RESUMO
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.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Canalículos Biliares/ultraestrutura , Resistência a Múltiplos Medicamentos/genética , Fígado/citologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/análise , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Trifosfato de Adenosina/fisiologia , Adulto , Sequência de Aminoácidos , Animais , Ânions/metabolismo , Sequência de Bases , Canalículos Biliares/metabolismo , Transporte Biológico/genética , Western Blotting , Membrana Celular/metabolismo , DNA Complementar/genética , Imunofluorescência , Expressão Gênica/fisiologia , Humanos , Membranas Intracelulares/metabolismo , Leucotrieno C4/metabolismo , Fígado/fisiologia , Fígado/ultraestrutura , Masculino , Microscopia Confocal , Pessoa de Meia-Idade , Dados de Sequência Molecular , Mutação/fisiologia , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , Ratos , Ratos Wistar , TrítioRESUMO
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.
Assuntos
Trifosfato de Adenosina/fisiologia , Proteínas de Transporte/metabolismo , Leucotrieno C4/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP , Sequência de Aminoácidos , Proteínas de Transporte/química , Membrana Celular/metabolismo , Humanos , Leucemia/metabolismo , Glicoproteínas de Membrana/química , Dados de Sequência Molecular , Proteínas de Neoplasias/química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Células Tumorais Cultivadas , Vacúolos/metabolismoRESUMO
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.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Glucuronatos/metabolismo , Glutationa/metabolismo , Sulfatos/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Antineoplásicos/metabolismo , Transporte Biológico , Resistência a Múltiplos Medicamentos , Estrogênios Conjugados (USP)/metabolismo , Técnicas de Transferência de Genes , Ácido Glucurônico , Células HeLa , HumanosRESUMO
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.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/química , Proteínas de Membrana Transportadoras , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Linhagem Celular , Membrana Celular/química , Resistência a Múltiplos Medicamentos , Regulação da Expressão Gênica , Humanos , Isoenzimas/química , Icterícia Idiopática Crônica/genética , Cinética , Fígado/metabolismo , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Especificidade por SubstratoRESUMO
Amphiphilic carboxylates acting as peroxisome proliferators and hypolipidemic drugs induce enzymes of peroxisomal lipid beta-oxidation, certain drug-metabolizing enzymes in the liver, and a number of additional proteins. The peroxisome proliferators represent a well-established class of non-genotoxic hepatocarcinogens. In this study we characterized the hepatic elimination of the peroxisome proliferator nafenopin. In the rat in vivo, 1 hr after intravenous administration of [3H]nafenopin, approx. 40% of injected radioactivity was recovered in bile. HPLC analysis of bile samples revealed that only about 10% of the radioactivity recovered in bile was associated with non-metabolized nafenopin and approx. 90% with more polar metabolites. One of the main metabolites formed in the liver and excreted into bile was identified as nafenopin glucuronide by beta-glucuronidase-catalysed reconversion to nafenopin. In mutant rats deficient in the canalicular transport of leukotriene C4 and related amphiphilic anion conjugates, recovery of [3H]nafenopin-derived radioactivity in bile was reduced to 4% of the injected dose. Although nafenopin glucuronide could not be detected in bile, it was a major metabolite in the liver from these mutant rats. Using membrane vesicles enriched in bile canalicular membranes from normal rats, transport of nafenopin glucuronide was shown to be a primary-active ATP-dependent process which was inhibited by leukotriene C4 and S-dinitrophenyl glutathione with IC50 values of 0.2 and 12 microM, respectively. ATP-dependent transport was not detectable for non-conjugated nafenopin. In canalicular membrane vesicles prepared from the mutant rats, the rate of ATP-dependent transport of nafenopin glucuronide was less than 10% of the transport observed in vesicles from normal rats. These data indicate that conjugation and subsequent transport by the ATP-dependent export carrier for leukotriene C4 and related conjugates is a major pathway for the elimination of nafenopin and structurally-related peroxisome proliferators.
Assuntos
Canalículos Biliares/metabolismo , Fígado/metabolismo , Nafenopina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Bile/metabolismo , Canalículos Biliares/ultraestrutura , Transporte Biológico , Glucuronatos/metabolismo , Masculino , Mutação , Ratos , Ratos Wistar , TrítioRESUMO
Cyclosporins are potent tools to inhibit several primary-active, ATP-dependent export carriers. This has been demonstrated in membrane vesicle transport assays for CsA and for its non-immunosuppressive analog PSC 833. Inhibition in the low micromolar and in the nanomolar concentration range is shown for the three distinct ATP-dependent export carriers in the liver canalicular membrane mediating the secretion into bile of leukotrienes (LTC4, other cysteinyl leukotrienes, and related conjugates), bile salts (taurocholate), and amphiphilic, mostly cationic substances (daunorubicin and other P-glycoprotein substrates). Competitive inhibition by cyclosporins is most potent for ATP-dependent taurocholate transport with Ki values of 0.2 and 0.6 microM for CsA and PSC 833, respectively. This inhibition is in agreement with in vivo studies in the rat demonstrating a block at the canalicular membrane in the hepatobiliary elimination of labeled taurocholate. The data suggest that cholestasis, as a side effect during CsA therapy, is largely due to inhibition of the ATP-dependent bile salt export carrier in the canalicular membrane. Inhibition by cyclosporins is less effective with respect to ATP-dependent leukotriene transport, both during biosynthetic release from mastocytoma cells and during hepatobiliary excretion. The Ki values for the former were 4.5 and 30 microM, and the Km/Ki ratios only 0.015 and 0.002 for CsA and PSC 833, respectively. Distinct transporters are inhibited by the cyclosporins with different potency and structurally modified cyclosporins may serve to induce preferential inhibition of a selected transporter. This is illustrated by the inhibition of the multidrug export carrier with daunorubicin as substrate using PSC 833 as inhibitor with a Ki value of 0.3 microM in an in vitro membrane transport system.
Assuntos
Trifosfato de Adenosina/metabolismo , Canalículos Biliares/metabolismo , Proteínas de Transporte/metabolismo , Membrana Celular/metabolismo , Ciclosporinas/farmacologia , Animais , Bile/metabolismo , Canalículos Biliares/citologia , Transporte Biológico Ativo/efeitos dos fármacos , Proteínas de Transporte/efeitos dos fármacos , Ciclosporina/farmacologia , Ciclosporinas/uso terapêutico , Daunorrubicina/metabolismo , Resistência a Múltiplos Medicamentos/fisiologia , Terapia de Imunossupressão/efeitos adversos , Leucotrieno C4/metabolismo , Masculino , Ratos , Ratos Wistar , Ácido Taurocólico/metabolismoRESUMO
The MRP gene encodes a 190-kDa integral membrane glycoprotein which functions as a primary-active ATP-dependent export pump for amphiphilic anions. The MRP gene-encoded conjugate export pump and its canalicular isoform represent the transport activity which has been described earlier as multispecific organic anion transporter, non-bile acid organic anion transporter, glutathione S-conjugate export pump, or leukotriene export pump. Analyses of the substrate specificity of the human MRP pump were performed in plasma membrane vesicles from MRP-overexpressing drug-selected cells (7) and cells transfected with an MRP expression vector (8). Substrates for MRP include thioether-linked conjugates of lipophilic compounds with glutathione, cysteinyl glycine, cysteine, and N-acetyl cysteine, but also glutathione disulfide, and glucuronate conjugates such as etoposide glucuronide. This broad-specificity ATP-dependent export pump is not only overexpressed in several multidrug resistant tumor cells and tissues, but is also present in most normal cells and tissues. The expression of cMRP and MRP in human liver and of cMrp and its homolog Mrp in rat liver was demonstrated by reverse transcription PCR, cDNA sequencing, and immunoblotting (13). The important function of the cMRP gene-encoded broad-specificity conjugate export pump in hepatobiliary excretion is illustrated by the selective absence of this canalicular isoform from the hepatocyte canalicular membrane in transport-deficient mutant rats. This altered lack of cMrp is the basis for the hereditary detect of the hepatobiliary excretion of anionic conjugates in the mutant animals (13). The absence of this canalicular Mrp in the mutants is analogous to the defect in the human Dubin-Johnson syndrome which is characterized by an impaired excretion of conjugated anions across the canalicular membrane.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Resistência a Múltiplos Medicamentos/fisiologia , Preparações Farmacêuticas/metabolismo , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Sequência de Aminoácidos , Animais , Proteínas de Transporte de Ânions , Bile/metabolismo , Proteínas de Transporte/metabolismo , Etoposídeo/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Leucotrieno C4/metabolismo , Fígado/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Ratos , Ratos Endogâmicos , Especificidade por Substrato , Transfecção/genéticaRESUMO
Several members of the multidrug resistance protein (MRP) family mediate the ATP-dependent transport of amphiphilic anions across membranes. The substrate specificity of recombinant human MRP1 has been most extensively defined by use of inside-out membrane vesicles. Substrates include the glutathione S-conjugate leukotriene C4, 17 beta-glucuronosyl estradiol, glucuronosyl bilirubin, glutathione disulfide, in addition to the fluorescent lipophilic anion Fluo-3. These substances are also substrates for the apical isoform MRP2, also termed canalicular multispecific organic anion transporter, cMOAT, which shares only 49% amino acid sequence identity with MRP1. The K(m) of leukotriene C4 for MRP2 is 10-fold higher than for MRP1, and the K(m) of 17 beta-glucuronosyl estradiol is 4.8-fold higher for MRP2 than for recombinant human MRP1. Human as well as rat MRP2 confer multidrug resistance to polarized MDCKII cells permanently expressing the recombinant glycoprotein in their apical plasma membrane. Resistance of cells transfected with human and rat MRP2 to etoposide was enhanced 5-fold and 3.8-fold, and resistance to vincristine was enhanced 2.3-fold and 6.0-fold, respectively. Conjugate-transporting members of the MRP family with a related sequence and a similar function have been detected recently. In addition to several MRP isoforms (MRP1-6) and orthologs in mammals (human, rat, rabbit, mouse), MRP family members have been identified in the nematode Caenorhabditis elegans, in the yeast Saccharomyces cerevisiae, and in the plant Arabidopsis thaliana. These conjugate export pumps of the MRP family play a widespread role in detoxification, drug resistance, and, because of the role in the export of glutathione disulfide by MRP1 and MRP2, in the defense against oxidative stress.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Genes MDR , Trifosfato de Adenosina/metabolismo , Animais , Proteínas de Transporte de Ânions , Linhagem Celular , Cães , Resistência a Múltiplos Medicamentos/genética , Resistência a Múltiplos Medicamentos/fisiologia , Humanos , Técnicas In Vitro , Transporte de Íons , Cinética , Camundongos , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Filogenia , Coelhos , Ratos , TransfecçãoRESUMO
Transport processes control not only synthesis and release of LTC4 but also the elimination and excretion of LTC4 and its metabolites. (i) A primary-active ATP-dependent export carrier mediates the release of LTC4 from a leukotriene-generating cell, as exemplified by mastocytoma cells, and as measured in mastocytoma plasma membrane vesicles (2). (ii) Release of cysteinyl leukotrienes into the blood circulation is followed by a rapid elimination with an initial half-life of 38 sec in rats and 4.0 min in man, as measured with the labeled, representative LTC4 catabolite, N-acetyl-LTE4. (iii) 11C-labeled N-acetyl-LTE4 can serve for non-invasive studies on cysteinyl leukotriene elimination and excretion by the liver and kidney in the intact organism using positron emission tomography. An impairment of leukotriene transport from the liver across the canalicular membrane into bile, studied in mutant rats and in extrahepatic cholestasis, leads to a compensatory diversion of cysteinyl leukotriene elimination to the kidney. N-Acetyl-LTE4 labeled with a short-lived positron-emitting isotope provides quantitative insight into the pathways of cysteinyl leukotriene elimination in vivo. (iv) Cysteinyl leukotriene export from the liver into bile is mediated by an ATP-dependent primary-active export carrier. This decisive step in cysteinyl leukotriene elimination has been characterized in hepatocyte canalicular membrane vesicles (3). The leukotriene exporter is deficient in transport mutant rats. The leukotriene carrier is distinct from other ATP-dependent export carriers identified in this membrane domain, such as the ATP-dependent bile salt export carrier (25) and the multidrug export carrier (27).
Assuntos
Leucotrieno E4/análogos & derivados , SRS-A/análogos & derivados , Trifosfato de Adenosina/farmacologia , Bile/metabolismo , Transporte Biológico Ativo , Membrana Celular/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Fígado/metabolismo , Masculino , Taxa de Depuração Metabólica , Pessoa de Meia-Idade , SRS-A/sangue , SRS-A/metabolismo , Tomografia Computadorizada de Emissão/métodosRESUMO
Bilirubin conjugates are secreted from hepatocytes into bile. Monoglucuronosyl bilirubin is an endogenous substrate for the multidrug resistance protein 2, which is located in rat hepatocyte canalicular membrane. We have characterized this ATP-dependent transport using rat canalicular membrane vesicles. Monoglucuronosyl bilirubin, 3H-labeled in the glucuronosyl moiety, was synthesized enzymatically using recombinant UDP-glycosyltransferase 1A1, and was stabilized with ascorbate. The rate for ATP-dependent transport of monoglucuronosyl bilirubin (at 0.5 µM) was 7.3+/-1.1 pmol mg protein(-1) min(-1) and the K(m) value was 1.3+/-0.4 µM. This is the first time to demonstrate this kinetic constant of monoglucuronosyl bilirubin for the rat hepatocyte canalicular membrane. The K(m) value is similar to one for recombinant rat multidrug resistance protein 2.
RESUMO
The membrane proteins mediating the ATP-dependent transport of glutathione S-conjugates and related amphiphilic anions have been identified as the multidrug resistance proteins MRP1 and MRP2. These 190-kDa membrane glycoproteins were cloned in recent years and shown to be unidirectional, ATP-driven, export pumps with an amino acid identity of 49% in humans. MRP1 is detected in the plasma membrane of many cell types, including erythrocytes; whereas MRP2, also termed canalicular MRP (cMRP) or canalicular multispecific organic anion transporter (cMOAT), has been localized to the apical domain of polarized epithelia, such as the hepatocyte canalicular membrane and kidney proximal tubule luminal membrane. Physiologically important substrates of both transporters include glutathione S-conjugates, such as leukotriene C4, as well as bilirubin glucuronides. 17 beta-glucuronosyl estradiol and glutathione disulfide. Both transporters have been associated with multiple drug resistance of malignant tumors because of their capacity to pump drug conjugates and drug complexes across the plasma membrane into the extracellular space. The substrate specificity of MRP1 and MRP2 studied in inside-out oriented membrane vesicles is very different from MDR1 P-glycoprotein. MRP1 and MRP2 may be termed conjugate transporting ATPases, functioning in detoxification and, because of their role in glutathione disulfide export, in the defense against oxidative stress.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Trifosfato de Adenosina/metabolismo , Glutationa/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Proteínas de Transporte de Ânions , Transporte Biológico Ativo , Proteínas de Transporte/metabolismo , Resistência a Múltiplos Medicamentos , Humanos , Inativação Metabólica , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Mutação , Estresse Oxidativo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoAssuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Leucotrieno C4/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/isolamento & purificação , Trifosfato de Adenosina/metabolismo , Transporte Biológico , Fracionamento Celular/métodos , Linhagem Celular , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Humanos , Cinética , Técnica de Diluição de Radioisótopos , Especificidade por Substrato , TrítioAssuntos
Transportadores de Cassetes de Ligação de ATP/genética , Resistência a Múltiplos Medicamentos/genética , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Transporte Biológico , Linhagem Celular , Humanos , Dados de Sequência Molecular , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
The search for the membrane proteins mediating the ATP-dependent transport of conjugates with glutathione, glucuronate, or sulfate has led to the identification of the multidrug resistance proteins MRP1 and MRP2. Both 190-kDa membrane glycoproteins were cloned in the recent years and shown to be unidirectional ATP-driven export pumps with an amino acid identity of 49% in human. MRP1 is detected in the plasma membrane of many cell types, including erythrocytes, whereas MRP2, also termed canalicular MRP (cMRP) or canalicular multispecific organic anion transporter (cMOAT), has been localized to the apical domain of polarized epithelia, particularly to the hepatocyte canalicular membrane. Physiologically important substrates of both transporters include glutathione S-conjugates such as leukotriene C4, bilirubin glucuronides, 17 beta-glucuronosyl estradiol, dianionic bile salts such as 6 alpha-glucuronosyl hyodeoxycholate, and glutathione disulfide. Both transporters have been associated with multiple drug resistance of malignant tumors because of their capacity to pump drug conjugates and drug complexes across the plasma membrane into the extracellular space. The substrate specificity of MRP1 and MRP2 is very different from MDR1 P-glycoprotein. MRP1 and MRP2 may be termed conjugate transporting ATPases functioning in detoxification and, because of their role in glutathione disulfide export, in the defense against oxidative stress.
Assuntos
Citocinas/metabolismo , Glucuronatos/metabolismo , Glutationa/metabolismo , Proteínas Inflamatórias de Macrófagos , Proteínas de Membrana Transportadoras , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Animais , Quimiocinas CC , Citocinas/genética , Resistência a Múltiplos Medicamentos/genética , Dissulfeto de Glutationa/metabolismo , Humanos , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Estresse Oxidativo/fisiologiaRESUMO
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.
Assuntos
Carcinoma Hepatocelular/metabolismo , Leucotrienos/metabolismo , Neoplasias Hepáticas Experimentais/metabolismo , Neoplasias Hepáticas/metabolismo , Fígado/metabolismo , Marcadores de Afinidade , Animais , Ligação Competitiva , Humanos , Cinética , Leucotrieno B4/metabolismo , Leucotrieno E4 , Fotoquímica , Ratos , Ratos Sprague-Dawley , SRS-A/análogos & derivados , SRS-A/metabolismo , Sulfobromoftaleína/farmacologia , Trítio , Células Tumorais CultivadasRESUMO
The biosynthesis of leukotriene C4 (LTC4) must be followed by an export of this mediator into the extracellular space where it interacts with receptors. Using mastocytoma cells we have demonstrated the existence of a primary-active, ATP-dependent transport mediating this export of LTC4 [Schaub, T., Ishikawa, T. & Keppler, D. (1991) FEBS Lett. 279, 83-86]. The following inhibitors served to characterize further this transport system in plasma membrane vesicles from mastocytoma cells: Probenecid, an inhibitor of organic anion transport, induced half-maximal inhibition of the ATP-dependent LTC4 transport at 71 microM. Cyclosporin A and its non-immunosuppressive analog PSC 833 inhibited the ATP-dependent transport with Ki values of 4.5 microM and 30 microM, respectively. The LTD4 receptor antagonist 3-([(3-(2-[7-chloro-2-quinolinyl]ethenyl)phenyl)-[(3-dimethylamino-3- oxopropyl)-thio]-methyl]thio)propanoic acid (MK 571) was the most potent competitive inhibitor of the export carrier with a Ki value of 0.8 microM. The transport inhibitor MK 571 served as competitor in the photoaffinity labeling of LTC4-binding membrane proteins using [3H]LTC4 as the photolabile ligand. Proteins with molecular masses of about 190 kDa and 35 kDa were predominantly labeled. In addition, a minor [3H]LTC4 labeling was observed in the molecular mass range of 100 kDa. The [3H]LTC4 labeling of the 190-kDa protein was competed for by MK 571. The labeled proteins resisted extraction from the membrane with 2% sodium taurocholate suggesting that they are integral membrane proteins. Treatment of the membrane proteins with peptide N-glycosidase F resulted in the appearance of an additional labeled polypeptide of about 140 kDa suggesting that the 190-kDa protein is a glycoprotein. Photoaffinity labeling with 8-azido[alpha-32P]ATP predominantly labeled the LTC4-binding 35-kDa protein. The [3H]LTC4-labeled 190-kDa protein showed a mean isoelectric point at pH 6.3 with a range of pH 5.8-6.7, while the 35-kDa protein had an isoelectric point at pH 6.8. Specific labeling of a 190-kDa membrane glycoprotein by the glutathione conjugate LTC4, which is competed for by a potent inhibitor of the ATP-dependent LTC4 export carrier, pinpoints its involvement in the ATP-dependent transport of LTC4 and related conjugates.
Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas de Transporte/metabolismo , Leucotrieno C4/metabolismo , Sarcoma de Mastócitos/metabolismo , Glicoproteínas de Membrana/metabolismo , Trifosfato de Adenosina/análogos & derivados , Marcadores de Afinidade/metabolismo , Animais , Azidas/metabolismo , Proteínas de Transporte/isolamento & purificação , Linhagem Celular , Membrana Celular/metabolismo , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Cinética , Glicoproteínas de Membrana/isolamento & purificação , Camundongos , Camundongos Endogâmicos BALB C , Peso Molecular , Células Tumorais CultivadasRESUMO
BACKGROUND: Para-aminohippurate (PAH), a widely used model substrate for organic anion transport in proximal tubule epithelia, was investigated as a substrate for the apical multidrug resistance protein MRP2 (symbol ABCC2). This ATP-dependent export pump for anionic conjugates and additional amphiphilic anions was cloned recently and localized to the apical membrane of proximal tubules in human and rat kidney. METHODS: Membrane vesicles from HEK-MRP2 cells containing recombinant human MRP2 and from control vector-transfected HEK-Co cells were incubated with various concentrations of [3H]PAH, and the net ATP-dependent transport into inside-out vesicles was determined. Comparative studies were performed with membrane vesicles containing recombinant human MRP1. RESULTS: Transport rates at 10 micromol/L PAH were 21.9 +/- 1.9 and 1.6 +/- 0.4 pmol x mg protein-1 x min-1 (means +/- SEM, N = 10) with membrane vesicles from HEK-MRP2 and HEK-Co cells, respectively. The Km value for PAH was 880 micromol/L. The high-affinity substrate leukotriene C4 and the inhibitor of MRP-mediated transport, MK571, inhibited MRP2-mediated transport of PAH (100 nmol/L) with IC50 values of 3.3 and 4.0 micromol/L, respectively. The nephrotoxic mycotoxin ochratoxin A inhibited MRP2-mediated PAH transport with an IC50 value of 58 micromol/L. Ochratoxin A was itself a substrate for MRP2. CONCLUSIONS: PAH is a good substrate for the ATP-dependent export pump MRP2. The localization and function of MRP2 indicate that this unidirectional transport protein contributes to the secretion of PAH and other amphiphilic anions into the lumen of kidney proximal tubules.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Trifosfato de Adenosina/fisiologia , Proteínas de Membrana Transportadoras , Ácido p-Aminoipúrico/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Transporte Biológico/fisiologia , Linhagem Celular , Membrana Celular/metabolismo , Células HeLa , Humanos , Cinética , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Ocratoxinas/metabolismo , Ocratoxinas/farmacologia , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Ácido p-Aminoipúrico/antagonistas & inibidoresRESUMO
omega-Oxidation with subsequent beta-oxidation from the omega-end is the major pathway for inactivation and degradation of leukotrienes. Oxidative degradation of leukotriene E4 (LTE4), N-acetyl-LTE4, and LTB4 was inhibited by the omega-trifluoro analogs of LTE4, omega-trifluoro-LTE4 (omega-F3-LTE4), and (1S,2R)-5-(3-[1-hydroxy-15,15,15-trifluoro-2-(2-1H- tetrazol-5-ylethyl-thio)pentadeca-3(E),5(Z)-dienyl+ ++]phenyl)-1H-tetrazole (LY 245769). The latter substance inhibited the oxidative degradation of LTE4 and N-acetyl-LTE4 in the rat in vivo by 50% at a dose of 7 mumol/kg body weight. In rat hepatocyte cultures both omega-trifluoro analogs interfered with the omega-oxidation of N-acetyl-LTE4 and LTB4 with IC50 values of about 4 microM. Both analogs inhibited the omega-hydroxylation in isolated rat liver microsomes with IC50 values between 16 and 37 microM. This inhibition is apparently competitive. In addition, in liver cytosol, the conversion of the omega-hydroxylated leukotrienes to omega-carboxy-LTE4 and omega-carboxy-LTB4 was inhibited by both compounds. omega-Trifluoro analogs of leukotrienes provide a new tool for interfering with the inactivation of leukotrienes in the omega-oxidation pathway.
Assuntos
Leucotrieno B4/metabolismo , Fígado/efeitos dos fármacos , SRS-A/análogos & derivados , Sulfetos/farmacologia , Tetrazóis/farmacologia , Animais , Bile/química , Células Cultivadas , Leucotrieno E4 , Fígado/metabolismo , Masculino , Microssomos Hepáticos/metabolismo , Oxirredução , Ratos , Ratos Endogâmicos , SRS-A/química , SRS-A/metabolismo , SRS-A/farmacologia , Sulfetos/química , Tetrazóis/químicaRESUMO
The multidrug resistance-associated protein (MRP) is the product of an ATP-binding cassette transporter gene overexpressed in some tumor cells resistant to antineoplastic agents. We studied the transport function of MRP in membrane vesicles prepared from HeLa cells transfected with an MRP expression vector and overexpressing this 190-kDa membrane glycoprotein. ATP-dependent primary-active transport into the vesicles was demonstrated for leukotriene C4 (LTC4), LTD4, LTE4, and S-(2,4-dinitrophenyl)glutathione with relative rates, at a substrate concentration of 50 nM, of 1.0, 0.27, 0.14, and 0.16, respectively. The endogenous glutathione conjugate LTC4 had the highest affinity for this transporter with a Km of 97 nM. The Km for ATP was 19 microM. Direct photoaffinity labeling with [3H]LTC4 labeled a 190-kDa membrane protein predominantly in the MRP-transfected HeLa cells. ATP-dependent LTC4 transport was effectively inhibited by the LTD4 receptor antagonist MK 571, whereas cyclosporin A and, particularly, its analog PSC 833 were much less potent. The respective Ki values were 0.6, 5, and 27 microM, respectively. In addition, MK 571 preferentially inhibited photoaffinity labeling of the 190-kDa protein in the MRP transfectants. Our results provide direct evidence that the MRP gene encodes a primary-active ATP-dependent export pump for conjugates of lipophilic compounds with glutathione and several other anionic residues. We conclude that the biosynthetic release of LTC4 from cells is mediated by the 190-kDa product of the MRP gene.
Assuntos
Transportadores de Cassetes de Ligação de ATP/biossíntese , Trifosfato de Adenosina/metabolismo , Resistência a Múltiplos Medicamentos/genética , Leucotrieno C4/metabolismo , Receptores de Leucotrienos , Transportadores de Cassetes de Ligação de ATP/isolamento & purificação , Transportadores de Cassetes de Ligação de ATP/metabolismo , Transporte Biológico , Membrana Celular/metabolismo , Ciclosporina/farmacologia , Células HeLa , Humanos , Cinética , Antagonistas de Leucotrienos , Leucotrieno D4/metabolismo , Leucotrieno E4/metabolismo , Proteínas de Membrana/biossíntese , Proteínas de Membrana/isolamento & purificação , Proteínas de Membrana/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Propionatos/farmacologia , Quinolinas/farmacologia , Especificidade por Substrato , TransfecçãoRESUMO
BACKGROUND/AIMS: Inhibition of bile salt transport across the hepatocyte during cholestasis induced by cyclosporin A has been shown. However, the contribution of the different bile salt transport systems in liver to cholestasis has remained controversial. METHODS: The sensitivity of different bile salt transport systems in liver to cyclosporin-induced inhibition was determined by transport assays in plasma membrane vesicles and by in vivo studies in the rat. RESULTS: Cyclosporin A--induced inhibition of sodium-dependent uptake of bile salts across the sinusoidal membrane, of potential-dependent, and of adenosine triphosphate (ATP)-dependent bile salt transport across the canalicular membrane exhibited inhibition constants (Ki) of 5, 70, and 0.2 mumol/L, respectively. The nonimmunosuppressive cyclosporin analogue PSC 833 also preferentially inhibited the ATP-dependent bile salt transport with an inhibition constant of 0.6 mumol/L. Cyclosporin A and its analogue PSC 833 [(3'-oxo-4-butenyl-4-methyl-Thr1)-(Val2)-cyclosporin] (25 mg/kg each) served as tools to interfere with [14C]taurocholate secretion into bile in vivo, causing an accumulation of [14C]-taurocholate in liver and reducing bile flow to 50%. In mutant rats deficient in the transport of leukotriene C4 and related conjugates across the canalicular membrane, bile flow was reduced to 14%. CONCLUSIONS: The cyclosporins preferentially inhibit the ATP-dependent bile salt export carrier in the canalicular membrane. This inhibition reduces bile salt-dependent bile flow and causes intrahepatic cholestasis.