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.     

Peroxisomal degradation of leukotrienes by beta-oxidation from the omega-end.

Chain shortening via beta-oxidation from the omega-end has been recognized as the major pathway for the degradation of cysteinyl leukotrienes as well as leukotriene B4 (LTB4). The metabolic compartmentation of this pathway was studied using peroxisomes purified from normal and clofibrate-treated rat liver. beta-Oxidation products of omega-carboxy-LTB4, including omega-carboxy-dinor-LTB4 identified by gas chromatography-mass spectrometry, were formed by the isolated peroxisomes. The reaction was dependent on CoA, ATP, and NAD and was stimulated by FAD. NADPH was necessary for the further metabolism of omega-carboxy-dinor-LTB4. Together with microsomes a degradation of omega-carboxy-LTB4 also proceeded in isolated mitochondria in the presence of CoA, ATP, and carnitine. beta-Oxidation of the cysteinyl leukotriene omega-carboxy-N-acetyl-leukotriene E4 was observed only with isolated peroxisomes in combination with lipid-depleted microsomes. Direct photoaffinity labeling using omega-carboxy-[3H] LTB4 and omega-carboxy-N-[3H]acetyl-LTE4 served to identify peroxisomal leukotriene-binding proteins. The bifunctional protein (EC 4.2.1.17 and 1.1.1.35) and 3-ketoacyl-CoA thiolase (EC 2.3.1.16) of the peroxisomal beta-oxidation system were the predominantly labeled polypeptides as revealed by precipitation with monospecific antibodies. In vivo studies with N-acetyl-[3H2]LTE4, N-acetyl-[3H8]LTE4, and N-[14C]acetyl-LTE4 after treatment with the peroxisome proliferator clofibrate indicated formation and biliary excretion of large amounts of metabolites more polar than omega-carboxy-tetranor-N-acetyl-LTE3 including omega-carboxy-tetranor-delta 13-N-acetyl-LTE4 and omega-carboxy-hexanor-N-acetyl-LTE3. Increased formation of beta-oxidized catabolites of N-acetyl-LTE4 and LTB4 was also observed in hepatocytes isolated after clofibrate treatment. Our results indicate that peroxisomes play a major role in the beta-oxidation of leukotrienes from the omega-end. Whereas omega-carboxy-LTB4 was beta-oxidized both in isolated peroxisomes and mitochondria, the cysteinyl leukotriene omega-carboxy-N-acetyl-LTE4 was exclusively degraded in peroxisomes.     

Metabolism of cysteinyl leukotrienes in monkey and man

The proinflammatory cysteinyl leukotrienes are inactivated in primates by (a) intravascular degradation, (b) hepatic and renal uptake from the blood circulation, (c) intracellular metabolism of leukotriene E4 (LTE4), and (d) biliary and renal excretion of LTC4 degradation products. We have analyzed cysteinyl leukotriene metabolites excreted into bile and urine of the monkey Macaca fascicularis and of man. In both species, hepatobiliary leukotriene elimination predominated over renal excretion. In a representative healthy human subject at least 25% of the administered radioactivity were recovered from bile and 20% from urine within 24 h. In monkey and man intravenous administration of 14,15-3H2-labeled LTC4 resulted in the biliary and urinary excretion of labeled LTE4, omega-hydroxy-LTE4, omega-carboxy-LTE4, omega-carboxy-dinor-LTE4, and omega-carboxy-tetranor-dihydro-LTE4. Small amounts of N-acetyl-LTE4 were detected in human urine only. Oxidative metabolism of LTE4 proceeded more rapidly in the monkey resulting in the formation of higher relative amounts of omega-oxidized leukotrienes in this species as compared to man. [3H]H2O amounted to less than 2% of the administered dose in monkey and human bile and urine samples. Incubation of isolated human hepatocytes with [3H2]LTC4, [3H2]LTD4, and [3H2]LTE4 showed that only [3H2]LTE4 underwent intracellular oxidative metabolism resulting in the formation of omega- and beta-oxidation products. N-Acetylated LTE4 derivatives were not detected as products formed by human hepatocytes. By a combination of reversed-phase high-performance liquid chromatography and radioimmunoassay, endogenous LTE4 and N-acetyl-LTE4 were detected in human urine in concentrations of 220 +/- 40 and 24 +/- 3 pM, corresponding to 12 +/- 1 and 1.5 +/- 0.2 nmol/mol creatinine, respectively (mean +/- SEM; n = 10). Endogenous LTD4 and LTE4 were detected in human bile (n = 3) in concentrations between 0.2-0.9 nM. Our results demonstrate that LTD4 and LTE4 are major LTC4 metabolites in human bile and/or urine and may serve as index metabolites for the measurement of endogenously generated cysteinyl leukotrienes. Moreover, omega-oxidation and subsequent beta-oxidation from the omega-end contribute to the metabolic degradation of LTE4 not only in monkey but also in man.     

Metabolism of cysteinyl leukotrienes in non-recirculating rat liver perfusion. Hepatocyte heterogeneity in uptake and biliary excretion

1. The uptake, metabolism and biliary excretion of the cysteinyl leukotrienes LTC4, LTD4 and LTE4, were studied in a non-recirculating rat liver perfusion system at constant flow in both antegrade (from the portal to the caval vein) and retrograde (from the caval to the portal vein) perfusion directions. During a 5-min infusion of [3H]LTC4, [3H]LTD4 and [3H]LTE4 (10 nmol/l each) in antegrade perfusions single-pass extractions of radioactivity from the perfusate were 66%, 81% and 83%, respectively. Corresponding values for LTC4 and LTD4 in retrograde perfusions were 83% and 93%, respectively, indicating a more efficient uptake of cysteinyl leukotrienes in retrograde than in antegrade perfusions. The concentrations of unmetabolized leukotrienes in the effluent perfusate were 8-12% in antegrade and 2-4% in retrograde perfusions. [14C]Taurocholate extraction from the perfusate was inhibited by LTC4 by only 3%, suggesting that an opening of portal-venous/hepatic-venous shunts does not explain the effects of perfusion direction on hepatic LTC4 uptake. 2. Following infusion of [3H]LTC4 and [3H]LTD4, in the antegrade perfusion direction, about 80% and 87%, respectively, of the radiolabel taken up by the liver was excreted into bile. In retrograde perfusions, however, only 40% and 57%, respectively, was excreted into bile and the remainder was slowly redistributed into the perfusate, indicating that leukotrienes were taken up into a hepatic compartment with less effective biliary elimination or converted to metabolites escaping biliary excretion. The metabolite pattern found in bile was not affected by the direction of perfusion. Biliary products of LTC4 were polar metabolites (31-38%), LTD4 (27-30%), LTE4 (about 1%) and N-acetyl-LTE4 (3-4%) in addition to unmodified LTC4 (17-18%). 3. LTC4 was identified as a major metabolite of [3H]LTD4 in bile, amounting to about 20% of the total radioactivity excreted into bile. This is probably due to a gamma-glutamyltransferase-catalyzed glutamyl transfer from glutathione in the biliary compartment, as demonstrated in in vitro experiments. The presence of sinusoidal gamma-glutamyltransferase activity in perfused rat liver was shown in experiments on the hydrolysis of infused gamma-glutamyl-p-nitroanilide. 90% inhibition of this enzyme activity by AT-125 did not affect the metabolism of LTC4. 4. When [3H]LTE4 was infused in the antegrade perfusion direction, biliary metabolites comprised N-acetyl-LTE4 (24%) and polar components (60%).(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

ATP-dependent primary active transport of cysteinyl leukotrienes across liver canalicular membrane. Role of the ATP-dependent transport system for glutathione S-conjugates

The liver is the major organ which eliminates leukotriene C4 (LTC4) and other cysteinyl leukotrienes from the blood circulation into bile. Transport of LTC4 was studied using inside-out vesicles enriched in canalicular and sinusoidal membranes from rat liver. The incubation of canalicular membrane vesicles with [3H]LTC4 in the presence of ATP resulted in an uptake of LTC4 into vesicles. The initial rate of ATP-stimulated LTC4 uptake was about 40-fold higher in canalicular than in sinusoidal membrane vesicles. When liver plasma membrane vesicles were incubated in the absence of ATP, an apparent transient uptake of LTC4 was observed which was temperature-dependent and not affected by the osmolarity. This indicates that LTC4 was bound to proteins on the surface of plasma membrane vesicles. Two proteins with relative molecular weights of 17,000 and 25,000 were detected by direct photoaffinity labeling as major LTC4-binding proteins. One protein (Mr 25,000) was ascribed to subunit 1 (Ya) of glutathione S-transferase which was associated with the membrane. LTD4, LTE4, N-acetyl-LTE4, and omega-carboxy-N-acetyl-LTE4 were also transported into liver plasma membrane vesicles in an ATP-dependent manner with initial rates relative to LTC4 (1.0) of 0.46, 0.11, 0.35, and 0.22, respectively. Mutual competition between the cysteinyl leukotrienes and S-(2,4-dinitrophenyl)-glutathione for uptake indicated that they are transported by a common carrier. Apparent Km values of the transport system for LTC4, LTD4, and N-acetyl-LTE4 were 0.25, 1.5, and 5.2 microM, respectively. The ATP-dependent transport of LTC4 into vesicles was not inhibited by doxorubicin, daunorubicin, or verapamil, or by the monoclonal antibody C219, suggesting that the transport system differs from P-glycoprotein. Liver plasma membrane vesicles prepared from mutant rats deficient in the hepatobiliary excretion of cysteinyl leukotrienes lacked the ATP-dependent transport of cysteinyl leukotrienes and S-(2,4-dinitrophenyl)-glutathione. These results demonstrate that the ATP-dependent carrier system is responsible for the transport of cysteinyl leukotrienes and glutathione S-conjugates from the hepatocytes into bile.     

Carboxypeptidase H. A regulatory peptide-processing enzyme produced by human hepatoma Hep G2 cells

Human hepatoma (Hep G2) cells have been shown to secrete nanogram quantities of carboxypeptidase N (Grimwood, B. G., Plummer, T. H., Jr., and Tarentino, A. (1988) J. Biol. Chem. 263, 14397-14401). A second carboxypeptidase with an acidic pH optimum (pH 5.5) is also secreted at levels 2-3-fold greater than carboxypeptidase N. This enzyme was partially purified from the conditioned medium and compared with pure bovine pituitary carboxypeptidase H. The two enzymes behaved in a similar fashion in DE52 ion-exchange chromatography and on gel filtration, with the Hep G2 enzyme being slightly larger than the bovine pituitary enzyme (52-54 versus 50-52 kDa). Both enzymes hydrolyzed COOH-terminal basic amino acids from typical synthetic substrates as well as from natural leuenkephalin peptides and were identical based on pH activity profiles, inhibition by EDTA or guanidinoethyl mercaptosuccinic acid, and stimulation by Co2+ ions. Inhibition of enzyme secretion from Hep G2 cells by tunicamycin indicated that the Hep G2 enzyme was glycosylated. This finding was confirmed by a parallel deglycosylation of the Hep G2 and bovine pituitary carboxypeptidase H enzymes with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F. Immunoblots using mouse antiserum to bovine pituitary carboxypeptidase H revealed that the Hep G2 enzyme was immunocross-reactive with the bovine enzyme but was slightly larger in size (54 versus 52 kDa). Continuous [35S]methionine labeling and purification to near homogeneity using an affinity matrix corroborated the observations that the secreted Hep G2 carboxypeptidase H was slightly larger than bovine pituitary carboxypeptidase H. The Hep G2-secreted enzyme in pulse-chase experiments was initially detected intracellularly after a 15-min pulse as a single protein of about 54 kDa and was present in the 30-min chase medium with no evidence for pre- or postsecretion proteolytic processing. The human adrenergic cell line IMR-32 continuously labeled with [35S]methionine also secreted carboxypeptidase H of the same size as the Hep G2 enzyme.     

Inhibition of leukotriene D4 catabolism by D-penicillamine

Inhibition of the catabolism of the most biologically potent cysteinyl leukotriene, LTD4, was studied in rat hepatoma cells in vitro and in the rat in vivo. LTD4 dipeptidase, an ectoenzyme on the surface of AS-30D hepatoma cells, exhibited an apparent Km value of 6.6 microM for LTD4. D-Penicillamine and L-penicillamine inhibited this enzyme activity with apparent Ki values of 0.46 mM and 0.21 mM respectively. Bestatin, an inhibitor of the aminopeptidase activity of hepatoma cells, did not affect LTD4 hydrolysis at concentrations as high as 5 mM, indicating that the aminopeptidase did not contribute to LTD4 catabolism. In the rat in vivo, D-penicillamine also inhibited LTD4 catabolism. After intravenous injection of [3H]LTC4 an accumulation of [3H]LTD4 and a retarded formation of [3H]LTE4 were observed in the circulating blood after D-penicillamine pretreatment. Within 1 h after intravenous [3H]LTC4 injection, about 80% of the administered radioactivity was recovered in bile. After D-penicillamine pretreatment [3H]LTD4 was the major biliary leukotriene metabolite, whereas in untreated controls leukotriene metabolites more polar than LTC4 predominated in bile. After stimulation of endogenous leukotriene production in vivo by platelet-activating factor, N-acetyl-LTE4 was the major cysteinyl leukotriene detected in bile. D-Penicillamine treatment prior to platelet-activating factor resulted in the accumulation of LTD4, which under these circumstances was the major endogenous leukotriene metabolite detected in bile.     

Targeted inhibition of transferrin-mediated iron uptake in Hep G2 hepatoma cells

We have used a model system consisting of two human hepatoma cell lines, Hep G2, representing well differentiated normal hepatocytes, and PLC/PRF/5, representing poorly differentiated malignant hepatocytes, to demonstrate that the differential presence of asialoglycoprotein receptor activity in these cell lines can be used to influence transferrin-mediated iron uptake. We based our experiments on the following facts: Hep G2 cells possess receptors that bind, internalize, and degrade galactose-terminal (asialo-)glycoproteins; PLC/PRF/5 cells have barely detectable asialoglycoprotein receptor activity; both cell lines possess active transferrin-mediated iron uptake; transferrin releases iron during acidification of intracellular vesicular compartments; primary amines, e.g. primaquine, inhibit acidification and iron release from transferrin. When added to culture medium, [55Fe]transferrin delivered 55Fe well to both cell lines. As expected, in the presence of [55Fe]transferrin, free primaquine caused a concentration-dependent decrease in 55Fe uptake in both cell lines. To create a targetable conjugate, primaquine was covalently coupled to asialofetuin to form asialofetuin-primaquine. When PLC/PRF/5 (asialoglycoprotein receptor (-)) cells were preincubated with this conjugate, transferrin-mediated 55Fe uptake was unaffected. However, transferrin-mediated 55Fe uptake by Hep G2 (asialoglycoprotein receptor (+)) cells under identical conditions was specifically decreased by 55% compared to control cells incubated without the conjugate.     

Conjunctival goblet cell secretion stimulated by leukotrienes is reduced by resolvins D1 and E1 to promote resolution of inflammation

The conjunctiva is a mucous membrane that covers the sclera and lines the inside of the eyelids. Throughout the conjunctiva are goblet cells that secrete mucins to protect the eye. Chronic inflammatory diseases such as allergic conjunctivitis and early dry eye lead to increased goblet cell mucin secretion into tears and ocular surface disease. The purpose of this study was to determine the actions of the inflammatory mediators, the leukotrienes and the proresolution resolvins, on secretion from cultured rat and human conjunctival goblet cells. We found that both cysteinyl leukotriene (CysLT) receptors, CysLT(1) and CysLT(2,) were present in rat conjunctiva and in rat and human cultured conjunctival goblet cells. All leukotrienes LTB(4), LTC(4), LTD(4), and LTE(4), as well as PGD(2), stimulated goblet cell secretion in rat goblet cells. LTD(4) and LTE(4) increased the intracellular Ca(2+) concentration ([Ca(2+)](i)), and LTD(4) activated ERK1/2. The CysLT(1) receptor antagonist MK571 significantly decreased LTD(4)-stimulated rat goblet cell secretion and the increase in [Ca(2+)](i). Resolvins D1 (RvD1) and E1 (RvE1) completely reduced LTD(4)-stimulated goblet cell secretion in cultured rat goblet cells. LTD(4)-induced secretion from human goblet cells was blocked by RvD1. RvD1 and RvE1 prevented LTD(4)- and LTE(4)-stimulated increases in [Ca(2+)](i), as well as LTD(4) activation of ERK1/2. We conclude that cysteinyl leukotrienes stimulate conjunctival goblet cell mucous secretion with LTD(4) using the CysLT(1) receptor. Stimulated secretion is terminated by preventing the increase in [Ca(2+)](i) and activation of ERK1/2 by RvD1 and RvE1.     

Fatty acid uptake by human hepatoma cell lines represents a carrier-mediated uptake process

Cellular influx kinetics of a representative long chain fatty acid, [3H]oleate, were examined in monolayer cultures of three different human hepatoma cell lines (Hep G2; PLC/PRF 5; Mz-Hep-1). The cultures were incubated with 173 microM [3H]oleate in the presence of various concentrations of albumin which served to modulate the unbound oleate concentration in the medium. For all [3H]oleate-albumin complexes incubated, it was shown that cellular uptake of [3H]oleate over the initial 30 s incubation period was maximal, linear and independent of intracellular fatty acid metabolism, representing cellular influx. With increasing unbound oleate concentrations in the medium cellular influx by all three cell lines revealed similar saturation kinetics with Km values of 112.6 +/- 14.5 nM and Vmax values of 7.19 +/- 0.32 nmol.min-1 per mg cell protein. When these hepatoma cell lines were pretreated with the IgG fraction of a monospecific antibody to the rat liver membrane fatty acid binding protein (MFABP), initial uptake of [3H]oleate was selectively inhibited compared to controls pretreated with the IgG fraction of the preimmune serum. Furthermore, immunoblot analysis with the monospecific antibody to the rat MFABP revealed reactivity with a single 40 kDa protein in the homogenates of all three cell lines. These data suggest that uptake of fatty acids by human hepatoma cells may be mediated by a specific membrane fatty acid binding protein.     

Characterization of leukotriene C4 binding in anterior pituitary membrane preparations.

Pituitary cells produce leukotrienes (LTs) and respond to exogenous administration of LTs by releasing gonadotropins. Specific high affinity leukotriene C4 (LTC4) binding has been found in membrane preparations of bovine anterior pituitaries. Unlabelled LTC4 displaced specific [3H]LTC4 binding. Other leukotrienes (LTB4, LTD4, LTE4, LTF4) did not compete with [3H]LTC4 for binding sites when administered at increasing concentrations together with a constant amount of radioligand indicating that the binding is highly specific for LTC4. Scatchard analysis of binding data obtained from saturation studies revealed a single binding site for [3H]LTC4 with a Kd of 8.95 +/- 5.53 nM and a B max of 15.44 +/- 6.93 pmol per mg of membrane protein. Glutathione S-transferase, a possible LTC4 binding site, did not display activity in the membrane fraction although the two glutathione derivates S-octylglutathione and S-decylglutathione competed with LTC4 in binding experiments. As leukotrienes are potent stimulators of gonadotropin secretion and modulators of gonadotropin-releasing hormone (GnRH)-induced gonadotropin release it is concluded that leukotrienes may be involved in the signal transduction pathway of GnRH and that they may act via a specific and high affinity receptor.     

Characteristics of sinusoidal uptake and biliary excretion of cysteinyl leukotrienes in perfused rat liver

In single-pass perfused rat liver, the sinusoidal uptake of infused 3H-labelled leukotriene (LT) C4 (10 nmol.l-1) was inhibited by sulfobromophthalein. Inhibition was half-maximal at sulfobromophthalein concentrations of approximately 1.2 mumol.l-1 in the influent perfusate and leukotriene uptake was inhibited by maximally 34%. Sulfobromophthalein (20 mumol.l-1) also decreased the uptake of infused [3H]LTE4 (10 nmol.l-1) by 31%. Indocyanine green (10 mumol.l-1) inhibited the sinusoidal [3H]LTC4 uptake by 19%. Replacement of sodium in the perfusion medium by choline decreased the uptake of infused [3H]LTC4 (10 nmol.l-1) by 56%, but was without effect on the uptake of sulfobromophthalein. The canalicular excretion of LTC4, LTD4 and N-acetyl-LTE4 was inhibited by sulfobromophthalein. In contrast, the proportion of polar omega-oxidation metabolites recovered in bile following the infusion of [3H]LTC4 was increased. Taurocholate, which had no effect on the sinusoidal leukotriene uptake, increased bile flow and also the biliary elimination of the radioactivity taken up. With increasing taurocholate additions, the amount of LTD4 recovered in bile increased at the expense of LTC4. Following the infusion of [3H]LTD4 (10 nmol.l-1), a major biliary metabolite was LTC4 indicating a reconversion of LTD4 to LTC4. In the presence of taurocholate (40 mumol.l-1), however, this reconversion was completely inhibited. The findings suggest the involvement of different transport systems in the sinusoidal uptake of cysteinyl leukotrienes. LTC4 uptake is not affected by bile acids and has a sodium-dependent and a sodium-independent component, the latter probably being shared with organic dyes. Sulfobromophthalein also interferes with the canalicular transport of LTC4, LTD4 and N-acetyl-LTE4, but not with the excretion of omega-oxidized cysteinyl leukotrienes. The data may be relevant for the understanding of hepatic leukotriene processing in conditions like hyperbilirubinemia or cholestasis.     

Platelet-activating factor induces the production of leukotrienes by human monocytes

The aim of this study was to evaluate the role of platelet-activating factor (PAF) as a stimulator of leukotriene production by human monocytes. The production of leukotrienes was time- and concentration-dependent. Release of leukotrienes was half-maximal after 2 min and reached a maximum after 10 min. At a concentration of 10(-8) M, PAF induced the production of 0.14 +/- 0.01 ng LTB4/10(6) cells (mean +/- S.E., n = 8). At concentrations of 10(-6) M, PAF induced the production of 1.0 +/- 0.04 ng LTB4 and 0.22 +/- 0.03 ng peptidoleukotrienes (mean +/- S.E., n = 16). There was no metabolism of LTB4 as judged from stability of [3H]LTB4 added to the incubations. LTC4 was slowly metabolized by human monocytes to LTD4 and LTE4. The two specific PAF-receptor antagonists BN 52021 and WEB 2086 in concentrations of 10(-4) and 10(-6) M, respectively, inhibited the PAF (10(-6) M) stimulated LTB4 production completely. In this study, we demonstrate that nanomolar concentrations of PAF can stimulate the production of LTB4 and peptidoleukotrienes in human monocytes by a receptor-mediated mechanism.     

Identification of cyclosporin binding sites in rat liver plasma membranes, isolated hepatocytes, and hepatoma cells by photoaffinity labeling using [3H]cyclosporin-diaziridine

[3H]Cyclosporin diaziridine, a new photoaffinity label, enters rat liver cells in the dark. Photoaffinity labeling of isolated rat liver-cell plasma membranes with this probe modifies several polypeptides with molecular mass of 200, 85, 54, 50, 34 kDa. The major labeled protein of 85 kDa represents 2% of the total plasma membrane protein. A 50 kDa protein is heavily labeled in freshly isolated rat hepatocytes at low temperature and after short incubation in the dark. The 85 kDa protein becomes substituted after longer preincubation periods at temperatures above 10 degrees C. This suggests a localisation at the cytoplasmic side of the membrane. Several controls point to a specific interaction with the above mentioned proteins. Comparison of [3H]cyclosporin-diaziridine- and isothiocyanatobenzamido[3H] cholic acid-labeled membrane proteins reveals identity of binding proteins with the exception of the 85 kDa protein. However, the interaction of bile acids with the 85 kDa protein became apparent at higher concentrations as demonstrated by the differential photoaffinity labeling experiments. In the cytosol of rat liver cells, further [3H]cyclosporin-diaziridine binding proteins could be identified. In particular, a 17 kDa polypeptide was found which appears similar to cyclophilin, a protein known to be present in T-lymphocytes (R. Handschumacher et al. (1984) Science 226, 544-547: Cyclophilin. A specific cytosolic binding protein for cyclosporin A). Proteins with molecular mass of 90, 56, 30, 24, 20 kDa are labeled in AS-30D ascites hepatoma cells and those with molecular mass of 200, 150, 80, 70, 42, 25 kDa in Ehrlich ascites tumor cells.     

Leukotriene C4 action and metabolism in the isolated perfused bullfrog heart

The effects of leukotrienes (LTs) have been widely studied in the isolated perfused mammalian heart; however, little is known about the effect or metabolism of LTs in the isolated bullfrog heart. Isolated perfused bullfrog hearts were administered randomized doses of LTC4, LTD4, or LTE4. The cardiac parameters of heart rate, developed tension, and its first derivative (dT/dt) were recorded. LTC4 was the most potent of the leukotrienes tested in eliciting positive inotropic effects. LTD4 and LTE4 were equally effective but about one order of magnitude less potent than LTC4. None of the LTs showed any chronotropic effects in this preparation. A series of [3H]LTC4 metabolism experiments were carried out using whole perfused hearts and minced bullfrog heart tissue. Isolated perfused bullfrog hearts administered [3H]LTC4 converted significant amounts to [3H]LTD4, and to a lesser degree, [3H]LTE4, during the 6-min course of collection. Both minced atrial and ventricular tissue converted [3H]LTC4 to radioactive metabolites that co-migrated with authentic LTD4 and LTE4 standards. In both tissues, the major product was [3H]LTD4, with smaller amounts of [3H]LTE4 produced. The atrium converted significantly more [3H]LTC4 to its metabolites than did the ventricle. The metabolism of [3H]LTC4 to [3H]LTD4 by both tissues was virtually abolished in the presence of serine borate. Cysteine had no effect on [3H]LTE4 production. The data in this study demonstrate that leukotrienes have the opposite inotropic effect on the heart when compared with mammals. Also in contrast to mammals, frogs metabolize LTC4 to a less potent compound and may use the LTC4 to LTD4 conversion as a mechanism of LTC4 inactivation.     

Transport functions of the liver. Lack of correlation between hepatocellular ouabain uptake and binding to (Na+ + K+)-ATPase

Ouabain uptake was studied on isolated rat hepatocytes. Hepatocellular uptake of the glycoside is saturable (Km = 348 mumol/l, Vmax = 1.4 nmol/mg cell protein per min), energy dependent and accumulative. Concentrative ouabain uptake is not present on permeable hepatocytes, Ehrlich ascites tumor cells and AS-30D ascites hepatoma cells. There is no correlation between ouabain binding to rat liver (Na+ + K+)ATPase and ouabain uptake into isolated rat hepatocytes. While ouabain uptake is competitively inhibited by cevadine, binding to (Na+ + K+)-ATPase is not affected by the alkaloid. Although the affinities of digitoxin and ouabain to (Na+ + K+)-ATPase are similar, digitoxin is 10000-times more potent in inhibiting [3H]ouabain uptake as compared to ouabain. That binding to (Na+ + K+)-ATPase appears to be no precondition for ouabain uptake was also found in experiments with plasmamembranes derived from Ehrlich ascites tumor cells and AS-30D hepatoma cells. While tumor cell (Na+ + K+)-ATPase is ouabain sensitive, the intact cells are transport deficient. Hepatic ouabain uptake might be related to bile acid transport. Several inhibitors of the bile acid uptake system also inhibit ouabain uptake.     

Enhanced levels of leukotriene B(4) in synovial fluid in Lyme disease

The purpose of this study was to evaluate the potential role of LTB(4) and cysteinyl leukotrienes in Lyme disease (LD). Therefore, a total number of 34 patients divided into four groups was studied. The patients were classified as having Lyme arthritis (n = 7) or Lyme meningitis (n = 10), and as control groups patients with a noninflammatory arthropathy (NIA) (n = 7) and healthy subjects (n = 10). LTB(4) as well as LTC(4) secretion from stimulated polymorphonuclear leukocytes (PMNL) from all groups of patients showed no statistical differences. LTB(4) levels in synovial fluid were significantly increased in patients with Lyme arthritis (median 142 ng/ml, range 88-296) when compared to the control subjects with NIA (median 46 ng/ml, range 28-72) (p < 0.05). No statistical difference of urinary LTE(4) levels between all the different groups of patients was observed. These results show that cysteinyl leukotrienes do not play an important role in the pathogenesis of LD. In contrast to previous findings in rheumatoid arthritis, LTB(4) production from stimulated PMNL was not found to be increased in LD. However, the significantly elevated levels of LTB(4) in synovial fluid of patients with Lyme arthritis underline the involvement of LTB(4) in the pathogenesis of this disease.     

Characteristics of formation and further metabolism of leukotrienes in the chopped human lung

The bronchoconstrictive leukotrienes (LTs) LTC4, LTD4 and LTE4 (cysteinyl-LTs) and the chemoattractant LTB4 were formed in chopped human lung stimulated by the calcium ionophore A23187, or supplied with the precursor LTA4. In contrast, challenge with anti-IgE exclusively induced release of cysteinyl-LTs, indicating that LTB4 is not released as a primary consequence of IgE-mediated reactions in the human lung. Furthermore, several differences were observed with respect to formation and further conversion of LTB4 and LTC4 in the chopped lung preparation. Thus, exogenous [1-14C]arachidonic acid was dose-dependently converted to radioactive LTB4, whereas the cysteinyl-LTs released were not radiolabeled and the amounts of LTC4, D4 and E4 were not influenced by addition of increasing concentrations of arachidonic acid. LTC4 was rapidly and completely converted into LTD4 and LTE4, with no further catabolism of LTE4 within 90 min. The metabolism of LTB4 was much slower than that of LTC4. Thus, following a 60 min incubation approx. 25% of the material remained as LTB4, whereas 35% was omega-oxidized and 40% eluted on RP-HPLC as two unidentified peaks.     

Metabolism and analysis of cysteinyl leukotrienes in the monkey

Predominant hepatobiliary elimination from blood and subsequent enterohepatic circulation of cysteinyl leukotrienes is demonstrated in the monkey Macaca fascicularis. From intravenous [3H]leukotriene C4, about 40% were recovered as metabolites in bile and about 20% in urine within 5 h. [3H]Leukotriene E4 was a predominant metabolite of defined structure in blood plasma, bile, and urine. From intraduodenal [3H]leukotriene C4, about 5% were recovered as metabolites in bile and about 8% in urine within 8 h. Endogenous cysteinyl leukotrienes generated in vivo were measured after implantation of a subcutaneously looped biliary bypass. Tapping of the loop allowed access to bile and prevented interference by leukotrienes produced by surgical trauma (Denzlinger, C., Rapp, S., Hagmann, W., and Keppler, D. (1985) Science 230, 330-332). Endogenous cysteinyl leukotrienes were analyzed in bile, urine, and blood plasma by the sequential use of high-performance liquid chromatography and a radioimmunoassay that was optimized for leukotriene E4 as a predominant metabolite detected in the tracer studies. Biliary leukotriene E4 rose from less than 0.2 to 9 nmol/liter, when leukotriene synthesis was elicited in anesthesized monkeys by staphylococcal enterotoxin B administered intragastrically. This study provides an approach to the analysis of cysteinyl leukotrienes in primates and serves to define the role of these mediators under pathophysiological as well as physiological conditions in vivo.