Eur. J. Biochem. 209,281 -289 (1992) 1 0 FEBS 1992

Leukotriene uptake by hepatocytes and hepatoma cells Inka LEIER, Michael MULLER, Gabriele JEDLITSCHKY and Dietrich KEPPLER Division of Tumor Biochemistry, Deutsches K rebsforschungs7entrum, Heidelberg, Federal Republic of Germany (Received May 18/July 10, 1992) - EJB 92 0676

The uptake of tritiated cysteinyl leukotrienes (LTC,, LTD,, LTE,) and LTB, was investigated in freshly isolated rat hepatocytes and different hepatoma cell lines under initial-rate conditions. Leukotriene uptake by hepatocytes was independent of an Na’ gradient and a K diffusion potential across the hepatocyte membranes as established in experiments with isolated hepatocytes and plasma membrane vesicles. Kinetic experiments with isolated hepatocytes indicated a low-K, system and a non-saturable system for the uptake of cysteinyl leukotrienes as well as LTB, under the conditions used. AS-30D hepatoma cells and human Hep G2 hepatoma cells were deficient in the uptake of cysteinyl leukotrienes, but showed significant accumulation of LTB,. Moreover, only LTB, was metabolized in Hep G2 hepatoma cells. Competition studies on the uptake of LTE4 and LTB, (10 nM each) indicated inhibition by the organic anions bromosulfophthalein, S-decyl glutathione, 4,4‘diisothiocyanato-stilbene-2,2’-disulfonate,probenecid, docosanedioate, and hexadecanedioate (100 pM each), but not by taurocholate, the amphiphilic cations verapamil and N-propyl ajmaline, and the neutral glycoside ouabain. Cholate and the glycoside digitoxin were inhibitors of LTB, uptake only. Bromosulfophthalein, the strongest inhibitor of leukotriene uptake by hepatocytes, did not inhibit LTB, uptake by Hep G2 hepatoma cells under the same experimental conditions. Leukotriene-binding proteins were analyzed by comparative photoaffinity labeling of human hepatocytes and Hep G2 hepatoma cells using [3H]LTE4and [3H]LTB4as the photolabile ligands. Predominant leukotriene-binding proteins with apparent molecular masses in the ranges of 48 58 kDa and 38 - 40 kDa were labeled by both leukotrienes in the particulate and in the cytosolic fraction of hepatocytes, respectively. In contrast, no labeling was obtained with [3H]LTE4in Hep G2 cells. With [’HILTB, a protein with a molecular mass of about 48 kDa was predominantly labeled in the particulate fraction of the hepatoma cells, whereas in the cytosolic fraction a labeled protein in the range of 40 kDa was detected. Our results provide evidence for the existence of distinct uptake systems for cysteinyl leukotrienes and LTB4 at the sinusoidal membrane of hepatocytes; however, some of the inhibitors tested interfere with both transport systems. Only LTB,, but not cysteinyl leukotrienes, is taken up and metabolized by the transformed hepatoma cells. +

Effective elimination from the blood circulation and metabolic inactivation of leukotrienes are important for regulating the concentration of the biologically active mediators at their site of action. During its short half-life in the blood circulation, LTC, undergoes rapid intravascular metabolism yielding LTD, and LTE, [I - 51. These cysteinyl leukotrienes are rapidly rcmoved from the circulation, predominantly by hepatocellular uptake and elimination [2- lo]. The liver is also a major site for the in vivo catabolism of LTB, [Ill. Catabolism of LTB4 was shown in granulocytes [12-151, in the isolated perfused liver [ll], and in intact animals [16]. Pioneering kinetic studies by Ormstad et al. [17] and Uehara et al. [18] indicated cysteinyl leukotriene uptake into hepatocytes by an active and temperature-sensitive transport mechanism specific for cysteinyl leukotrienes. Recently, it was shown that uptake of cysteinyl leukotrienes by the isolated Correspondencu to I. Leier, Division of Tumor Biochemistry, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, W-6900 Heidelberg, Federal Republic of Germany Ahhrevialions. RSP, bromosulfophthalein; DIDS, 4,4’-diisothiocyanatostilbene-2,2’-disulfonate; LT, leukotriene.

perfused liver is inhibited by bromosulfophthalein (BSP) and indocyanine green [19]. In contrast to hepatocytes, AS-30D hepatoma cells are deficient in the uptake of cysteinyl leukotrienes [20]. Cysteinyl-leukotriene-binding proteins in the particulate and cytosolic fraction of hepatocytes have been identified by direct photoaffinity labeling, using radioactive LTE, as the photolabile ligand. One of the mainly labeled proteins in the sinusoidal membrane of hepatocytes was a protein with an apparent molecular mass of 48 kDa [21, 221. In hepatocarcinogenesis numerous liver functions are altered or lost. Alterations of the cell surface membrane [23 -261 and of transport functions occur upon transformation of normal cells into neoplastic cells. Several investigations demonstrated the loss of bile salt transport in a number of hepatoma cells [27,28] and the insensitivity of hepatoma cells to phallotoxins due to a lack of uptake [29, 301. This study was aimed at a detailed characterization of leukotriene uptake by hepatocytes under initial-rate conditions, comparing cysteinyl leukotriene uptake with that of LTB4. The results were essential for comparative investigations on hepatoma cells employing kinetic as well as ph otoaffi nity labeling techniques.

282 EXPERIMENTAL PROCEDURES Materials [3H2]LTC4(1420 TBq/mol), C3H2]LTD4(1450 TBq/mol), ['H2]LTE4 (1480 I'Bq/mol), and [3H2]LTB4(1200 TBq/mol) were obtained from New England Nuclear/DuPont (Boston, MA, USA). N-Acetylation of ['Hz]LTE4 and LTE4 was performed with acetic anhydride [9]. Unlabeled leukotrienes were from Amersham Buchler (Braunschweig, FRG). BioluteS and Quicksint 501 were purchased from Zinsser Analytic (Frankfurt. FRG). All other substances were obtained from commercial sources at the highest purity available. The LTD4/ LTE4 receptor antagonist (1S,2R)-5-{3-[l-hydroxy-15,15,15trifluoro - 2 - (2 - 1H-tetrazol- 5 - ylethyl- thio)pentadeca - 3(E), S(Z)-dienyll-pheny1)-1H-tetrazol (LY 245769) was kindly provided by Eli Lilly (Lilly Research Center, Earl Wood Manor, Windlesham, UK). Animals and hepatoma cells Sprague-Dawley rats (Zentralinstitut fur Versuchstierzuchtung, Hannover, FRG) weighed 250 - 300 g and were maintained on a standard diet (Altromin 300(@,Altromin, Lage, FRG). Hep G2 hepatoma cells and the AS-30D ascites hepatoma cells were from the tumor cell collection of the Deutsches Krebsforschungszentrum, Heidelberg. Cell isolation and culture Hepatocytes from male Sprague-Dawley rats were isolated by recirculating collagenase perfusion [31]. Adult human hepatocytes were prepared from surgical liver biopsies [32]. After washing, the cells were stored on ice in modified Krebs/ Henseleit bicarbonate buffer containing 118 mM NaCl, 4.7 mM KCI. 0.6 mM KH2P04, 0.6 mM Na2HP04, 25 mM NaHC03, 5.5 mM glucose, 1.2 mM MgC12 and 1.3 mM CaC12 (pH 7.4). The viability of the rat and human hepatocytes was > 85% and 6O%, respectively, as judged from trypan blue exclusion. The contamination with nonparenchymal cells was less than 2% as determined by light microscopy. For metabolism studies with hepatocyte cultures, the cells were plated on 35-mm tissue-culture dishes (1.5 x lo6 cells/dish) in 2 ml Ham's F 12 medium (Boehringer Mannheim, Mannheim, FRG) containing 10% fetal bovine serum and 20 Ujl insulin. Cultures were mslintained at 37 'C in the presence of 95% air and 5% C 0 2 ;4 h after plating, the culture medium was changed to the Krebs/Henseleit incubation buffer (500 pl/dish). The transplantable AS-30D ascites hepatoma cell line [33] was carried in female Sprague-Dawley rats. The tumor cells were transplanted at weekly intervals by intraperitoneal injection of 0.5 ml ascitcs fluid. For uptake measurements, tumor cells were collected, washed and suspended in the incubation buffer. Hep G2 hepatoma cells were plated on 35-mm culture tissues in 2 ml Dulbecco's minimum essential medium containing 10% fetal bovine serum, 100 IU/ml penicillin and 100 pg/ml streptomycin and maintained at 37°C in the presence of 95% air and 5% C02. After 24 h the hepatoma cell cultures reprehented confluent monolayers and the culture medium was changed to the incubation buffer. Leukotriene metabolism in hepatocytes and Hep 6 2 hepatoma cells Human and rat hepatocyte suspensions (3 x lo9 cells/l) were incubated with the ['H2]leukotrienes (10 nM) in incu-

bation buffer at 37°C in the presence of 95% air and 5% COz. At the indicated times, the incubations were stopped by addition of 4 vol. ice-cold methanol containing 1 mM 4hydroxy-2,2,6,6-tetramethylpiperidine-l-oxyl. Hep G2 hepatoma cells in culture were incubated with [3H2]LTB4or ['H2]LTE4 (10 nM final concentration) for 30 min at 37'C in the presence of 95% air and 5% COz. After this, the supernatants were removed and the cells washed twice with incubation buffer. For deproteinization, 2 ml ice-cold methanol containing 1 mM 4-hydroxy-2,2,6,6-tetramethyl-piperidine-loxyl was added to the cells and supernatants. Separation of leukotriene metabolites Precipitated proteins from samples containing 80% niethanol were removed by centrifugation. The samples were dried, redissolved in 30% aqueous methanol and analysed by reverse-phase HPLC in a system containing 100% water, switching at 5 min to a linear gradient of 40-80% aqueous methanol within 35 min, followed by 10 min of 80% aqueous methanol and a linear gradient of 80- 100% aqueous methanol within 5 min, and additionally by 20 inin of 100% methanol. All solvents contained 0.1YOacetic acid and the pH was adjusted to 5.0 with ammonium hydroxide. Retention times of the standards are indicated in the figures by arrows. Measurement of leukotriene uptake by isolated hepatocytes and hepatoma cells Hepatocytes and AS-30D hepatoma cells in suspension were preincubated for 15 min in incubation buffer at 37°C. In thc case of incubations for determining the temperature dependence, cells were preincubated at 4"C, 10°C and 20°C. Leukotrienes were diluted in 100 pl incubation buffer (final concentration 10 nM) and the uptake initiated by adding the cell suspension (lo6 cells/900 PI). In the case of inhibition studies, the inhibitor was added prior to the cell suspension, As potential inhibitors the organic anions, tetrabromosulfophthalein, S-decyl glutathione, 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS), probenecid, hexadecanedioate, docosanedioate, cholate, taurocholate, the amphiphilic cations N-propyl ajmaline and verapamil, and the neutral glycosides digitoxin and ouabain were used. When hexadecanedioate or docosanedioate were used, the incubation buffer contained, in addition, 3.7 pM fatty-acid-free bovine serum albumin. In the case of studies for testing the sodium dependence, NaCl was exchanged by choline chloride and all other sodium salts by the respective potassium salt. In order to evaluate the influence of chloride ions on leukotriene uptake, aliquots of the cell suspensions were centrifuged prior to incubation and resuspended in a buffer containing 135 mM NaCI, 1.2 mM MgC12, 0.8 mM MgS04, 27.8 mM glucose, 2.5 mM CaCI,, and 25 mM Hepes pH 7.4, or the corresponding gluconate salts instead of chloride [34]. Aliquots of 100 pl were taken every 15 s for 2 min and uptake rates were determined using a rapid filtration procedure. The radioactivity associated with the cell pellet was determined with a liquid scintillation counter after the cell pellets had been lysed by Biolute-S. Aliquots of the remaining suspensions were used for protein determination. Leukotriene uptake by Hep G2 cell cultures was initiated by adding the leukotrienes (final concentration 10 nM) and, in the case of inhibition studies, simultaneously the inhibitor. When palmitate was added, the incubation buffer contained 3.7 pM bovine serum albumin (fatty-acid-free) in addition. After incubation, the buffer was

283 I

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INCUBATION TIMES (s)

Fig. 1. Comparison of the time-dependent uptake of [3H]leukotrienesby freshly isolated rat hepatocytes, AS30D hepatoma cells in suspension, and Hcp G2 hepatoma cells in culture. Cells were prepared as described under Experimental Procedures. Hepatocytes ( 0 )and AS-30D cells (0)were incubated for 2 min at 3?"C with 10 nM [3H2]LTC4,[3HZ]LTD4,[3H2]LTE4or [3H2]LTB4(as indicated, from left to right). Aliquots of 100 PI were taken at intervals of 15 s. The cells were separated and the initial uptake rates were calculated from the cellular radioactivity. Hep G2 cultures (M)were incubated with 10 nM [3H2]LTE4or [3Hz]LTB4for 15. 30,60, and 120 s. At the indicated time points, incubations were stopped, the cells washed three times with incubation buffer and the cellular radioactivity was determined. The curves present values from one cell preparation with duplicate determinations.

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RETENTION TIMES ( m i d Fig. 2. LTB4 metabolites formed in short-term incubations with hepatocytes. Freshly isolated rat hepatocytes were incubatcd with [3HZ]LTB4(10 nM) under the experimental conditions of uptake experiments for 30 s (lop) or 120 s (bottom). Mctabolites were separated by reverse-phasc HPLC as described under Experimental Procedures. Thc retention times of the synthetic standards are indicated by arrows.

removed, the cells were washed several times and lysed by 1 M NaOH with 0.5% SDS. Aliquots of the lysate were used for the determination of the radioactivity and the protein concentration. For determination of the initial uptake rates, the differences of the values at 1 min and at 15 s were used. Protein concentrations were dctermined by a modified biuret method using bovine serum albumin as a standard [35].

Preparation of plasma membrane vesicles from rat liver and measurement of leukotriene and taurocholate uptake by plasma membrane vesicles

Plasma membrane vesicles were prepared and characterized as described [36, 371. The protein concentration was 5.5 mg/ml buffer (250 mM sucrose and 10 mM Tris/HCl pH 7.4) Uptake studies for testing the sodium dependence were carried out at 37 "C and started by adding [3H2]LTE4 (10 nM), [3H2]LTB4(10 nM), or [3H]taurocholate (4.4 pM), to the incubation medium (85 pl: 180 mM sucrose, 0.2 mM CaC12, 10 mM MgC12, and 10 mM TrisjHepes adjusted to pH 7.4, and 100 mM NaCl or KCI). Previously the medium was preincubated at 37 "C with 20 ~1 of the vesicle suspension for about 5 min. Aliquots of 20 p1 were taken at 0, 10, 15, 30, and 60 s. The eflect of K' on the uptake of LTE4 and LTB4 into membrane vesicles was investigated according to [38]. Vesicle suspensions (20 1.11) were preincubated at 37 "C with 3.3 p1 valinomycin solution (10 pg/mg protein in 0.3% ethanol) or ethanol alone for 2 min followed by addition or t3H2]LTE4or t3H2]LTB4(10 nM) in 85.7 pl incubation buffer (100 mM KCI, 180 m M sucrose, 0.2 mM CaCI2, 10 mM MgCI2, 10 mM Tris/Hepes, adjusted to pH 7.4, at 37°C). Aliquots of 20 p1 were taken every 5 s, 30 s, and at 0, 1,2, 5 , and 10 rnin during total incubation periods of 20 s, 2 m n , and 10 min, respectively. Radioactivity incorporated into the vesicles was measured by rapid filtration using Millipore membrane filters (0.22 pm, type GSWP, Millipore, Bedford, MA). Photoaffinity labeling with leukotrienes

Freshly isolated human hepatocytes (10' cells, 1.6 mg protein) were incubated with 110 kBq [3H]LTE4and [3H]LTR4 (final concentrations 230 nM and 300 nM, respectively) a1 37°C for 2 min. After incubation, the samples were shockfrozen by transfer into quartz test tubes precooled in liquid nitrogen. The frozen samples were irradiated at 300 nm for

284

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0.1

- 0.05

Table 1. Kinetic parameters for hepatocellular uptake of leukotricncs. Cell$ were preincubated at 37°C for 15 rnin and incubated with [3HZ]LTC4,[31-I,]LT,TD4, I3HZ]LTE4.and [3H2]LTB, in a range of 5 -200 nM. Relativc uptake rates were calculated from the velocity determined between 15 -- 60 s as described under Experimental Procedures. The kinetic parametcrs were calculated graphically by Lineweaver-Burk plots. Mean values from two determinations except for LTE4 (n = 6 , mean valueb f SD). Leukotriene

LTB4 LTC4 LTD, LTE4

Km

v,,,

nM

pmol x mg-' x min-'

115

6.4 12.0 10.0 7.4 f 4

200 150 109 & 31

0

0

30

60

0

30

60

INCUBATION TIMES (s)

Fig. 3. Na+ dependencc of taurocholate and LTE4 transport by hepatocyte plasma membrane vesicles. Membrane suspension (20 pl, 110 pg protein) was preincubatcd at 37°C in Tris/NaCl (@) or ?'ris/ KCl ( W ) buffer for 5 min as described under Experimental Procedures. The transport was started by addition of ['H*]LTE4 (10 nM) (right) or ['H]taurocholate (4.4 pM) (left). Aliquots were taken at the times indicated and the membrane-associated radioactivity determined. Similar rcsults were obtaincd in three separate cxperiments.

-

0.20

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a

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L

heated at 95°C for 5 min and centrifuged at 16000 x g for 5 min in order to remove non-soluble particles. Aliquots of each supernatant (200 pg protein) were used for SDS gel electrophoresis using an 8 - 20% polyacrylamide gradient 121, 391. Hep G2 hepatoma cells in culture (1.5 x lo6 cells/dish) were used for labeling 24 h after plating. For photoaffinity labeling with ['H]LTE4 and ['H]LTB4 (final concentrations 140 nM and 180 nM, respectively) the culture medium was removed and the cells washed three times with incubation buffer. Incubations were performed at room temperature for 2 min. Irradiation was performed for 5 min at 300 nm. Incubation buffer was removed, the cells scraped off and suspended in 500 p1 buffer A. Sample preparation for electrophoresis was as described above.

Q

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RESULTS Leukotriene uptake into rat hepatocytes

Y

Uptake of ['H2]leukotrienes into freshly isolated rat hepatocytes was studied during short-term incubations = 0.10 (Fig. 1). LTE4 and LTB4 showed the highest uptake velocities 0 30 6 0 0 30 6 0 compared to the other leukotrienes as indicated by the relative uptake rates: LTE4 (155+47%) > LTD4 (129*24%) > INCUBATION TIMES (s) LTC4 (100+12°/~) (mean values SD, n = 10, with cells Fig.4. Comparison of leukotriene uptake by freshly isolated rat hepatocytes in the presence or absence of chloride in the incubation from two separate cell preparations). The uptake rate of LTB4 medium. Hepatocytes, suspended either in a buffer containing chloride (1 59 45%) was comparable to that of LTE4. The uptake rates for LTE4 and LTB4 were significantly higher than those ( 0; CL-) or gluconatc (A,Glue-) (as described under Experimental Procedures), were incubated for 1 rnin at 37°C with 10 nM [3H2]LTE4 for LTD4 and LTC4 ( P < 0.05). The uptake rates for LTE4 (left) or [3H2]LTB4(right). Aliquots were taken at the times indicated. and LTD4 differed only by P < 0.1. Mean valucs from duplicate determinations, deviation I 10Oh. During the short-term incubations of freshly isolated rat hepatocytes with [3Hz]LTE4and [3Hz]LTB4only a negligible extent of metabolism was observed reaching a maximum of 5 min, whereby the test tubes were cooled in liquid nitrogen 7 % for both leukotrienes within 2 rnin. The limited extent of every 30 s 1211. Subsequent to photolysis, the samples were [3Hz]LTB4w-oxidation is shown in Fig. 2 . The initial rates of thawed and diluted with a buffer containing 10 mM Tris/HCl uptake were calculated from the slope of the uptake curvc pH 7.4, with 0.5 mM phenylmethylsulfonyl fluoride, 5 mM within the first 60 s. The temperature dependence of the uptake of [3H2]LTE4 EDTA, 1 mM leupeptin, 1 mM benzamidine (buffer A) to a total volume of 1 ml and stored for 1 h on ice. The particulate and [3Hz]LTB4by freshly isolated rat hepatocytes was comfractions were collected by centrifugation at 100000 x g for pared. A reduction of the incubation temperature from 37'C 30 min. The resulting pellets were resuspended in buffer A and to 24°C decreased the initial velocity of LTE4 and LTB4 upcentrifuged at 16000 x g for 30 min at 4°C before solubiliza- take to 45% and 53%, respectively. At I O T , a decrease to tion in buffer B (62.5 mM Tris/HCl pH 6.8) containing 10% 32% and 34% was detected for the LTE4 and LTB4 uptake, glycerol, 5% SDS, 5% 2-mercaptoethanol, and 0.001% bro- respectively, when compared to 37'C. At 4°C no significant mophenol blue. The supernatants were concentrated and the transport was determined with LTE4, whereas with LTB, a protein concentrations were adjusted to 3 g/l. Samples were low increase in cell- associated radioactivity was detected.

F a

+

+

285 Table 2. Inhibition of the hepatocellular uptake of LTE4 and LTB4 by several arnphiphilic compounds. Hepatocyte supensions were incubated with 10 nM ['HILTE, or [3H]LTB4at37°C. The concentration ofeach inhibitor was 100 pM. Relative uptake rates were calculated from the initial velocity determined between 15-60 s of incubation as described under Experimental Procedures. Data represent the percentage inhibition of the initial uptake rate relative to control (mean values f SD; n = 6 from three separate cell preparations with duplicate detcrmination). The uptake velocities of controls amounted to 0.58f0.24 pmolxmg protein-Ixmin-' and 0.77f0.32 pmol (mean values f SD, n = 32 from 14 separx mg protein x min ate experiments) for LTE4 and LTB4, respcctivcly. Thc corrcsponding leukotriene uptakc rates in controls were in the range of 0.31.3 pmoljmg protein and 0.34- 1.4 pmol/mg protein for LTE4 and LTB4, respectively. Inhibitor

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BSP

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S-DECYL-GSH

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Inhibition of uptake of

IC, 0C I,

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Anionic Bromosulfophthalein S-Decyl glutathione DIDS Probenccid Hcxadecanedioate Docosanedioate Cholate Taurocholatc

90+ 2 73+ 9 61 f 16 47 16 35 13 2 3 f 10 < 10

< 10

79* 9 72+ 8 48 f 16 43f11 40+15 46+31 63 13 < 10

Basic N-Propyl ajmalin Verapamil

< 10 < 10

< 10 < 10

Neutral Digitoxin Ouabain

< 10 < 20

57+ 16 < 10

+

*

The role of the sodium gradient as a possible driving force for leukotriene uptake was investigated in suspensions of freshly isolated rat hepatocytes as well as by use of hepatocyte plasma membrane vesicles. Substitution of sodium by choline ions had no effect on the uptake of cysteinyl leukotrienes and LTB4 into isolated hepatocytes. The relative rates of uptake determined in these experiments were 117f 11%, 96f 12%, 112*15% and 104f28% (2 SD, n = 4) for 1 0 n M [3Hz]LTC4,[3H2]LTD4,[3H2JLTE4,and [3H2]LTB,, respectively, expressed as a percentage of the sodium control. As a control for a sodium-dependent uptake, hepatocyte plasma membrane vesicles were incubated with [3H]taurocholate. In the presence of the Tris/NaCl buffer the characteristic overshoot of taurocholate uptake was determined with a maximum at 10 s. In the presence of the Tris/KCl buffer only 29&6% ( fSD, IZ = 3, using vesicles from two separate preparations) of the uptake determined in the presence of NaCl was determined (Fig. 3 ) . However, no significant uptake of [3H2]LTE4 and [3H2]LTB4was observed in these vesicles with either incubation buffer (Fig. 3). A K + diffusion potential was excluded as a driving force for leukotriene uptake by incubating [3Hz]leukotrienes with membrane vesicles preincubated for 10 min with valinomycin. No initial increase of membraneassociated radioactivity was detected during short-term incubations up to 2 min, nor a gradual increase within 10 min. A chloride activation of the hepatocellular uptake, as described for cholate [40] and BSP [34, 41, 421 with a decrease of the uptake of these substances into hepatocytes in the absence of

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25 50 INHIBITOR (uM)

-

8 LIM 11 UM I

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75

100

Fig. 5. Interference of bromosulfophthalein (BSP)and S-decyl glutathione (S-decyl-GSH) of the hepatocellular uptake of leukotrienes. Freshly isolated rat hepatocytcs were incubated at 37°C with 1 0 n M [3H2]LTE4(top) and [3H2]LTB4(bottom) in the presence of BSP (m) or S-decyl-glutathione ( 0 ) .Aliquots were taken after 15, 30,45, and 60 s. The cells were separated, and the initial uptake rates were calculated from the cellular radioactivity. Data for each inhibitor concentration represent percentage inhibition of uptake as compared to controls in the absence of inhbitor. The IC50values were defined as the concentrations at which the remaining uptake rates amounted to 50% of thc controls. Mean values & SD, n = 4.

chloride, was not observed for [3H2]LTE4or ['H2]LTB4, as shown by chloride replacement by gluconate in the medium (Fig. 4). The concentration dependence of leukotriene uptake by freshly isolated rat hepatocytes, determined with LTC,, LTD4, LTE,, and LTB, in the range from 8 - 200 nM, showed a nonlinear relation between 1/Vand [l/S]. Apparent rC, values for the low-K, system were approximated from the slope of the curve in the low-concentration range after substraction of the non-saturable part (Table 1). In addition, kinetic studies with LTE, and LTB, were performed in a higher concentration range (25 - 1250 nM). Even with these substrate concentrations, the uptake was not saturable. The substrate specifity of leukotriene uptake was studied by the interactions between different leukotrienes. No mutual uptake inhibition occurred between cysteinyl leukotrienes and LTB, in a concentration excess up to 1000-fold. An inhibition of the initial [3H2]LTE4 uptake (10nM) of 60+13%, 69+12%,67f380/u,and63+_23%(meanvalues +_ SD;n = 4 from two separate cell preparations) was achieved by LTC4, LTD4, LTE4, and LTB,, respectively, at a 5000-fold higher concentration. An analogous inhibition was not seen for [3H2]LTB4uptake. In addition, the effect of different substrates of hepatocellular uptake systems at a concentration of 100 pM on the uptake of 10 nM [3H2]LTE4and [3H2JLTB, was studied at 37°C (Table 2). No inhibition was found by the amphiphilic basic compounds verapamil and N-propyl ajmaline, nor by the glycoside ouabain. In the case of digitoxin, no inhibition of the LTE, uptake was observed but

286 .

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HUMAN HEPATOCYTES

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TOTAL INCUBATE^

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HUMAN HEPATOCYTES

[TOTAL INCUBATE] 12

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RETENTION TIMES (rnin)

Fig. 6. Metabolism of LTE4 and LTB4 by human hepatocytes and Hep G2 hepatoma cells. Cells were prepared and incubated with [3H2]LTE4 or [3H2]LTB4(10 nM) for 30 min at 37°C as described under Experimenlal Procedures. Leukolrienes were separated by reverse-phase HPLC. (A, D) Metabolites of LTE4 (A) and LTB4 (D), after incubation with human hcpatocytes. (B, E) Supernatants of the Hep G2 cell incubations with LTEl (B) and LTB4 (E). (F) LTB4 metabolites in the hepatoma cells. (C) Total incubate of the LTE4 incubation with the hepatoma cells. The retention times of the synthetic standards are indicated by arrows

a 60% inhibition of LTB4 uptake. Among the organic anions, taurocholate was without effect on both LTE4 and LTB4 uptake. BSP, S-decyl glutathione, DIDS, probenecid, hexadecanedioate, and docosanedioate significantly inhibited the initial uptake of LTB4 and LTE4 (Table 2). values for the uptake inhibition by S-decyl glutathione and BSP were 16 pM and about 2 pM with LTE4 and 11 pM and 8 pM with LTB4, respectively, as substrate (Fig. 5). Leukotriene uptake by AS30D rat hepatoma cell and human Hep G2 hepatoma cells in culture Uptake of cysteinyl leukotrienes by AS-30 D and Hep G2 hepatoma cells was absent under initial-rate conditions at 37°C. however, both cell lines showed significant uptake of LTR4 under the same conditions (Fig. 1). The uptake rates were 0.22k0.13 pmol x mg protein-' xmin-I (mean f SD, n = 6) and 0.30 f0.14 pmol x mg protein- x min- (mean SD, n = 10) for AS-30 D ascites cells and Hep G2 hepatoma cells, respectively. The LTB4 uptake by Hep G2 hepatoma cells was not inhibited by BSP or palmitate up to 10000fold excess in concentration. The pattern of metabolites derived from [3H2]LTB4in Hep G2 hepatoma cells (Fig. 6 E, F) was different from that observed in human hepatocytes (Fig. 6 D), and the degradation of LTB4 was not inhibited by ethanol (50 mM) or LY 245769 (50 pM) which are effective inhibitors of leukotrienc o-oxidation in hepatocytes [43, 441.

Photoaffinity labeling Freshly isolated human hepatocytes were used for direct photoaffinity labeling with [3H2]LTE4and [3H2]LTB4.With both leukotrienes a similar labeling pattern was obtained (Fig. 7). Proteins with molecular masses of 68, 48-50 and 38 -40 kDa were significantly labeled in the particulate fraction (A, C). In the cytosolic fraction leukotriene-binding proteins with apparent molecular masses of 60, 50 and 3840 kDa were significantly labeled (Fig. 7 B, D). Photoaffinity labeling of Hep G2 hepatoma cells with ['HILTE, indicated no leukotriene-binding proteins in the cytosolic nor in the particulate fraction (Fig. 8 C, D). With [3H2]LTB4,however, proteins with apparent molecular masses of 67, 48, 40 and 30 kDa were labeled in the particulate fraction (Fig. 8 A). In the cytosolic fraction of these hepatoma cells a 40-kDa protein was labeled (Fig. X B).

DISCUSSI 0N Using kinetic and competition experiments (Table 2), we provide evidence in this study for the existence of distinct uptake systems for cysteinyl leukotrienes and LTB4 at the sinusoidal membrane of hepatocytes. This result is in line with the original proposal by Uehara et al. [18]. Because of the rapid leukotriene metabolism and the transport of metabolites

287

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out of the cells into the incubation medium [45], we determined uptake rates within the initial 60 s of incubation. The driving force for the uptake of both types of leukotriene has not been fully elucidated in our uptake studies. In earlier work a decrease in cysteinyl leukotriene uptake by hepatocytes was detected after ATP depletion induced by carbonyl cyanide p-trifluoromethoxy-phenylhydrazide (FCCP) [17,18]. LTC4 uptake by the isolated perfused rat liver was decreased in the absence of sodium ions [19]. However, a sodium dependence of the cysteinyl leukotriene or of LTB4 uptake was not detected in our experiments with isolated rat hepatocytes. In addition, plasma membrane vesicles were used to analyze the driving force for the leukotriene uptake. Measurement of sodium-dependent taurocholate uptake served as a positive control of this system, since taurocholate transport into hepatocytes had been established as a process predominantly dependent on the presence of sodium. However, for leukotriene uptake neither sodium nor a potassiumdiffusion potential were effective as driving forces (Fig. 3). The driving force for leukotriene uptake, e.g. binding to intracellular proteins, was absent in the experimental system using plasma membrane vesicles. Based on these negative results, one may conclude that leukotriene uptake is not mediated by a sodium-dependent cotransport nor by a potassium-diffusion potential. The kinetic studies indicated the involvement of a low-K, system and a non-saturable system for the initial uptake of both types ofleukotrienes under the conditions used (Table 1). With respect to the physiological leukotriene concentrations, which are in the nanomolar and subnanomolar range [2, 31, only the Iow-K, system may be the biologically relevant one. This system has a high affinity for the leukotrienes as indicated by the relatively high inhibitor concentrations required for inhibition of uptake-(Table 2).

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MIGRATION DISTANCE ( cm Fig. 8. Photoaffinity labeling of human Hep G2 hepatoma cells with (3HJleukotrienes.Distribution of radioactivity after SDS/PAGE of the particulate (A, C) and cytosolic (B, D) fractions. Hep Ci2 cultures werc incubated with [3FT,]LTE4 (C, D) and [3H2]LTB4(A, B) (1 10 kBy) for 2 min. Irradiation was performed for 5 min at 300 nm. lncubatjon buffer was removed and cells scraped off. Particulate and cytosolic fractions

were prcpared as described undcr Experimental Procedures.

288 LTB4 caused significant inhibition of LTE, uptake in our studies, as opposed to earlier work [18]. However, no inhibition of LTB, uptake was caused by the cysteinyl leukotrienes. The identical lipophilic domain between C13 and C20 ofthe leukotriene molecules could allow LTB4 to interact with the cysteinyl leukotriene uptake system and to inhibit the LTE4 uptake. In contrast, the polar amino acid residue of LTE, may prevent its interaction with the LTB4 transport system. Alternatively, LTB, might inhibit the uptake in a more specific manner. Different organic amphiphilic anions interact with the hepatocellular uptake of LTE4 and LTB, as shown in competition studies (Table 2). In contrast, taurocholate was without inhibitory action. The hepatocellular uptake of taurocholate itself occurs by sodium-dependent as well as by sodium-independent transport systems [46, 471 although the latter only accounts for a minor part. Further indications for an involvement of different transport systems for uptake of LTE, and LTB, were obtained by the cholate and digitoxin inhibition (Table 2). Both were without effect on LTE, uptake but significantly inhibited LTB, transport. For digitoxin also a strong inhibition of the cholate uptake by hepatocytes has been shown [48]. In contrast to taurocholate, cholate is taken up by hepatocytes predominantly by a sodium-independent transport system [49]. Ouabain, another neutral compound tested, did not affect LTE, uptake nor the transport of LTB4. Earlier work showed that ouabain also does not inhibit cholate uptake [50],but uptake of ouabain into liver cells was inhibited by cholate [48, 511 as well as taurocholate [48, 51, 521. Additional differences of the LTE, and LTB4 uptake process were seen in the AS-30D rat hepatoma cells and the human hepatoma cell line Hep G2. These cells were deficient in the uptake of cysteinyl leukotrienes but showed accumulation of LTB4 (Fig. 1). The latter was not due to binding to the cell membrane. In Hep G2 cells a conversion of LTB4 to intracellular metabolites was shown (Fig. 6F). These were not identical with known o-oxidation and fi- oxidation products generated in hepatocytes, since they showed longer HPLC retention times (Fig. 6) and their formation was not diminished by inhibitors of the leukotriene o-oxidation. Comparative photoaffinity labeling studies with human hepatocytes (Fig. 7) and Hep G2 hepatoma cells (Fig. 8) using the photolabile [3H2]LTE4and [3H2]L.TB4[21] support our conclusion that LTB, is taken up by hepatoma cells, based both on the uptake and the metabolism data. The labeled 40kDa protein in the cytosolic fraction of Hep G2 hepatoma cells provides additional evidence for LTB4 uptake by these cells. The labeling studies with [3H2]LTE4 indicated that labeling can occur only from the intracellular side since leukotriene-binding proteins were not detected in the particulate nor in the cytosolic fraction of Hep G2 cells. The process responsible for LTB, uptake by hepatoma cells seems to be different from the above-mentioned low-K,,, system because of the lack of inhibition by BSP. Because of this and because of the lipophilic structure of the LTB, molecule, its uptake by simple diffusion cannot be excluded. This process may contribute to the uptake by hepatocytes since LTB4 is taken up slowly even at 4°C. Studies on BSP uptake also revealed differences between its transport into hepatocytes and hepatoma cells [33, 41, 531. In hepatocytes, uptake systems with different affinities exist, whereas in hepatoma cells only the low-affinity system is active. Several groups described polypeptides with an apparent molecular mass of 55 kDa. uossiblv involved in the RSP uptake by hepatocytes and hepatoma cells [54,55].Photoafinit; labeling ofpolypep-

tides in this size range was not observed after incubation of Hep G2 cells with [3Hz]LTB4(Fig. 8). The data of this study show that the cysteinyl leukotrienes and LTB4 are taken up by different mechanisms. Some compounds, however, inhibit both transport processes. In addition, evidence is provided that LTB,, in contrast to cysteinyl leukotrienes, is taken up by hepatoma cells and degraded intracellulary. We are indebted to Dr. J. R. Boot, Lilly Research Centre Ltd, Eli Lilly and Company (Erl Wood Manor, Windlesham, UK) for providing the LTDA/LTE4 receptor antagonist LY 245769 as an inhibitor of leukotrienc a-oxidation [44]. We thank Dr. Michael Huber for his helpful support, Ulrike Rerger for help with SDSjPAGE analysis, and Dr. Toshihisa Tshikawa and Cornelia Klunemann for their support in the preparation of hepatocyte plasma membranes and in vesicle transport studies. Part of this work was supported by thc Deutsche Forschungsgemeinschu~~ through SFB 352, Heidelberg.

REFERENCES 1. Hammarstrom, S., Bernstrom, K., Orning, L., Dahlen, S.-E., Hedqvist, P., Smedegard, G. & Reveals, B. (1981) Biochem. Biophys. Res. Commun. 101. 1109-1115. 2. Denzlinger, C., Rapp, S., Hagmann, W. & Keppler, D. (1985) Science 230. 330 - 332. 3. Denzlinger, C., Guhlmann, A,. Scheuber, P. IT., Wilker, D., Hanimer, D. K. & Keppler, D. (1986) J . Biol. Ckem. 261, 35601 15606. 4. Huber, M., Guhlmann, A., Jansen, P. L. M. & Keppler, D. (1987) Hepatology 7, 224 -227. 5. Huber, M. & Keppler, D. (1987) W r . J. Riochem. 167, 73 -79. 6. Appelgren, L.-E. & Hammarstrom, S. (1982) J . Bid. Clzem. 257, 531 -535. 7. Hagmann, W., Denzlinger, C. & Keppler, D. (1984) Circ. Shock 14.223-235. 8. Hagmann, W., Denzlinger. C. & Keppler, D. (1985) FEBS Letr. 180, 309-3313, 9. Hagmann, W., Dcnzlinger, C., Rapp, S., Weckbecker., G. & Keppler, D. (1986) Prostaghndins 31,239 - 251. 10. Fostcr, A,, Fitzsimmons, B., Rockach, J. & Letts, L. G. (1987) Biochim. Biophys. Actu 921,486-493. 11. Hagmann, W. & Korte, M. (1990) Biochem. J. 267,467-470. 12. Hansson, G., Lhdgren, J. A., DablCn, J. E., Hedquist, P. & Sarnuelsson, B. (1981) FEBS Lett. 130, 107- 112. 13. Shak, S. &Goldstein, I. A. (1985) 9. Clin. Invest. 76, 1218-1228. 14. Soberman, R. J., Sutyak, J. P., Okita, R. T., Wendelborn, D. F., Roberts, L. J. & Austen, K. F. (1988) J. Bid. Chem. 263,7996 8002. 15. Gotoh, Y., Sumimoto, H. & Minakami, S. (1989) Eur. J . Biochem. 179, 315-321. 16. Serafin, W. E., Oates, J. A. & Hubbard, W. C. (1984) Pvostugkundins27, 899-911. 17. Ormstad, K., Uehara, N., Orrenius, S., Orning, L. & Hammarstrom, S. (1982) Biochem. Biophys. Res. Commun. 104, 1434-1441. 18. Uehara, N., Ormstad, K., Orning, L. & Hammdrstrom, s. (1983) Biochim. Biophys. Acta 732, 69- 74. 19. Wettstein, M., Gerok, W. & Haussinger, D. (1990) Eur. J. Biochem. 191,251-255. 20. Wcckbccker, G . & Keppler, D. (1986) Eur. J Bioclzem. 154, 559562. 21. Falk, E., Muller, M.. Huber, M., Keppler, D. & Kurz, G . (1989) Eur. J . Biochem. 186,741 - 747. 22. Muller, M., Falk, E., Sandbrink, R., Berger, U., Leier, I., Jedlitschky, G., Huber, M., Kurz, G. & Keppler. D. (1990) Adv. Prostaglandin Tlzromboxanc Leukotriene Res. 21, 395 - 398. 23. Koizumi, K., Tamiya-Koizumi, K., Fujii, T., Okuda, J. & Kohima, K . (1980) Cuncer Res. 40, 909-913. 24. Hixson. D. C., Allison, J. P., Chesner, J. E., Leger, M. J., Ridge, L. L. & Walborg, E. F. jr. (1983) Cuncer Rcs. 43, 3874-33884.

289 25. Hixson, D. C., McEntire, K. D. & Obring, B. (1985) Cancer Res. 45, 3742-3749. 26. Becker, A,. Neumeier, R., Heidrich, C., Loch, N., Hartel, S . & Rcuttcr, W. (1986) Biol. Chem. Hoppe-Seyler 367, 681 -688. 27. Kroker, R., Anwer, M. S. & Hegner, D. (1978) NazmnynSchmiedeberg’s Arch. Pharmacol. 303, 299 - 301. 28. von Dippe, P. & Levy. D. (1983) J . B i d Chem. 258,8896 - 8901. 29. Frimmer, M., Petzinger, E., Rufeger, U . & Veil, L. B. (1977) Naunyn-Schmiedeberg’s Arch. Pharmacol. 301,145- 247. 30. Grundmann, E., Petzinger, E., Frimmer, M. & Boschek, C. B. (1978) Nuz~n~M-Schmiedeberg’s Arch. Pharmacol. 305, 253 259. 31. Berry, M. N. & Friend, D. S. (1969) J . Cell Biol. 43, 506-529. 32. Huber, M., Miiller, J., Leier, I., Jedlitschky, G., Ball, H. A,, Moore. K., Taylor, G. W.. Williams, R . & Keppler, D. (1990) EUP.J . Biochem. 194, 309-315. 3 3 . Smith, D. F., Walborg, E. F. Jr. & Chang, J. P. (1970) Cancer Res. 30, 705-711. 34. Min, A. D., Johansen, K. L., Campbell, G. & Wolkoff, A. W. (1991) J . Clin. Invest. 87, 1496- 1502. 35. Hiibscher, G., West, G. R. & Brindley, D. N. (1965) Biochem. J . 97,629-642. 36. Meier, P. J. & Boyer, J. L. (1990) Meth0d.s Enzymol. f92, 53454s. 37. Ishikawa, T., Mulfer, M., Kliinemann, C., Schaub. T. & Keppler, D. (1990) J. Biol.Chem. 265,19279-19286. 38. Kobayashi, K., Sogame, Y., Hard, H. & Hayashi, K. (1990) J . Biol. Chem. 265,7731 - 7’741. 39. Llimmli, U . K. (1970) Nature (Lond.) 227,680-685. 40. Pelzinger, E. & Frimmer, M. (1 984) Biochim. Biophys. Acta 778, 539 - 548. 41. Wolkoff, A. W., Samuelson, A. C., Johansen, K. L. Nakata, R., Withers. D. M. & Sosiak, A. (1987) J . Clin. Invest. 7Y, 12591268.

42. Jacquemin, E., Hagenbuch, B., Stieger, B., Wolkoff, A. W. & Meier, P. J. (1991) J . Clin. Invest. 88,2146-2149. 43. Baumert, T., Huber, M., Mayer, D. & Keppler, D. (1989) Eur. J . Biochem. 182, 223 - 229. 44. Jedlitschky, G., Leier, I., Huber, M., Mayer, D. & Keppler, D. (1990) Arch. Biochem. Biophys. 282, 333 -339. 45. Keppler, D., Miiller, M., Klunemann, C., Guhlmann, A., Krauss, K., Miiller, J.; Berger, U., Leier, T. & Mayatepek, E. (1992) Adv. Enzyme Regul. 32, 107-116. 46. Anwer, M. S . & Hegner, D. (1978) Hoppe-Seyler’s Z . Physiol. Chern. 359,181 192. 47. Anwer, M. S . & Hegner, D. (1978) Hoppe-Seyler’s 2. Physiol. Chem. 359,1027 - 1030. 48. Pctzingcr, E.: Fischer, K . & Fasold, H. (1986) in: Cardiac ,&cosidPs 1785-1985 (E. Erdmann, K. Greef & J. C. Skou, eds) pp. 297- 304, Steinkopff Verlag, Darmstadt. 49. Van Dyke, R. W., Stephens, J. E. & Scharschmidt, B. F. (1982) Am. J . Physiol. 243,484-492. 50. Anwer, M. S., Kroker, R. & Hegner, D. (1976) Hoppe-Seyler’s 2. Physiol. Chem. 357,1471- 1486. 51. Eaton D. L. & Klaassen, C. D. (1978) J . Pharmacol. Exp. Ther. 204,480-488. 52. Schwenk, M., Wiedemann. T. & Remmer, H. (1981) NaunynSchrniedeherg’.sArch. Pharmacol. 316, 340 - 344. 53. Min, A. D., Goeser, T., Liu, R., Campbell, C. G., Novikoff, P. M. & Wolkoff, A. W. (1991) Hepatology 14,1217-1223. 54. Wolkoff, A. W. &Chung, C. T. (1980) J . Clin. Invest. 65, 11521161. 55. Stremmel, W. &Diede, H. E. (1990) J . Hepatol. 10, 99-104. ~~