Leukotriene D4 and E4 formation by plasma membrane bound enzymes

The homogenate of rat basophilic leukemia cells produces both the dihydroxy-leukotrienes and the peptido-leukotrienes (LT) C₄, D₄ and E₄. The enzymes responsible for the formation of LTA₄ and LTB₄ are in the soluble fraction while the enzymes for LTC₄, LTD₄ and LTE₄ are particulate (10, 000 × g pellet). Centrifugation of the 10, 000 × g pellet over a sucrose gradient resulted in two subfractions, a membrane fraction and a pellet (sucrose pellet.) The fractions were incubated with LTC₄, and the products were identified by bioassay, HPLC and UV spectra. The membrane fraction contained the enzymes γ-glutamyl transpeptidase and amino peptidase which convert LTC₄ to LTD₄ and LTD₄ to LTE₄, respectively. When incubated with LTC₄, the membrane fraction showed a dose dependent formation of LTD₄ and a time course which reached a plateau at 30 to 45 minutes. Addition of serine borate blocked the formation of LTD₄, and cysteine blocked LTE₄. We conclude that the γ-glutamyl transpeptidase and the amino peptidase which produce LTD₄ and LTE₄ respectively are plasma membrane bound.     

Bioconversion of leukotriene C4 by rat glomeruli and papilla

Since leukotriene C4 (LTC4) may be locally synthesized by bone marrow-derived cells infiltrating the kidney in inflammatory renal diseases we examined the in vitro metabolism of exogenously added [3H] LTC4 by rat glomeruli and papilla using radiometric HPLC. Homogenized as well as intact glomeruli converted [3H] LTC4 mainly into [3H] LTE4 (83%) and, at a smaller extent, into [3H] LTD4 (4%). Intact [3H] LTC4 represented 13% of the sum of radioactive leukotrienes. Addition of L-cysteine resulted in accumulation of LTD4. In contrast, there was nearly no conversion of [3H] LTC4 (87% intact) in the presence of homogenized papilla. The metabolism of [3H] LTC4 by the glomeruli was time- and temperature-dependent. The 10,000 g supernatant and pellet of homogenized glomeruli both retained the ability to metabolize [3H] LTC4. The papillary 10,000 g supernatant was inactive, as found for the total homogenate, whereas the papillary 10,000 g pellet separated from its supernatant could transform [3H] LTC4 into its metabolites, LTD4 and LTE4. Addition of increasing amounts of papillary 10,000 g supernatant to homogenized glomeruli progressively protected [3H] LTC4 from its bioconversion. These results demonstrate that both glomeruli and papilla possess the gamma-glutamyl transpeptidase and dipeptidase necessary to process LTC4. However, the enzyme activity of the papilla is unmasked only when the inhibitor present in the 10,000 g supernatant is separated from the enzyme present in the pellet.     

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)     

Leukotriene C4 and D4 formation by particulate enzymes

The homogenate of rat basophilic leukemia cells, when incubated with arachidonic acid, glutathione, and calcium, formed 3 isomers of 5,12-dihydroxyeicosatetraenoic acid and 2 isomers of 5,6-dihydroxyeicosatetraenoic acid, as well as leukotriene (LT) C4 and D4. The products were identified by high pressure liquid chromatography, ultraviolet spectral analysis, co-migration with standards, bioassay, and gas chromatography-mass spectrometry. The enzymes responsible for the formation of LTC4 and LTD4 from LTA4 were found in the 10,000 x g pellet and, therefore, appear to be particulate. The possibility that these enzymes are bound to the cell membrane suggest that the formation of these leukotrienes might be important in the basophil and mast cells release reaction.     

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)     

Leukotriene C4 is an essential 5-lipoxygenase intermediate in A23187-induced macrophage cytostatic activity against P815 tumor cells

Resident peritoneal macrophages incubated with 3.5 x 10(-7) M Calcium ionophore A23187 in tumor cell growth medium (TGM) release large amounts of leukotriene (LT)E4 and an unidentified 5-lipoxygenase product, whereas A23187-stimulated macrophages produce in serum free medium LTD4, predominately. LTC4 and 3H-LTC4 incubated for 20 min at 37 degree C in serum containing TGM, convert into LTE4 and 3H-LTE4, respectively. Thus, LTC4 released from A23187-stimulated macrophages is an intermediate in TGM which rapidly converts into LTE4, probably because of the presence of gamma-glutamyl transpeptidase and cystenylglycinase in TGM. Macrophages express antitumor cytostatic activity towards P815 cells (49-53%) in a cocultured ratio (macrophage: tumor cell) 2:1 when stimulated with 3.5 x 10(-7) M A23187 in TGM. The 5-lipoxygenase inhibitor AA861 reverses the cytostatic activity by 42-58% and it inhibits also the formation of A23187-induced 5-lipoxygenase products from macrophages. Restoration of 38% macrophage- antitumor cytostatic activity by exogenous LTC4 (10(-8) M) indicates that LTC4 is an essential 5-lipoxygenase intermediate in the pathway of required signals underlying A23187-induced macrophage antitumor cytostatic activity. Macrophages not stimulated by A23187 do not express cytostatic activity in the presence of LTC4. This implies that besides LTC4, increased cytosolic [Ca2+] is required for A23187 induction of macrophage cytostatic activity.     

Leukotriene C4 metabolism by hepatoma cells deficient in the uptake of cysteinyl leukotrienes

Uptake and metabolism of the cysteinyl leukotrienes C4 and E4 (LTC4 and LTE4) were studied in AS-30D hepatoma cell suspensions and compared with rat hepatocytes. The hepatoma cells were deficient in the uptake of [3H]LTC4 and [3H]LTE4 but took up, in control experiments, L-[14C]glutamine and [14C]adenosine in a time-dependent manner. By contrast, isolated hepatocyte suspensions incubated under the same conditions took up [3H]LTC4 and [3H]LTE4 as well as L-[14C]glutamine and [14C]adenosine. The hepatoma cells deficient in the uptake of cysteinyl leukotrienes metabolized extracellular [3H]LTC4 to [3H]LTD4 and to [3H]LTE4. Addition of acivicin, an inhibitor of gamma-glutamyltransferase, largely prevented metabolism of [3H]LTC4 by the hepatoma cells. Sonication of the cells did not enhance the formation of [3H]LTD4 and [3H]LTE4 from [3H]LTC4. We conclude that ectoenzymes of AS-30D hepatoma cells catalyze the conversion of LTC4 to LTE4 via LTD4. As compared to hepatocytes, these neoplastic cells have lost the uptake system for cysteinyl leukotrienes and may serve in studies on leukotriene metabolism by cell-surface enzymes.     

Discriminative effect of gamma-glutamyl transpeptidase inhibitors on metabolism of leukotriene C4 in peritoneal cells

Leukotrienes are naturally-occurring metabolites of arachidonic acid that are formed via the 5-lipoxygenase pathway in several tissues. Rat peritoneal cells (RPC) can produce leukotrienes C4, D4 and E4 (LTC4, LTD4 and LTE4) in response to stimulation with the calcium ionophore A23187 (1,2). The mechanism of enzymatic conversion of LTC4 to LTD4 is presumed to be via the action of gamma-glutamyl transpeptidase (gamma-GTPase, Figure 1) and has been demonstrated with purified enzymes from rat and porcine kidneys (3-6). We report that RPC contain gamma-GTPase-like activity that catalyzes the liberation of p-Nitroaniline (p-NA) from the chromophoric substrate gamma-glutamyl-p-nitroanilide (gamma-GpNA) in the presence of the acceptor molecules glycylglycine and L-cysteine. Furthermore, we demonstrate that under similar conditions, this preparation catalyzes the conversion of LTC4 to LTD4. Activity with gamma-GpNA is inhibited by D,L-gamma-glutamyl(o-carboxy)-phenylhydrazide (GOP) and serine-borate complex, (competitive inhibitors of kidney gamma-GTPase), and 6-diazo-5-oxo-L-norleucine (DON) and o-diazo-acetyl-L-serine (AZA), (irreversible inhibitors of kidney gamma-GTPase). In contrast, conversion of both endogenously-generated or exogenous LTC4 into LTD4 by RPC is inhibited only by serine-borate complex. These results suggest that RPC contain at least two distinct forms of gamma-GTPase; one capable of recognizing gamma-GpNA and susceptible to inhibition by all four compounds, and a second form utilizing also LTC4 as substrate, and is not inhibited by high concentrations of several "classic" gamma-GTPase inhibitors.     

Pharmacologic characterization of the contractile activity of peptide leukotrienes in guinea-pig pulmonary artery

The actions of the peptide leukotrienes (LT) LTC4, LTD4 and LTE4 and phenylephrine (PE) were studied in isolated left branches of the guinea-pig pulmonary artery (GPPA). Indomethacin 5 x 10(-6) M enhanced both the potency and maximal response of all agonists, but the effect on LTD4 and LTE4 was larger. The influence of indomethacin suggests the release of an endogenous vasodilating cyclooxygenase product in GPPA. In the presence of indomethacin the rank-order of potency was LTC4 greater than LTD4 greater than LTE4 greater than or equal to PE with respective pD2 values of 7.65, 7.39, 6.35 and 6.26. All further studies were carried out in the presence of 5 x 10(-6) M indomethacin. Removal of the endothelium further increased both potency (greater than 3-fold) and the maximal response of all agonists tested, indicating that a non-cyclooxygenase endothelium-dependent relaxing factor may be present in GPPA. In separate studies, GPPA was demonstrated capable of metabolizing 3H-LTC4 to 3H-LTD4 by an L-serine borate inhibitable gamma-glutamyl transpeptidase. In contrast, relatively little formation of 3H-LTE4 was apparent either from 3H-LTC4 or 3H-LTD4. The LTD4-selective antagonists, LY 171,883 and ICI 198,615 had -log molar KB values of 6.07 +/- 0.14 and 9.38 +/- 0.32, respectively, against LTD4 in the absence of endothelium. The ability of LY 171,883 to antagonize LTC4 was eliminated in the presence of 45 mM serine borate in endothelium denuded tissues. LT receptors in GPPA appear to be heterogeneous and similar to guinea pig airway receptors.     

Synthesis and metabolism of leukotrienes in gamma-glutamyl transpeptidase deficiency

Leukotrienes (LTs) are active lipid mediators derived in the 5-lipoxygenase pathway. LTC(4), the primary cysteinyl LT, is cleaved by gamma-glutamyl transpeptidase (GGT), resulting in LTD(4). We studied the synthesis and metabolism of LTs in three patients with GGT deficiency. LTs were analyzed in urine, plasma, and monocytes after HPLC separation by enzyme immunoassays, radioactivity detection, and electrospray tandem mass spectrometry. Analysis of LTs in urine revealed increased concentrations of LTC(4) (12.8-17.9 nmol/mol creatinine; controls, <0.005 nmol/mol creatinine), whereas LTE(4) was below the detection limit (<0.005 nmol/mol creatinine; controls, 32.2 +/- 8.6 nmol/mol creatinine). In plasma of one patient, LTC(4) was found to be increased (17.3 ng/ml; controls, 9.6 +/- 0.4 ng/ml), whereas LTD(4) and LTE(4) were below the detection limit (<0.005 ng/ml). LTB(4) was found within normal ranges. In contrast to controls, the synthesis of LTD(4) and LTE(4) in stimulated monocytes was below the detection limit (<0.1 ng/10(6) cells; controls, 37.1 +/- 4.8 cells and 39.4 +/- 5.6 ng/10(6) cells, respectively). The formation of [(3)H]LTD(4) from [(3)H]LTC(4) in monocytes was completely deficient (<0.1%; controls, 85 +/- 7%). Our data demonstrate a complete deficiency of LTD(4) biosynthesis in patients with a genetic deficiency of GGT. GGT deficiency represents a new inborn error of cysteinyl LT synthesis and provides a unique model in which to study the pathobiological coherence of LT and glutathione metabolism.     

Catabolism of leukotriene A4 into B4, C4, and D4 by rat liver subcellular fractions

[3H]Leukotriene A4 was incubated with various subcellular fractions of rat liver homogenates. After solvent extraction and purification on C18 Sep-Pak cartridges, tritiated products migrating on reversed-phase HPLC with authentic unlabelled leukotriene C4, D4 and B4 were observed. The identity of leukotriene C4 was confirmed through enzymatic conversion into D4 by gamma-glutamyl transpeptidase as well as by bioassay on the rat stomach fundus after HPLC purification. The contractile response to the extracted material was blocked by the SRS antagonist, FPL 55712. Leukotriene B4 synthesis was located in the 100 000 X g supernatant, while C4 synthesis was present in the corresponding pellet. Leukotriene C4 formation was enhanced when reduced glutathione was supplemented in the incubation medium. These results demonstrate the presence in rat liver of various enzymatic steps in leukotriene A4 catabolism.     

Metabolism of LTC4 to LTD4 and LTE4 in isolated guinea pig lung lobes

Leukotrienes are known to be easily metabolized to other substances. But the metabolic fates of LTC4 and LTD4 have not been established in the intact lung. In this investigation we perfused isolated guinea pig lung lobes and injected synthesized LTC4 and LTD4. The effluent was assayed by HPLC. LTD4 and LTE4 were detected following perfusion of LTC4, and LTE4 was detected following perfusion of LTD4. These results suggest that perfused guinea pig lung lobes may metabolize LTC4 to LTD4 and LTE4, and LTD4 to LTE4.     

Transport of amino acids in gamma-glutamyl transpeptidase-implanted human erythrocytes

gamma-Glutamyl transpeptidase purified from hog kidney cortex was implanted in the human erythrocyte membrane by incubation of erythrocytes at 37 degrees c with gamma-glutamyl transpeptidase-incorporated dipalmitoyl phosphatidylcholine vesicles. Membranes prepared from these implanted cells exhibited 4- to 5-fold increase in gamma-glutamyl transpeptidase activity. The association/insertion of gamma-glutamyl transpeptidase into erythrocyte membrane was further demonstrated by antibody to gamma-glutamyl transpeptidase. Implantation of gamma-glutamyl transpeptidase into erythrocyte membrane led to stimulation of uptake of glutamate and alanine, which are normally transported at a slow rate in human erythrocytes. The uptake of these amino acids in the implanted system was inhibited by inhibitors (serine-borate and azaserine) of transpeptidase activity as well as by antibody to gamma-glutamyl transpeptidase. These results in the implanted human erythrocytes demonstrate that gamma-glutamyl transpeptidase enzyme can mediate the translocation of amino acids and provide further evidence in support of its postulated role in the transport of amino acids in natural membranes.     

Conversion of leukotriene C4 to leukotriene D4 by a cell-surface enzyme of rat macrophages

Leukotriene (LT) C4-metabolizing enzyme was studied using rat leukocytes. Neutrophils and lymphocytes hardly metabolized LTC4, whereas macrophages rapidly converted LTC4 to LTD4. The LTC4-metabolizing enzyme of macrophages was present in the membrane fraction but not in the nuclear, granular and cytosol fractions. When macrophages were modified chemically with diazotized sulfanilic acid, a poorly permeant reagent which inactivates cell-surface enzymes selectively, the LTC4-metabolizing activity of macrophages decreased significantly (greater than 90%). These findings suggest that rat macrophages possess the LTC4-metabolizing enzyme which converts LTC4 to LTD4, on the cell surface membrane.     

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.     

Relationship between leukotriene A4 metabolism and biological activity

The data on the pharmacology of leukotrienes showed that LTA4, LTC4 and LTD4 were equipotent on the guinea-pig lung parenchyma whereas LTB4 was slightly less active. However, on the trachea, the myotropic activity of LTC4 and LTD4 was equivalent and higher than LTB4 and LTA4. The potency of these compounds was also different on the ileum where LTD4 was more active than LTC4; at the concentration used, LTA4 and LTB4 were inactive on this tissue. These results suggested that the transformation of leukotrienes by the smooth muscle preparations was a prerequisite for its biological activity. To verify this hypothesis, LTA4 (100 ng) was incubated for 10 min. with 20,000 g supernatants of homogenates of guinea-pig lung parenchyma, trachea and ileum; the metabolites were analysed by bioassay using strips of guinea-pig ileum and lung parenchyma in a cascade superfusion system and by RP-HPLC. Homogenates of lung parenchyma rapidly transformed LTA4 to LTB4, LTC4, LTD4 and LTE4, which is in agreement with the myotropic potency of these leukotrienes on the lung parenchymal strip. Conversely, incubation of LTA4 with homogenates of guinea-pig ileum showed the formation of LTB4 and its isomers which are inactive on this preparation. Similarly, incubation of homogenates of trachea with LTA4 led to the formation of LTB4; this finding is again in agreement with the potency of these two leukotrienes on the trachea. Our results suggest that the myotropic activity and potency of LTA4 is related to the tissue levels of enzymes which catalyse its transformation.     

Release of peptide leukotrienes from rat Kupffer cells

Kupffer cells isolated from the normal rat liver were incubated with calcium ionophore A23187, and the levels of peptide leukotrienes (LTC4, LTD4, and LTE4) contained in the culture supernatant were determined by the combined technique of reverse-phase high-performance liquid chromatography and radioimmunoassay. In response to A23187, Kupffer cells released LTC4, LTD4, and LTE4. After 10 min-preincubation of Kupffer cells with AA861, a 5-lipoxygenase inhibitor, the generation of LTC4, LTD4, and LTE4 from A23187-stimulated Kupffer cells was significantly suppressed. Platelet activating factor (PAF), a phospholipid mediator, significantly enhanced the release of LTC4, LTD4, and LTE4 from Kupffer cells stimulated with A23187. These results suggested that Kupffer cells may participate in inflammatory and immunologic events in the liver tissue by the release of peptide leukotrienes.     

The in vivo production of peptide leukotrienes after pulmonary anaphylaxis in the rat

Inbred hyper-reactive rats, actively sensitized to OVA, were anesthetized, cannulated, and ventilated with room air. Tracheal instillation of Ag (OVA) resulted in an elevation of airways pressure (14.4 +/- 0.6 cm H2O). Measurement of biliary peptide leukotriene levels before and after Ag challenge using reverse phase HPLC and RIA techniques showed significant elevations in leukotriene (LT) levels, the amounts released being LTC4 (3.65 +/- 0.78), LTD4 (2.8 +/- 1.11), and N-Ac LTE4 (3.87 +/- 1.15) expressed as ng/100 g of body weight, n = 13. Identification of these metabolites were confirmed by HPLC/RIA techniques and LTC4 was further characterized by UV spectroscopy and its enzymatic conversion by gamma-glutamyl transpeptidase to LTD4. [3H]LTC4 (16 ng) administration by tracheal instillation resulted in a 31.4 +/- 4.3% recovery of radioactivity through the bile over 4 h (n = 3) with the major identified metabolite being N-Ac LTE4. [3H]LTC4 (16 ng) plus synthetic LTC4 (5 micrograms) showed a 30.8 +/- 3.1% recovery through the bile after tracheal instillation (3-h collection, n = 4) with significant amounts of LTC4 as well as N-Ac LTE4 present. [3H]LTC4 administration by the portal vein resulted in a 37.4 +/- 8.8% biliary recovery over 60 min (n = 6), the metabolites present in the bile being LTC4, LTD4, LTE4, and N-Ac LTE4. Pretreatment with the 5-lipoxygenase inhibitor L-656,224 (15 mg/kg, 3.5 h pre-p.o.) before Ag challenge resulted in a significant inhibition (greater than 90%, p less than 0.05) of biliary leukotriene levels in this model. Our study demonstrates that peptide leukotrienes are produced in the anesthetized rat after pulmonary anaphylaxis and that biliary leukotriene measurement is suitable for showing the biochemical efficacy of leukotriene inhibitors in vivo. In vivo tracer experiments suggest that the biliary metabolic profile of the peptide leukotrienes is dependent on the site and levels of release as well as the efficiency of the vascular clearance of the various metabolites.     

Leukotriene C4 binding to rat lung membranes

A high affinity binding site for leukotriene C4 (LTC4), one component of slow reacting substance of anaphylaxis, has been identified in a membrane preparation from rat lung. As measured by a filtration technique, [3H]LTC4 binding was saturable, specific, reversible, and heat-sensitive. In the presence of 20 mM CaCl2, the dissociation constant (KD) was 41 +/- 9 nM and the maximum number of binding sites (Bmax) was 31 +/- 10 pmol/mg of protein. Specificity was demonstrated by competition studies in which LTC4 had a Ki of 40 nM against specifically bound [3H]LTC4, whereas leukotriene D4 (LTD4) had a Ki of 4 microM. The stereoisomers (5R, 6R) LTC4, (5S, 6S) LTC4, and (5R, 6S) LTC4 had Ki values 3-, 15-, and 25-fold higher than that of natural (5S, 6R) LTC4. Leukotrienes E4 and B4, several prostaglandins and fatty acids, glutathione, and platelet activating factor were even less effective with Ki values above 10 microM. A slow reacting substance of anaphylaxis antagonist, FPL 55712, which, in some systems, distinguishes LTC4- from LTD4-induced contractions, was a weak competitor with a Ki of 16 microM. Serine-borate complex which inhibits gamma-glutamyl transpeptidase, an enzyme responsible for LTC4 metabolism, did not alter binding. In addition, 100 microM FPL 55712 did not reduce metabolism. These observations suggest that the binding observed for LTC4 may represent association with a physiological receptor for this molecule which has a relatively low affinity for LTD4.     

Identification and characterization of leukotriene C4 and D4 receptors on a cultured smooth muscle cell line, BC3H-1

We studied the characteristics of the leukotriene (LT) C4 and D4 receptors on a cultured smooth muscle cell line, BC3H-1. Specific [3H]LTC4 binding to the cell membrane was greater than 80% of total binding and saturable at a density of 3.96 +/- 0.39 pmol/mg protein, with an apparent dissociation constant (Kd) of 14.3 +/- 2.0 nM (n = 9). The association and dissociation of [3H]LTC4 binding were rapid and apparent equilibrium conditions were established within 5 min. Calculated Kd value of [3H]LTC4 binding from the kinetic analysis was 9.9 nM. From the competition analysis, calculated Ki value of unlabeled LTC4 to compete for the specific binding of [3H]LTC4 was 9.2 nM and was in good agreement with the Kd value obtained from the Scatchard plots or kinetic analysis. The rank order of potency of the unlabeled competitors for competing specific [3H]LTC4 binding was LTC4 much greater than LTD4 greater than LTE4 greater than FPL-55712. The maximum number of binding sites (Bmax) of [3H]LTD4 in the membrane of BC3H-1 cell line was about 11 times lower than that of the [3H]LTC4. The calculated values of Kd and Bmax of [3H]LTD4 binding were 9.3 +/- 0.8 nM and 0.37 +/- 0.04 pmol/mg protein, respectively (n = 3). The rank order of potency or the unlabeled competitors for competing specific [3H]LTD4 binding was LTD4 = LTE4 greater than FPL-55712 much greater than LTC4. These findings demonstrate that BC3H-1 cell line possess both LTC4 and LTD4 receptors with a predominance of LTC4 receptors. Thus BC3H-1 cell line is a good model to study the regulation of LTC4 and LTD4 receptors.