Cholestasis and the role of basolateral efflux pumps.

ATP-dependent transport of biliary constituents, such as bile acids, reduced glutathione, and bilirubin glucuronosides across the hepatocyte canalicular membrane into bile represents the decisive driving force for the formation of biliary fluid. Functional characterization, cloning, and localization of hepatocellular transporter proteins has provided a molecular understanding of the mechanisms underlying bile flow and intrahepatic cholestasis. Genetic variants in humans and genetic knockout in rodents, or transporter inhibition have indicated that both the conjugate export pump MRP2 (multidrug resistance protein 2; ABCC2) and the bile salt export pump BSEP (ABCB11) are major contributors to bile acid-independent and bile acid-dependent bile flow, respectively. In humans, genetic variants of BSEP, leading to an impaired transport activity or localization of the protein in the canalicular membrane, are associated with severe intrahepatic cholestasis. Efflux pumps of the basolateral hepatocyte membrane, particularly MRP3 (multidrug resistance protein 3; ABCC3) and MRP4 (multidrug resistance protein 4; ABCC4) pump substances from hepatocytes into sinusoidal blood. These efflux pumps have been recognized in recent years to play an important compensatory role in cholestasis and to contribute to the balance between uptake and efflux of substances during the vectorial transport from sinusoidal blood into bile. This sinusoidal efflux not only enables subsequent renal elimination, but also re-uptake of substances into neighboring and more centrally located hepatocytes in the sinusoid.

Prevention of endogenous leukotriene production during anaphylaxis in the guinea pig by an inhibitor of leukotriene biosynthesis (MK-886) but not by dexamethasone.

Leukotriene C4 (LTC4) underwent rapid elimination from the circulating blood and was extensively converted to LTD4 within the vascular space of the guinea pig. To mimic the elimination and metabolism of endogenous LTC4 generated during anaphylaxis, 14,15-3H-labeled LTC4 was infused intravenously over a period of 15 min, leading to a recovery in bile of 85% of the infused LT radioactivity within 2 h. Corresponding to the tracer studies, LTD4 and, to a lesser extent, LTC4 were the predominant endogenous cysteinyl LTs in guinea pig bile. The biliary production rate of endogenous LTD4 increased from 0.3 +/- 0.1 to 6.2 +/- 1.8 pmol x min-1 x kg-1 (p less than 0.001) during anaphylactic shock induced by intravenous injection of OVA (0.2 mg/kg) into sensitized guinea pigs. A novel LT biosynthesis inhibitor (MK-886; 10 mg/kg, i.v., 15 min before antigen challenge) suppressed the antigen-induced cysteinyl LT production by greater than 92% (p less than 0.001). This inhibition of systemic LTC4 formation was associated with a complete protection against lethal anaphylactic shock in animals pretreated in addition with the H1 receptor antagonist pyrilamine. Pretreatment with either the inhibitor of LT synthesis or the histamine receptor antagonist reduced the lethality during anaphylactic shock from 100 to 60 and 78%, respectively. In artificially ventilated, pyrilamine-pretreated animals, the antigen-induced decrease in dynamic lung compliance and the rise in hematocrit were significantly reduced (p less than 0.05) by pretreatment with the inhibitor of LT synthesis. Dexamethasone at high doses (10 mg/kg, i.p., once daily for 7 d, or in a single dose of 10 mg/kg, i.v., 3.5 h before challenge) had no inhibitory effect on LT generation during anaphylaxis in vivo. However, in resident peritoneal macrophages, harvested from these dexamethasone-treated sensitized guinea pigs and stimulated with zymosan, both cysteinyl LT and 6-keto-PGF1 alpha formation were strongly suppressed. These studies indicate an important role of cysteinyl LTs in systemic anaphylaxis in vivo and demonstrate the blockade of anaphylactic LT generation by a novel inhibitor of LT biosynthesis (MK-886) but not by dexamethasone.

Expression of the MRP gene-encoded conjugate export pump in liver and its selective absence from the canalicular membrane in transport-deficient mutant hepatocytes.

We have previously shown that the multi-drug resistance protein (MRP) mediates the ATP-dependent membrane transport of glutathione S-conjugates and additional amphiphilic organic anions. In the present study we demonstrate the expression of MRP in hepatocytes where it functions in hepatobiliary excretion. Analysis by reverse transcription-PCR of human and normal rat liver mRNA resulted in two expected cDNA fragments of MRP. Four different antibodies against MRP reacted on immunoblots with the glycoprotein of about 190 kD from human canalicular as well as basolateral hepatocyte membrane preparations. A polyclonal antibody directed against the carboxy-terminal sequence of MRP detected the rat homolog of MRP in liver. Double immunofluorescence microscopy and confocal laser scanning microscopy showed the presence of human MRP and rat Mrp in the canalicular as well as in the lateral membrane domains of hepatocytes. The transport function of the mrp gene-encoded conjugate export pump was assayed in plasma membrane vesicles with leukotriene C4 as a high-affinity glutathione S-conjugate substrate. The deficient ATP-dependent conjugate transport in canalicular membranes from TR- mutant rat hepatocytes was associated with a lack of amplification of one of the mrp cDNA fragments and with a selective loss of Mrp on immunoblots of canalicular membranes. Double immunofluorescence microscopy of livers from transport-deficient TR- mutant rats localized Mrp only to the lateral but not to the canalicular membrane. Our results indicate that the absence of Mrp or an isoform of Mrp from the canalicular membrane is the basis for the hereditary defect of the hepatobiliary excretion of anionic conjugates by the transport-deficient hepatocyte.

Leukotrienes as mediators in tissue trauma.

A significant increase in the production of cysteinyl leukotrienes was observed after mechanical or thermal trauma in the anesthesized rat. The amount of biliary N-acetyl-leukotriene E4, which represents a suitable indicator for blood plasma leukotrienes, was used as a measure of leukotriene generation. Cysteinyl leukotrienes were rapidly eliminated from blood plasma into bile where N-acetyl-leukotriene E4 was the major metabolite. Leukotrienes were at a much lower concentration in blood plasma than in bile and differed in the pattern of metabolites. The detected amounts of leukotrienes were sufficient to induce known phenomena associated with trauma, such as tissue edema and circulatory and respiratory dysfunction. Increased leukotriene generation appears to play an important role in the pathophysiology of tissue trauma.

Leukotrienes as mediators in ischemia-reperfusion injury in a microcirculation model in the hamster.

Leukotriene (LT)B4 promotes leukocyte chemotaxis and adhesion to the endothelium of postcapillary venules. The cysteinyl leukotrienes, LTC4, LTD4, and LTE4, elicit macromolecular leakage from this vessel segment. Both leukocyte adhesion to the endothelium and macromolecular leakage from postcapillary venules hallmark the microcirculatory failure after ischemia-reperfusion, suggesting a role of leukotrienes as mediators of ischemia-reperfusion injury. Using the dorsal skinfold chamber model for intravital fluorescence microscopy of the microcirculation in striated muscle in awake hamsters and sequential RP-HPLC and RIA for leukotrienes, we demonstrate in this study that (a) the leukotrienes (LT)B4 and LTD4 elicit leukocyte/endothelium interaction and macromolecular leakage from postcapillary venules, respectively, that (b) leukotrienes accumulate in the tissue after ischemia and reperfusion, and that (c) selective inhibition of leukotriene biosynthesis (by MK-886) prevents both postischemic leukotriene accumulation and the microcirculatory changes after ischemia-reperfusion, while blocking of LTD4/E4 receptors (by MK-571) inhibits postischemic macromolecular leakage. These results demonstrate a key role of leukotrienes in ischemia-reperfusion injury in striated muscle in vivo.

Impaired degradation of leukotrienes in patients with peroxisome deficiency disorders.

The degradation of leukotrienes by beta-oxidation from the omega-end proceeds in peroxisomes (Jedlitschky et al. J. Biol. Chem. 1991. 266:24763-24772). Peroxisomal degradation of leukotrienes was studied in humans by analyses of endogenous leukotrienes in urines from eight patients with biochemically established peroxisome deficiency disorder and eight age- and sex-matched healthy infant controls. Leukotriene metabolites were separated by high-performance liquid chromatography, quantified by radioimmunoassays, and identified as well as quantified by gas chromatography-mass spectrometry. Urinary leukotriene E4 (LTE4) and N-acetyl-LTE4 excretions, relative to creatinine, were increased > 10-fold in the patients in comparison to healthy infants. The beta-oxidation product omega-carboxy-tetranor-LTE3 averaged 0.05 mumol/mol creatinine in the controls but was not detectable in the patients. However, omega-carboxy-LTE4 (median 13.6 mumol/mol creatinine) was significantly increased in the patients' urine, whereas LTB4 (median 0.07 mumol/mol creatinine) and omega-carboxy-LTB4 were detected exclusively in the urines of the patients. These data indicate an impairment of the inactivation and degradation of both LTE4 and LTB4 in patients with peroxisomal deficiency. The increased levels of the biologically active, proinflammatory mediators LTE4 and LTB4 might be of pathophysiological significance in peroxisome deficiency disorders. This is the first and so far only condition with a pronounced urinary excretion of omega-carboxy-LTE4, omega-carboxy-LTB4, and LTB4. This impaired catabolism of leukotrienes and the altered pattern of metabolites may be of diagnostic value. These findings underline the essential role of peroxisomes in the catabolism of leukotrienes in humans.

Human multidrug resistance protein 8 (MRP8/ABCC11), an apical efflux pump for steroid sulfates, is an axonal protein of the CNS and peripheral nervous system.

Dehydroepiandrosterone 3-sulfate and other neurosteroids are synthesized in the CNS and peripheral nervous system where they may modulate neuronal excitability by interacting with ligand-gated ion channels. For this modulatory activity, neurosteroids have to be locally released from either neurons or glial cells. We here identify the integral membrane protein ABCC11 (multidrug resistance protein 8) as an ATP-dependent efflux pump for steroid sulfates, including dehydroepiandrosterone 3-sulfate, and localize it to axons of the human CNS and peripheral nervous system. ABCC11 mRNA was detected in human brain by real-time polymerase chain reaction. Antibodies raised against ABCC11 served to detect the protein in brain by immunoblotting and immunofluorescence microscopy. ABCC11 was preferentially found in the white matter of the brain and co-localized with neurofilaments indicating that it is an axonal protein. Additionally, ABCC11 was localized to axons of the peripheral nervous system. For functional studies, ABCC11 was expressed in polarized Madin-Darby canine kidney cells where it was sorted to the apical membrane. This apical sorting is in accordance with the localization of ABCC11 to the axonal membrane of neurons. Inside-out plasma membrane vesicles containing recombinant ABCC11 mediated ATP-dependent transport of dehydroepiandrosterone 3-sulfate with a Km value of 21 microM. This transport function together with the localization of the ABCC11 protein in vicinity to GABAA receptors is consistent with a role of ABCC11 in dehydroepiandrosterone 3-sulfate release from neurons to sites of dehydroepiandrosterone 3-sulfate-mediated receptor modulation. Our findings may provide a basis for the characterization of mutations in the human ABCC11 gene and their linkage with neurological disorders.

Effects of D-glucosamine and 6-azauridine on nucleotide contents, 5-fluorouridine uptake, and cytotoxicity in TA3 mammary tumor cells.

In TA3 mammary carcinoma cells in suspension culture, D-glucosamine X HCl (GlcN) induced a diversion of uridylate from UTP into UDP-N-acetylhexosamines, reducing the intracellular pool of UTP and eliciting an increased rate of de novo uridylate synthesis. This rise in de novo synthesis was completely suppressed by addition of 6-azauridine (6-AzaUrd) to the cell suspension in vitro or in the solid TA3 mammary tumor in NMRI mice in vivo. A synergistic depletion of UTP pools to less than 6% of the UTP in controls was observed in TA3 cell suspensions exposed to GlcN and 6-AzaUrd. In solid TA3 tumors in vivo, UTP was reduced by this combination to 19% of the control value. A high sensitivity of the solid tumor to inhibition of pyrimidine synthesis de novo was indicated by the reduction of the UTP content after administration of 6-AzaUrd alone. UTP deficiency in TA3 tumor cells was accompanied by CTP deficiency. In addition, 6-AzaUrd caused a lowering of GTP in the neoplastic tissue. Host liver was resistant to 6-AzaUrd but responded to treatment with GlcN with a decrease in UTP to 67%. Uridine-cytidine kinase was less inhibited in the presence of lowered UTP and CTP, which are potent feedback inhibitors of the enzyme, and enabled an enhanced formation of phosphorylated derivatives of 5-fluorouridine (FUrd). Aside from the formation of 5-fluoro-UTP, we have identified 5-fluoro-UDP-N-acetylhexosamines (FUDPHexNAc), which accumulated when FUrd and GlcN were sequentially administered. Treatment of TA3 cells with FUrd after a pretreatment with 6-AzaUrd and GlcN resulted in a 2.5-fold increase in [14C]FUrd uptake and a duplication of 5-fluorouridylate incorporation into the RNA. The proportion of FUDPHexNAc increased to 58% of the phosphorylated FUrd metabolites, as compared to 6% in TA3 cells exposed exclusively to FUrd. In vivo chemotherapy of mice bearing TA3 ascites tumors was most effective with respect to tumor growth inhibition and animal survival when GlcN and FUrd were combined.

Uridine triphosphate deficiency, growth inhibition, and death in ascites hepatoma cells induced by a combination of pyrimidine biosynthesis inhibition with uridylate trapping.

A selective deficiency of uridine triphosphate (UTP) was induced in AS-30D rat ascites hepatoma cells by the synergistic action of D-galactosamine and 6-azauridine. The resistance of these hepatoma cells to low concentrations of D-galactosamine (less than 2 mM) was due to their active de novo pyrimidine synthesis which compensated the trapping of uridylate in the form of uridine diphosphate-amino sugars derived from D-galactosamine. The additional blockage of de novo pyrimidine synthesis led to noncompensated uridylate trapping with a UTP content of less than 0.05 mmole/kg of cell wet weight as compared to the control level of 0.66 mmole/kg. The induction of UTP deficiency by incubating the cells with low concentrations of D-galactosamine and 6-azauridine (0.5 mM each) was not accompanied by significant changes in the content of adenine and guanine nucleotides, uridine diphosphate glucose, and uridine diphosphate galactose. The depletion of UTP pools could be reversed within 10 min by the addition of uridine; orotate or uracil were completely ineffective in these hepatoma cells. A UTP content in the range of 0.1 to 0.4 mmole/kg, induced by either 6-azauridine or D-galactosamine, was associated with a reversible depression of cell growth in suspension culture. A UTP content below 0.05 mmole/kg led to irreversible growth inhibition and to necrocytosis in culture, as well as to a loss of transplantability in vivo. Uridine reversal studies indicated that the percentage of cells able to resume growth in culture decreased with an increasing time period of UTP deficiency. The deficiency period required for irreparable or lethal damage in these hepatoma cells ranged from 3 to 20 hr. The principle of noncompensated uridylate trapping can be extended to other inhibitors of nucleotide synthesis combined with various nucleotide-trapping sugar analogs. Noncompensated nucleotide trapping may be useful for an induction of selective nucleotide deficiencies in tumor cells.

Immunohistochemical and biochemical study of a cathepsin B-like proteinase in human colonic cancers.

An immunohistological study was carried out on 51 human colorectal adenocarcinomas and eight samples of histologically normal colonic mucosa removed far from tumors, using anti-rabbit cathepsin B and anti-human cathepsin B immunoglobulins. Positive reactions were obtained on tumor cells and macrophage-like cells. However, as these immunoglobulins could not discriminate between cathepsin B and cathepsin B-like proteinases, and as they cross-reacted with cathepsins H and L, a partial characterization of the proteinase activities was performed in order to identify the type of enzyme present in the positive cells. The levels of cathepsins H and L were very low in extracts of colorectal tumors and normal colonic mucosa. A peculiar cathepsin B-like proteinase activity with pH optimum at 6.8 was found in tumor extracts together with the lysosomal cathepsin B, whereas normal colonic mucosa showed only cathepsin B activity (pH optimum, 6.0). These results indicate that lysosomal cathepsin B is responsible for staining of macrophage-like cells found in the lamina propria of colonic mucosa and in the peritumoral stroma. Immunohistochemical staining of colonic tumor cells observed in 29/51 cases seems on the other hand to be primarily due to a cathepsin B-like proteinase. Three colonic tumor cell lines, Colo-205, HT-29, and SW-1116, were also studied using the same methods. These cells produced a latent cathepsin B-like proteinase which, after activation, was similar to that found in tumor extracts. This latent proteinase was detected mainly in the culture media. The cultured colonic tumor cells, after staining by anti-cathepsin B antibodies, showed strongly positive granules. In conclusion, this work demonstrates that malignant colonic cells are the source of a cathepsin B-like proteinase, with optimal activity near neutrality. Its secretion into the extracellular space indicates furthermore, that it may be an important component of the "proteinase cascade" associated with tumor invasion and metastasis.

ATP-dependent transport of glutathione S-conjugates by the multidrug resistance-associated protein.

The ATP-dependent transport of the endogenous glutathione conjugate leukotriene C4 (LTC4) was more than 25-fold higher in membrane vesicles prepared from human leukemia cells (HL60/ADR) overexpressing the multidrug resistance-associated protein than from drug-sensitive parental HL60 cells or revertant cells. Similar results were obtained with S-(2,4-dinitrophenyl)glutathione as substrate. Photoaffinity labeling detected preferentially in the HL60/ADR membranes a 190-kilodalton protein binding [3H]LTC4 and 8-azido[alpha-32P]ATP. The [3H]LTC4-labeled 190-kilodalton protein was immunoprecipitated by an antiserum against the COOH-terminal sequence of multidrug resistance-associated protein. Our results indicate that multidrug resistance-associated protein mediates the ATP-dependent transport of LTC4 and structurally related anionic amphiphilic conjugates.

Transport of glutathione, glucuronate, and sulfate conjugates by the MRP gene-encoded conjugate export pump.

Previous studies have identified the ATP-dependent export of glutathione conjugates as a physiological function of the multidrug resistance protein (MRP). The involvement of MRP in the transport of endogenous and xenobiotic conjugates was investigated further using membrane vesicles from MRP-transfected HeLa cells. The ATP-dependent transport of the glutathione conjugates [(3)H]leukotriene C(4), S-(2,4-dinitrophenyl)-[(3)H]glutathione, and (3)H- labeled oxidized glutathione was characterized by determination of the transport efficiency V(max):K(m) amounting to 1031, 114, and 7.1 ml multiplied by min(-1), respectively. Additional endogenous substrates for MRP-mediated transport included the steroid conjugate 17 beta- glucuronosyl [(3)H]estradiol and the bile salt conjugates [6 alpha-(14)C]glucuronosylhyodeoxycholate and 3 alpha-sulfatolithocholyl [(3)H]taurine. The K(m) value of MRP for 17-beta-glucuronosyl [(3)H]estradiol was 1.5 +/- 0.3 microM, with a V(max):K(m) ratio of 42 ml multiplied by mg protein(-1) multiplied by min(-1), and a K(i) value of 0.7 microM for the leukotriene receptor antagonist MK 571. MRP-mediated ATP-dependent transport was observed for the anticancer drug conjugates glucuronosyl [(3)H]etoposide and monocholoro-mono[(3)H]glutathionyl melphalan, but not for unmodified [(14)C]doxorubicin, [(3)H]daunorubicin, or [(3)H]vinblastine. Our results establish that MRP functions as an ATP-dependent export pump not only for glutathione conjugates but also for glucuronidated and sulfated endogenous as well as exogenous compounds.

Inhibition of tyrosine aminotransferase induction by UTP deficiency and its reversal by 5-fluorouridine in cultured hepatoma cells.

Hepatoma tissue culture cells, grown in the presence of D-galactosamine and 6-azauridine, demonstrate a strong reduction of the intracellular UTP pool that can be replenished by formation of UTP from uridine and FUTP from 5-fluorouridine within 2 h. Concomitantly with the UTP deficiency, a decrease of dexamethasone-induced tyrosine aminotransferase activity occurs. 5-Fluorouridine, as compared to uridine, is even more efficient in restoring the activity of tyrosine aminotransferase. Treatment of the cells with D-galactosamine alone results in a minor lowering of UTP that is not followed by the inhibition of the enzyme induction. However, the administration of D-galactosamine, simultaneously or at any time up to 5 h before or after dexamethasone, leads to a 1.5- to 2-fold higher induction (superinduction) which appears 24 h later.

Conjugate export pumps of the multidrug resistance protein (MRP) family: localization, substrate specificity, and MRP2-mediated drug resistance.

The membrane proteins mediating the ATP-dependent transport of lipophilic substances conjugated to glutathione, glucuronate, or sulfate have been identified as members of the multidrug resistance protein (MRP) family. Several isoforms of these conjugate export pumps with different kinetic properties and domain-specific localization in polarized human cells have been cloned and characterized. Orthologs of the human MRP isoforms have been detected in many different organisms. Studies in mutant rats lacking the apical isoform MRP2 (symbol ABCC2) indicate that anionic conjugates of endogenous and exogenous substances cannot exit from cells at a sufficient rate unless an export pump of the MRP family is present in the plasma membrane. Several mutations in the human MRP2 gene have been identified which lead to the absence of the MRP2 protein from the hepatocyte canalicular membrane and to the conjugated hyperbilirubinemia of Dubin-Johnson syndrome. Overexpression of recombinant MRP2 confers resistance to multiple chemotherapeutic agents. Because of its function in the terminal excretion of cytotoxic and carcinogenic substances, MRP2 as well as other members of the MRP family, play an important role in detoxification and chemoprevention.

Latency of cathepsin B secreted by human colon carcinoma cells is not linked to secretion of cystatin C and is relieved by neutrophil elastase.

The lysosomal cysteine proteinase cathepsin B is shown to be secreted by ten human colon carcinoma cell lines and to accumulate in culture media as a latent enzyme. The cell lines also secrete a physiological inhibitor of cathepsin B, cystatin C. A significant correlation was found between secretion of the latent enzyme and the inhibitor (r = 0.755, P < 0.01). The aim of the present study was to modulate the respective secretion of the two antagonists to test whether or not latency of cathepsin B was due to the concomitant secretion of the inhibitor. SW480 colon carcinoma cells were treated with the acidotropic agent ammonium chloride, phorbol 12-myristate 13-acetate, and the inflammatory cytokines TGF-beta, TNF-alpha, and IL-1 beta. Ammonium chloride significantly increased latent cathepsin B levels without affecting the constitutive secretion of cystatin C. Phorbol 12-myristate 13-acetate induced a 4- to 5-fold increase in secreted latent cathepsin B, but did not alter significantly the accumulation of cystatin C in media. The cytokines, TGF-beta, TNF-alpha, and IL-1 beta, had no major effect on the expression of these two antagonists. Latent cathepsin B released from human carcinoma cells could be efficiently activated by neutrophil elastase at neutral pH. It is concluded that latent cathepsin B is a true proenzyme rather than an enzyme-inhibitor complex. In addition, our data from neutrophil elastase activation experiments indicate that a proteolytic system for activation of the tumor cell-secreted latent enzyme may exist in vivo.

Export pumps for glutathione S-conjugates.

The release of glutathione S-conjugates from cells is an ATP-dependent process mediated by integral membrane glycoproteins belonging to the recently discovered multidrug-resistance protein (MRP) family. Many lipophilic compounds conjugated with glutathione, glucuronate, or sulfate are substrates for export pumps of the MRP family. In humans six MRP isoforms encoded by different genes have been cloned. Orthologs of MRP have been identified in many species including yeast, plants, and nematodes. Human MRP1 and MRP2 are currently best characterized with respect to substrate specificity by measurements of ATP-dependent transport into inside-out membrane vesicles. High-affinity substrates include the glutathione S-conjugate leukotriene C4, S-(2,4dinitrophenyl)glutathione, bilirubin glucuronosides, and 17beta-glucuronosyl estradiol. In addition, glutathione disulfide is transported by MRP1 and MRP2. Reduced glutathione may be released from cells in a process directly or indirectly mediated by members of the MRP family. Proteins of the MRP family are indispensable for transport of glutathione S-conjugates and glutathione disulfide into the extracellular space and play, therefore, a decisive role in detoxification and defense against oxidative stress.

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.

Production of peptide leukotrienes in endotoxin shock.

Arachidonate metabolites are potent mediators generated in endotoxin shock. Following endotoxin administration (15 mg/kg) into unanesthetized rats, we found a rapid biliary secretion of peptide leukotrienes. Analysis of bile for peptide leukotrienes included organic solvent extractions, reversed phase-HPLC, radioimmunoassay (RIA), and spectrophotometry. The major immunoreactive endogenous leukotriene (LT) from bile was eluted between LTC4 and LTD4 in three chromatographic systems. It corresponded thereby to a biliary metabolite of injected LTC4 and LTD4 which in turn showed the ultraviolet spectrum of a peptide leukotriene. This demonstration of endotoxin-induced generation of peptide LTs in vivo was possible by sequential HPLC and RIA analyses in bile into which peptide LTs are eliminated from blood.

Differential inhibition by cyclosporins of primary-active ATP-dependent transporters in the hepatocyte canalicular membrane.

The distinct ATP-dependent transporters for taurocholate, leukotriene C4, and daunorubicin, studied in rat liver canalicular membrane vesicles, are sensitive to inhibition by cyclosporin A and its non-immunosuppressive analog PSC 833. Ki values for cyclosporin A were 0.2, 3.4 and 1.5 microM for the transport of taurocholate, leukotriene C4, and daunorubicin, respectively. The corresponding Ki values for PSC 833 were 0.6, 29, and 0.3 microM. Both inhibitors were competitive with respect to the three substrates. The cyclosporins serve as new and potent tools to interfere with different potency with the distinct ATP-dependent export carriers in the hepatocyte canalicular membrane.

ATP-dependent transport of amphiphilic cations across the hepatocyte canalicular membrane mediated by mdr1 P-glycoprotein.

The ATP-dependent transport of the three 3H-labeled, amphiphilic cations quinidine, N-(n-pentyl)-quinidinium, and N-(4',4'-azo-n-pentyl)-21-deoxyajmalinium was studied in rat canalicular plasma membrane vesicles. N-Alkylation of quinidine with an n-pentyl residue resulted in a permanently charged cationic substrate for ATP-dependent transport which exhibited a 10-fold higher transport rate relative to quinidine. The Km value was 0.4 microM for N-(n-pentyl)-quinidinium and 5 microM for quinidine. The permanently cationic and photolabile derivative of ajmaline, N-(4',4'-azo-n-pentyl)-21-deoxyajmalinium, was also an efficient substrate and served to label canalicular membrane proteins with molecular masses of 143 kDa and 108 kDa. ATP-dependent transport of the permanently charged amphiphilic cations was inhibited by the P-glycoprotein inhibitors and substrates quinidine, verapamil, and daunorubicin. The data demonstrate that N-alkylation of quinidine and ajmaline results in most efficient substrates for mdr1 P-glycoprotein-mediated ATP-dependent transport.