Evidence for leukotriene C4 transport mediated by an ATP-dependent glutathione S-conjugate carrier in rat heart and liver plasma membranes

Using rat heart sarcolemma and liver plasma membrane vesicles, it has been verified that the transport of leukotriene C4 (LTC4) across membranes is an ATP-dependent process; the apparent Km for LTC4 was 150 nM (heart sarcolemma) or 250 nM (liver plasma membrane). S-(2,4-dinitrophenyl)-glutathione (DNP-SG) inhibited LTC4 uptake into the vesicles dose-dependently (I50 = 25 microM for both heart sarcolemma and liver plasma membrane vesicles). Mutual inhibition between LTC4 and DNP-SG in uptake into the vesicles demonstrates that transport of LTC4 is mediated by an ATP-dependent glutathione S-conjugate carrier.     

ATP-dependent transport for glucuronides in canalicular plasma membrane vesicles

Using rat liver canalicular plasma membrane vesicles, it has been verified that the transport of p-nitrophenyl glucuronide (NPG) across membranes is an ATP-dependent process; the apparent Km for NPG was 20 microM. S-(2,4-dinitrophenyl)-glutathione (DNP-SG) inhibited NPG uptake dose-dependently, and NPG or testosterone glucuronide did ATP-dependent DNP-SG uptake similarly. These results suggest that transport of glucuronide is mediated by an ATP-dependent glutathione S-conjugate carrier.     

ATP-dependent leukotriene export from mastocytoma cells

The biosynthesis of leukotrienes (LT) C4 and B4 is followed by an export of these mediators into the extracellular space. This transport was characterized using plasma membrane vesicles prepared from mastocytoma cells and identified as an ATP-dependent primary active process. The apparent Km-values were 110 nM for LTC4 and 48 microM for ATP. The transport rate was highest for LTC4, whereas LTD4, LTE4, and N-acetyl-LTE4 were transported with relative rates of 31, 12 and 8%, respectively, at a concentration of 10 nM. LTB4 transport was also dependent on ATP. LTC4 transport was inhibited by LTD4 receptor antagonists (IC50 = 1.0 microM for MK-571 and 1.3 microM for LY245769) and by the inhibitor of leukotriene biosynthesis MK-886 (IC50 = 1.8 microM). The ATP-dependent export carrier for leukotrienes in leukotriene-synthesizing cells represents a novel member of the family of ATP-dependent exit pumps.     

ATP-dependent transport of taurocholate across the hepatocyte canalicular membrane mediated by a 110-kDa glycoprotein binding ATP and bile salt

Direct photoaffinity labeling of liver plasma membrane subfractions enriched in sinusoidal and canalicular membranes using [35S]adenosine 5'-O-(thiotriphosphate) ([35S]ATP gamma S) allows the identification of ATP-binding proteins in these domains. Comparative photoaffinity labeling with [35S]ATP gamma S and with the photolabile bile salt derivative (7,7-azo-3 alpha, 12 alpha-dihydroxy-5 beta-[3 beta-3H]-cholan-24-oyl-2'- aminoethanesulfonate followed by immunoprecipitation with a monoclonal antibody (Be 9.2) revealed the identity of the ATP-binding and the bile salt-binding canalicular membrane glycoprotein with the apparent Mr of 110,000 (gp110). The isoelectric point of this glycoprotein was 3.7. Transport of bile salt was studied in vesicles enriched in canalicular and sinusoidal liver membranes. Incubation of canalicular membrane vesicles with [3H] taurocholate in the presence of ATP resulted in an uptake of the bile salt into the vesicles which was sensitive to vanadate. ATP-dependent taurocholate transport was also observed in membrane vesicles from mutant rats deficient in the ATP-dependent transport of cysteinyl leukotrienes and related amphiphilic anions. Substrates of the P-glycoprotein (gp170), such as verapamil and doxorubicin, did not interfere with the ATP-dependent transport of taurocholate. Reconstitution of purified gp110 into liposomes resulted in an ATP-dependent uptake of [3H]taurocholate. These results demonstrate that gp110 functions as carrier in the ATP-dependent transport of bile salts from the hepatocyte into bile. This export carrier is distinct from hitherto characterized ATP-dependent transport systems.     

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.     

Uptake, production and metabolism of cysteinyl leukotrienes in the isolated perfused rat liver. Inhibition of leukotriene uptake by cyclosporine.

1. The isolated perfused rat liver efficiently takes up cysteinyl leukotrienes (LTs) C4, D4, E4 and N-acetyl-LTE4 from circulation. More than 70% of these cysteinyl LTs are excreted from liver into bile within 1 h of onset of a 5 min infusion, while about 5% remain in the liver. About 20% of infused N-acetyl-LTE4 escapes hepatic first-pass extraction under our conditions. 2. Metabolites of LTC4 appearing in bile within 20 min of the onset of infusion include mainly LTD4 and N-acetyl-LTE4, but also omega-hydroxy-N-acetyl-LTE4 and omega-carboxy-N-acetyl-LTE4. Metabolites generated from omega-carboxy-N-acetyl-LTE4 by beta-oxidation from the omega-end represent the major biliary LTs secreted at later times. 3. Stimulation of the isolated perfused liver by the combined infusion of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) and the Ca2+ ionophore A23187 results in a transient increase of endogenous cysteinyl LT production, which is independent of extrahepatic cells. 4. The immunosuppressive drug cyclosporine causes a dose-dependent inhibition of hepatobiliary cysteinyl LT excretion, probably by interference with the sinusoidal uptake system for cysteinyl LTs.     

Metabolism of cysteinyl leukotrienes by the isolated perfused rat kidney.

The metabolism of cysteinyl leukotrienes by the isolated perfused rat kidney was investigated. For this purpose LTC4, LTD4 or LTE4 were studied in separate experiments. The isolated perfused rat kidney metabolized all cysteinyl leukotrienes to the final metabolite N-acetyl-LTE4. In the presence of 5% albumin 50% of LTC4 was metabolized to LTD4 (22%), LTE4 (15%) and N-acetyl-LTE4 (13%) within 60 min. Excretion of radioactivity into urine was less than 1%. In contrast, in the absence of albumin, LTC4 was completely metabolized within 45 min to N-acetyl-LTE4, the sole and final metabolite of LTC4 found in the perfusion medium as well as in urine. After 60 min 19% and 42% of total radioactivity were found in the perfusion medium and in urine, respectively. Isolated glomeruli metabolized LTC4 to LTD4 and to LTE4 but not to N-acetyl-LTE4 at a rate comparable to the rate observed by the isolated perfused kidney in the absence of albumin. In contrast to isolated glomeruli isolated tubuli metabolized LTE4 to N-acetyl-LTE4 at a rate comparable to that observed by the isolated perfused kidney in the absence of albumin. The present study shows that the isolated perfused rat kidney metabolizes cysteinyl leukotrienes to the sole and final metabolite N-acetyl-LTE4. In the presence of albumin metabolism is slowed down and excretion of N-acetyl-LTE4 into urine is prevented.     

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)     

Inhibition of Mrp2- and Ycf1p-mediated transport by reducing agents: evidence for GSH transport on rat Mrp2

Mammalian Mrp2 and its yeast orthologue, Ycf1p, mediate the ATP-dependent cellular export of a variety of organic anions. Ycf1p also appears to transport the endogenous tripeptide glutathione (GSH), whereas no ATP-dependent GSH transport has been detected in Mrp2-containing mammalian plasma membrane vesicles. Because GSH uptake measurements in isolated membrane vesicles are normally carried out in the presence of 5-10 mM dithiothreitol (DTT) to maintain the tripeptide in the reduced form, the present study examined the effects of DTT and other sulfhydryl-reducing agents on Ycf1p- and Mrp2-mediated transport activity. Uptake of S-dinitrophenyl glutathione (DNP-SG), a prototypic substrate of both proteins, was measured in Ycf1p-containing Saccharomyces cerevisiae vacuolar membrane vesicles and in Mrp2-containing rat liver canalicular plasma membrane vesicles. Uptake was inhibited in both vesicle systems in a concentration-dependent manner by DTT, dithioerythritol, and β-mercaptoethanol, with concentrations of 10 mM inhibiting by ∼40%. DTT’s inhibition of DNP-SG transport was noncompetitive. In contrast, ATP-dependent transport of [³H]taurocholate, a substrate for yeast Bat1p and mammalian Bsep bile acid transporters, was not significantly affected by DTT. DTT also inhibited the ATP-dependent uptake of GSH by Ycf1p. As the DTT concentration in incubation solutions containing rat liver canalicular plasma membrane vesicles was gradually decreased, ATP-dependent GSH transport was now detected. These results demonstrate that Ycf1p and Mrp2 are inhibited by concentrations of reducing agents that are normally employed in studies of GSH transport. When this inhibition was partially relieved, ATP-dependent GSH transport was detected in rat liver canalicular plasma membranes, indicating that both Mrp2 and Ycf1p are able to transport GSH by an ATP-dependent mechanism.     

ATP-dependent S-(2,4-dinitrophenyl)glutathione transport in canalicular plasma membrane vesicles from rat liver

Uptake of the thioether S-(2,4-dinitrophenyl)glutathione (DNPSG) in canalicular plasma membrane vesicles from rat liver is enhanced in the presence of ATP and exhibits an overshoot with a transient 5.5-fold accumulation of DNPSG. Stimulation by ATP is not caused by the generation of a membrane potential, based on responses of the indicator dye oxonol V. ATP-dependent uptake has an apparent Km of 71 microM for DNPSG and a Vmax of 0.34 nmol.min-1.mg of vesicle protein-1. Protein thiol groups are essential for transport activity as indicated by the sensitivity of DNPSG transport to sulfhydryl reagents. There is competitive inhibition with other thioethers, S-hexylglutathione (Ki = 66 microM), the photoaffinity label S-(4-azidophenacyl)glutathione (Ki = 56 microM), as well as with glutathione disulfide (Ki = 0.44 mM) and with the bile acid taurocholate (Ki = 0.61 mM). GSH (2 mM) or cholate (0.4 mM) does not inhibit. Both glutathione disulfide and taurocholate show ATP-dependent transport in the canalicular membrane vesicles which is inhibited by DNPSG. No ATP-dependent transport is found for GSH. Transport of DNPSG is also inhibited competitively by alpha-naphthyl-beta-D-glucuronide (Ki = 0.42 mM) but not by alpha-naphthylsulfate (2 mM), and there is substantial inhibition with the glucuronides from ebselen and p-nitrophenol. The results indicate that the canalicular transport system for DNPSG is directly driven by ATP and that the biliary transport of other classes of compounds may also proceed via this system.     

Inhibition of Mrp2- and Ycf1p-mediated transport by reducing agents: evidence for GSH transport on rat Mrp2

Mammalian Mrp2 and its yeast orthologue, Ycf1p, mediate the ATP-dependent cellular export of a variety of organic anions. Ycf1p also appears to transport the endogenous tripeptide glutathione (GSH), whereas no ATP-dependent GSH transport has been detected in Mrp2-containing mammalian plasma membrane vesicles. Because GSH uptake measurements in isolated membrane vesicles are normally carried out in the presence of 5-10 mM dithiothreitol (DTT) to maintain the tripeptide in the reduced form, the present study examined the effects of DTT and other sulfhydryl-reducing agents on Ycf1p- and Mrp2-mediated transport activity. Uptake of S-dinitrophenyl glutathione (DNP-SG), a prototypic substrate of both proteins, was measured in Ycf1p-containing Saccharomyces cerevisiae vacuolar membrane vesicles and in Mrp2-containing rat liver canalicular plasma membrane vesicles. Uptake was inhibited in both vesicle systems in a concentration-dependent manner by DTT, dithioerythritol, and beta-mercaptoethanol, with concentrations of 10 mM inhibiting by approximately 40%. DTT's inhibition of DNP-SG transport was noncompetitive. In contrast, ATP-dependent transport of [(3)H]taurocholate, a substrate for yeast Bat1p and mammalian Bsep bile acid transporters, was not significantly affected by DTT. DTT also inhibited the ATP-dependent uptake of GSH by Ycf1p. As the DTT concentration in incubation solutions containing rat liver canalicular plasma membrane vesicles was gradually decreased, ATP-dependent GSH transport was now detected. These results demonstrate that Ycf1p and Mrp2 are inhibited by concentrations of reducing agents that are normally employed in studies of GSH transport. When this inhibition was partially relieved, ATP-dependent GSH transport was detected in rat liver canalicular plasma membranes, indicating that both Mrp2 and Ycf1p are able to transport GSH by an ATP-dependent mechanism.     

Identification of the major endogenous leukotriene metabolite in the bile of rats as N-acetyl leukotriene E4

Mercapturic acid formation, an established pathway in the detoxication of xenobiotics, is demonstrated for cysteinyl leukotrienes generated in rats in vivo after endotoxin treatment. The mercapturate N-acetyl-leukotriene E4 (N-acetyl-LTE4) represented a major metabolite eliminated into bile after injection of [3H]LTC4 as shown by cochromatography with synthetic N-acetyl-LTE4 in four different HPLC solvent systems. The identity of endogenous N-acetyl-LTE4 elicited by endotoxin in vivo was additionally verified by enzymatic deacetylation followed by chemical N-acetylation. The deacetylation was catalyzed by penicillin amidase. Endogenous cysteinyl leukotrienes were quantified by radioimmunoassay after HPLC separation. A N-acetyl-LTE4 concentration of 80 nmol/l was determined in bile collected between 30 and 60 min after endotoxin injection. Under this condition, other cysteinyl leukotrienes detected in bile by radioimmunoassay amounted to less than 5% of N-acetyl-LTE4. The mercapturic acid pathway, leading from the glutathione conjugate LTC4 to N-acetyl-LTE4, thus plays an important role in the deactivation and elimination of these potent endogenous mediators.     

Stimulatory effect of regucalcin on ATP-dependent calcium transport in rat liver plasma membranes

The effect of regucalcin, a calcium-binding protein isolated from rat liver cytoplasm, on ATP-dependent calcium transport in the plasma membrane vesicles of rat liver was investigated. (Ca2+-Mg2+)-ATPase activity in the liver plasma membranes was significantly increased by the presence of regucalcin (0.1-0.5 \sgmaelig;M) in the enzyme reaction mixture. This increase was completely inhibited by the presence of sulfhydryl group modifying reagent Nethylmaleimide (5.0 mM NEM) or digitonin (0.04%), which can solubilize the membranous lipids. When ATP-dependent calcium uptake by liver plasma membrane vesicles was measured by using 45CaCl2, the presence of regucalcin (0.1-0.5 \sgmaelig;M) in the reaction mixture caused a significant increase in the 45Ca2+ uptake. This increase was about 2-fold with 0.5 \sgmaelig;M regucalcin addition. An appreciable increase was seen by 5 min incubation with regucalcin addition. The regucalcin-enhanced ATP-dependent 45Ca2+ uptake by the plasma membrane vesicles was completely inhibited by the presence of NEM (5.0 mM) or digitonin (0.04%). These results demonstrate that regucalcin activates (Ca2+-Mg2+)-ATPase in the liver plasma membranes and that it can stimulate ATP-dependent calcium transport across the plasma membranes.     

Enhanced transport of anticancer agents and leukotriene C4 by the human canalicular multispecific organic anion transporter (cMOAT/MRP2).

We established stable human canalicular multispecific organic anion transporter (cMOAT/MRP2) cDNA transfectants, CHO/cMOAT from non-polarized Chinese hamster ovary (CHO)-K1 and LLC/cMOAT from polarized pig kidney epithelial LLC-PK1. Human cMOAT was mainly localized in the plasma membrane of CHO/cMOAT and in the apical membrane of LLC/cMOAT. The ATP-dependent uptake of leukotriene C4 (LTC4) into CHO/cMOAT membrane vesicles was enhanced compared with empty vector transfectants. Km values in CHO/cMOAT membrane vesicles were 0.24 microM for LTC4 and 175 microM for ATP. Drug sensitivity to vincristine and cisplatin in human cMOAT cDNA transfectants decreased, but not to etoposide. Cellular accumulation of vincristine and cisplatin in human cMOAT cDNA transfectants decreased, but not of etoposide. The uptake of LTC4 into CHO/cMOAT membrane vesicles was inhibited by exogenous administration of vincristine or cisplatin, but not that of etoposide. Moreover, this inhibition was more enhanced in the presence of glutathione. These consequences indicate that drug resistance to vincristine or cisplatin appears to be modulated by human cMOAT through transport of the agents, possibly in direct or indirect association with glutathione.     

Leukotriene uptake by hepatocytes and hepatoma cells.

The uptake of tritiated cysteinyl leukotrienes (LTC4, LTD4, LTE4) and LTB4 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-Km system and a non-saturable system for the uptake of cysteinyl leukotrienes as well as LTB4 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 LTB4. Moreover, only LTB4 was metabolized in Hep G2 hepatoma cells. Competition studies on the uptake of LTE4 and LTB4 (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 microM 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 LTB4 uptake only. Bromosulfophthalein, the strongest inhibitor of leukotriene uptake by hepatocytes, did not inhibit LTB4 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]LTE4 and [3H]LTB4 as 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]LTE4 in Hep G2 cells. With [3H]LTB4 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 LTB4, but not cysteinyl leukotrienes, is taken up and metabolized by the transformed hepatoma cells.     

Leukotriene C4 inhibits ATP-dependent transport of glutathione S-conjugate across rat heart sarcolemma. Implication for leukotriene C4 translocation mediated by glutathione S-conjugate carrier

Sarcolemmal vesicles prepared from rat heart exhibited ATP-dependent uptake of S-(2,4-dinitrophenyl)glutathione (DNP-SG), which obeyed Michaelis-Menten kinetics with an apparent Km of 21 microM for DNP-SG and a Vmax of 0.27 nmol.10 min-1.mg protein-1. Several model glutathione S-conjugates inhibited DNP-SG uptake, but leukotriene C4 inhibited uptake much more significantly even at lower concentrations (competitive inhibition, Ki = 1.5 microM). However, leukotrienes D4 and E4, which lack the gamma-glutamyl moiety, were less effective. The results suggest that the ATP-dependent transport system has a high affinity for leukotriene C4, and may be responsible for the translocation of this compound.     

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.     

ATP-dependent GSH and glutathione S-conjugate transport in skate liver: role of an Mrp functional homologue

Multidrug resistance-associated proteins 1 and 2 (Mrp1 and Mrp2) are thought to mediate low-affinity ATP-dependent transport of reduced glutathione (GSH), but there is as yet no direct evidence for this hypothesis. The present study examined whether livers from the little skate (Raja erinacea) express an Mrp2 homologue and whether skate liver membrane vesicles exhibit ATP-dependent GSH transport activity. Antibodies directed against mammalian Mrp2-specific epitopes labeled a 180-kDa protein band in skate liver plasma membranes and stained canaliculi by immunofluorescence, indicating that skate livers express a homologous protein. Functional assays of Mrp transport activity were carried out using (3)H-labeled S-dinitrophenyl-glutathione (DNP-SG). DNP-SG was accumulated in skate liver membrane vesicles by both ATP-dependent and ATP-independent mechanisms. ATP-dependent DNP-SG uptake was of relatively high affinity [Michaelis-Menten constant (K(m)) = 32 +/- 9 microM] and was cis-inhibited by known substrates of Mrp2 and by GSH. Interestingly, ATP-dependent transport of (3)H-labeled S-ethylglutathione and (3)H-labeled GSH was also detected in the vesicles. ATP-dependent GSH transport was mediated by a low-affinity pathway (K(m) = 12 +/- 2 mM) that was cis-inhibited by substrates of the Mrp2 transporter but was not affected by membrane potential or pH gradient uncouplers. These results provide the first direct evidence for ATP-dependent transport of GSH in liver membrane vesicles and support the hypothesis that GSH efflux from mammalian cells is mediated by members of the Mrp family of proteins.     

Bile salt excretion in skate liver is mediated by a functional analog of Bsep/Spgp, the bile salt export pump

Biliary secretion of bile salts in mammals is mediated in part by the liver-specific ATP-dependent canalicular membrane protein Bsep/Spgp, a member of the ATP-binding cassette superfamily. We examined whether a similar transport activity exists in the liver of the evolutionarily primitive marine fish Raja erinacea, the little skate, which synthesizes mainly sulfated bile alcohols rather than bile salts. Western blot analysis of skate liver plasma membranes using antiserum raised against rat liver Bsep/Spgp demonstrated a dominant protein band with an apparent molecular mass of 210 kDa, a size larger than that in rat liver canalicular membranes, approximately 160 kDa. Immunofluorescent localization with anti-Bsep/Spgp in isolated, polarized skate hepatocyte clusters revealed positive staining of the bile canaliculi, consistent with its selective apical localization in mammalian liver. Functional characterization of putative ATP-dependent canalicular bile salt transport activity was assessed in skate liver plasma membrane vesicles, with [(3)H]taurocholate as the substrate. [(3)H]taurocholate uptake into the vesicles was mediated by ATP-dependent and -independent mechanisms. The ATP-dependent component was saturable, with a Michaelis-Menten constant (K(m)) for taurocholate of 40+/-7 microM and a K(m) for ATP of 0.6+/-0.1 mM, and was competitively inhibited by scymnol sulfate (inhibition constant of 23 microM), the major bile salt in skate bile. ATP-dependent uptake of taurocholate into vesicles was inhibited by known substrates and inhibitors of Bsep/Spgp, including other bile salts and bile salt derivatives, but not by inhibitors of the multidrug resistance protein-1 or the canalicular multidrug resistance-associated protein, indicating a distinct transport mechanism. These findings provide functional and structural evidence for a Bsep/Spgp-like protein in the canalicular membrane of the skate liver. This transporter is expressed early in vertebrate evolution and transports both bile salts and bile alcohols.     

Sodium gradient-dependent L-glutamate transport is localized to the canalicular domain of liver plasma membranes. Studies in rat liver sinusoidal and canalicular membrane vesicles

The driving forces for L-glutamate transport were determined in purified canalicular (cLPM) and basolateral (i.e. sinusoidal and lateral; blLPM) rat liver plasma membrane vesicles. Initial rates of L-glutamate uptake in cLPM vesicles were stimulated by a Na+ gradient (Na+o greater than Na+i), but not by a K+ gradient. Stimulation of L-glutamate uptake was specific for Na+, temperature sensitive, and independent of nonspecific binding. Sodium-dependent L-glutamate uptake into cLPM vesicles exhibited saturation kinetics with an apparent Km of 24 microM, and a Vmax of 21 pmol/mg X min at an extravesicular sodium concentration of 100 mM. Specific anionic amino acids inhibited L-[3H]glutamate uptake and accelerated the exchange diffusion of L-[3H]glutamate. An outwardly directed K+ gradient (K+i greater than K+o) further increased the Na+ gradient (Na+o greater than Na+i)-dependent uptake of L-glutamate in cLPM vesicles, resulting in a transient accumulation of L-glutamate above equilibrium values (overshoot). The K+ effect had an absolute requirement for Na+. In contrast, in blLPM the initial rates of L-glutamate uptake were only minimally stimulated by a Na+ gradient, an effect that could be accounted for by contamination of the blLPM vesicles with cLPM vesicles. These results indicate that hepatic Na+ gradient-dependent transport of L-glutamate occurs at the canalicular domain of the plasma membrane, whereas transport of L-glutamate across sinusoidal membranes results mainly from passive diffusion. These findings provide an explanation for the apparent discrepancy between the ability of various in vitro liver preparations to transport glutamate and suggest that a canalicular glutamate transport system may serve to reabsorb this amino acid from bile.