ovulatory period in humans

Prostaglandins are involved in ovulation and in every mammal studied so far, ovulation has been inhibited by prostaglandin inhibition. Information regarding the role of leukotrienes and thromboxanes in ovulation is more limited. In order to study the production of eicosanoids in human pre-ovulatory follicular fluid, follicular aspiration was timed by means of serial ultrasound scans and human chorionic gonadotrophin (hCG) to be immediately pre-ovulatory. 11 women were studied and the eicosanoids measured by radioimmunoassay (RIA). The follicular fluid was found to contain leukotrienes (LT) B4, LTC4 (D4, E4), prostaglandin (PG) E2, PGF2 alpha 6 keto PGF1 alpha k and thromboxane (TX) B2. This is the first published report of leukotrienes in human follicular fluid in spontaneous cycles, and is one of the few reports showing prostaglandins and thromboxanes. The significance of demonstrating leukotrienes in human follicular fluid is discussed as is the correlation between individual eicosanoids in the human ovary.     

Evidence of in-vivo omega-oxidation of peptide leukotrienes in the rat: biliary excretion of 20-CO2H N-acetyl LTE4

In a previous study in our laboratory it was observed that after [3H] LTC4 administration (luCi/kg i.v.) to the anesthetized rat, significant amounts of injected radioactivity (approximately 25%) were associated with previously unidentified biliary polar metabolite(s). In the present study we describe the isolation and characterization of the predominant polar metabolite. Rats were injected with synthetic LTC4 (20 microgram/kg i.v.) and bile collected over 30 min. After extraction and purification (2 step RP-HPLC procedure), the retention time of the metabolite was compared (plus coinjections) and found to be identical with synthetic 20-CO2H N-Ac LTE4 in two RP-HPLC systems. Also, the UV spectrum of the biologically derived metabolite was compared and found identical to the synthetic material, giving a characteristic conjugated triene absorption in the UV with a max of 281 nm and shoulders at 270 and 290 nm. Further, the trimethyl ester derivative of the metabolite showed identical chromatographic behaviors in 2 reverse and 2 normal phase HPLC systems compared with synthetic 20-CO2H N-Ac LTE4 trimethyl ester. We conclude omega-oxidation of peptide leukotrienes occurs in the rat and that 20-CO2H N-Ac LTE4 is an in vivo product of LTC4 metabolism.     

Generation of leukotrienes by antigen and calcium ionophore A23187 stimuli from a mouse mastocytoma cell line, MMC-16

The generation of leukotrienes and histamine release by the mouse mastocytoma cell line MMC-16 was investigated. These cells produced leukotriene C4 (LTC4) and released histamine upon calcium ionophore A23187 and antigen stimulation. The ionophore also stimulated the biosynthesis of leukotriene B4 (LTB4) by MMC-16. Generation of LTC4 was confirmed by its characteristic UV absorption spectrum, fast atom bombardment-MS, equivalent HPLC retention time with an authentic standard and radioimmunoassay. Leukotriene B4 was characterized by its distinctive UV spectrum and HPLC retention time compared with synthetic material. IgE-mediated LTC4 generation was also observed in a dose dependent fashion with MMC-16 cells passively sensitized with monoclonal IgE specific for ovalbumin. LTC4 biosynthesis was effectively inhibited by the lipoxygenase inhibitor NDGA.     

Differentiation of the mechanisms by which leukotrienes C4 and D4 elicit contraction of the guinea pig trachea

The contractions elicited by leukotriene (LT) C4 and D4 in isolated guinea pig trachea were characterized under conditions in which LTC4 to LTD4 metabolism was blocked by the presence of 45 mM l-serine-borate complex (SB). The presence of SB caused a shift of the LTC4-concentration-response curve to the left by 7.5-fold, and blocked the bioconversion of LTC4 to LTD4 by the trachea as estimated by HPLC analysis of the LTs present in the tissue bath fluid. The potency of FPL 55712 as an antagonist of the LTC4-induced contractions in the presence of SB was 15-30-fold less than its potency as an antagonist of the LTD4-induced contractions. In contrast, another LT antagonist, SK&F 101132, equally antagonized the contractions elicited by LTC4 and LTD4 in either the presence or absence of SB. The differential antagonism of LTC4 and LTD4 implies the existence of multiple pharmacologic receptors for the LTs. The calcium channel entry blockers, nifedipine and verapamil, at concentrations as high as 10 microM, suppressed the maximal LTC4-induced contraction by no more than 20%, whereas the purported intracellular calcium antagonist, TMB-8, completely suppressed the LTC4 concentration-response curve in the presence of SB, a profile identical to that previously reported for LTD4. Thus, if multiple LT receptors exist, they appear to mobilize calcium in a qualitatively similar fashion following LT stimulation.     

Neurotensin elevates hematocrit and plasma levels of the leukotrienes, LTB4, LTC4, LTD4 and LTE4, in anesthetized rats.

The IV injection of neurotensin (NT) into anesthetized rats produced a marked increase in hematocrit, labored breathing and peripheral blood stasis with cyanosis. This effect could also be produced by the NT-related peptides, neuromedin-N and xenopsin; however, it was not observed when nine other biologically active peptides, including bradykinin and substance P, were tested. Associated with these responses were increases in the plasma levels of histamine (measured radioenzymatically) and the leukotrienes, LTB4, LTC4, LTD4, and LTE4 (measured by RIA and HPLC). The increment in hematocrit after varying doses of NT correlated to the increase in plasma levels of LTC4. Histamine and LTC4 were both capable of elevating hematocrit when given IV; however, LTC4 was approximately 1000 times more potent than histamine and active doses of histamine elevated LTC4 levels. Furthermore, the effects of NT on plasma LTC4 and hematocrit were reduced by pretreating animals with antagonists to histamine and serotonin. Pretreatment with the specific mast cell degranulating agent, compound 48/80, also blocked NT's ability to elevate plasma levels of histamine, LTB4 and LTC4 and prevented the increased hematocrit and cyanosis. These results indicate that NT-related peptides are very potent and specific stimulators of leukotriene release and that this action is mediated by mast cells and associated with loss of plasma volume and blood stasis. A working hypothesis is that histamine, released from mast cells in response to NT, stimulates LTC4 production by other cells.     

Measuring leukotrienes of slow reacting substance of anaphylaxis: development of a specific radioimmunoassay

Rabbits were immunized with leukotriene C4 (LTC4) coupled to thiolated keyhole limpet hemocyanin (KLH) by using 6-N-maleimidohexanoic acid as a spacer molecule. Immune serum was obtained with 7.9 nmol of LTC4-specific immunoglobulin per milliliter and a mean association constant of 2.1 X 10(9) M-1. A radioimmunoassay was developed that detected 0.1 pmol of LTC4 per 1-ml sample. LTD4 and LTE4, three isomers of LTC4, the sulfones of LTC4, LTD4, and LTE4, and one isomer of LTD4 reacted to varying degrees in the assay. A number of other structurally related compounds, such as LTB4 and 5-HETE, did not react. Conditions were established to determine LTC4 levels in human plasma without loss of LTC4 during sample preparation and without the need for extraction procedures before the measurement of LTC4.     

Inhibition of 5-lipoxygenase and leukotriene C4 synthase in human blood cells by thymoquinone

Black cumin seed, Nigella sativa L., and its oils have traditionally been used for the treatment of asthma and other inflammatory diseases. Thymoquinone (TQ) has been proposed to be one of the major active components of the drug. Since leukotrienes (LTs) are important mediators in asthma and inflammatory processes, the effects of TQ on leukotriene formation were studied in human blood cells. TQ provoked a significant concentration-dependent inhibition of both LTC4 and LTB4 formation from endogenous substrate in human granulocyte suspensions with IC50 values of 1.8 and 2.3 microM, respectively, at 15 min. Major inhibitory effect was on the 5-lipoxygenase activity (IC50 3 microM) as evidenced by suppressed conversion of exogenous arachidonic acid into 5-hydroxy eicosatetraenoic acid (5HETE) in sonicated polymorphonuclear cell suspensions. In addition, TQ induced a significant inhibition of LTC4 synthase activity, with an IC50 of 10 microM, as judged by suppressed transformation of exogenous LTA4 into LTC4. In contrast, the drug was without any inhibitory effect on LTA4 hydrolase activity. When exogenous LTA4 was added to intact or sonicated platelet suspensions preincubated with TQ, a similar inhibition of LTC4 synthase activity was observed as in human granulocyte suspensions. The unselective protein kinase inhibitor, staurosporine failed to prevent inhibition of LTC4 synthase activity induced by TQ. The findings demonstrate that TQ potently inhibits the formation of leukotrienes in human blood cells. The inhibitory effect was dose- and time-dependent and was exerted on both 5-lipoxygenase and LTC4 synthase activity.