RESUMO
AIMS: Estradiol 17ß-d-glucuronide (E217G) induces cholestasis by triggering endocytosis and further intracellular retention of the canalicular transporters Bsep and Mrp2, in a cPKC- and PI3K-dependent manner, respectively. Pregnancy-induced cholestasis has been associated with E217G cholestatic effect, and is routinely treated with ursodeoxycholic acid (UDCA). Since protective mechanisms of UDCA in E217G-induced cholestasis are still unknown, we ascertained here whether its main metabolite, tauroursodeoxycholate (TUDC), can prevent endocytosis of canalicular transporters by counteracting cPKC and PI3K/Akt activation. MAIN METHODS: Activation of cPKC and PI3K/Akt was evaluated in isolated rat hepatocytes by immunoblotting (assessment of membrane-bound and phosphorylated forms, respectively). Bsep/Mrp2 function was quantified in isolated rat hepatocyte couplets (IRHCs) by assessing the apical accumulation of their fluorescent substrates, CLF and GS-MF, respectively. We also studied, in isolated, perfused rat livers (IPRLs), the status of Bsep and Mrp2 transport function, assessed by the biliary excretion of TC and DNP-SG, respectively, and Bsep/Mrp2 localization by immunofluorescence. KEY FINDINGS: E217G activated both cPKC- and PI3K/Akt-dependent signaling, and pretreatment with TUDC significantly attenuated these activations. In IRHCs, TUDC prevented the E217G-induced decrease in apical accumulation of CLF and GS-MF, and inhibitors of protein phosphatases failed to counteract this protection. In IPRLs, E217G induced an acute decrease in bile flow and in the biliary excretion of TC and DNP-SG, and this was prevented by TUDC. Immunofluorescence studies revealed that TUDC prevented E217G-induced Bsep/Mrp2 endocytosis. SIGNIFICANCE: TUDC restores function and localization of Bsep/Mrp2 impaired by E217G, by preventing both cPKC and PI3K/Akt activation in a protein-phosphatase-independent manner.
Assuntos
Colestase , Endocitose , Estradiol , Hepatócitos , Fosfatidilinositol 3-Quinases , Transdução de Sinais , Ácido Tauroquenodesoxicólico , Animais , Colestase/metabolismo , Colestase/induzido quimicamente , Colestase/prevenção & controle , Ratos , Transdução de Sinais/efeitos dos fármacos , Estradiol/metabolismo , Estradiol/farmacologia , Estradiol/análogos & derivados , Hepatócitos/metabolismo , Hepatócitos/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Ácido Tauroquenodesoxicólico/farmacologia , Ácido Tauroquenodesoxicólico/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos Wistar , Feminino , Masculino , Proteína Quinase C/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismoRESUMO
The endogenous metabolite of estradiol, estradiol 17ß-D-glucuronide (E17G), is considered the main responsible of the intrahepatic cholestasis of pregnancy. E17G alters the activity of canalicular transporters through a signaling pathway-dependent cellular internalization, phenomenon that was attributed to oxidative stress in different cholestatic conditions. However, there are no reports involving oxidative stress in E17G-induced cholestasis, representing this the aim of our work. Using polarized hepatocyte cultures, we showed that antioxidant compounds prevented E17G-induced Mrp2 activity alteration, being this alteration equally prevented by the NADPH oxidase (NOX) inhibitor apocynin. The model antioxidant N-acetyl-cysteine prevented, in isolated and perfused rat livers, E17G-induced impairment of bile flow and Mrp2 activity, thus confirming the participation of reactive oxygen species (ROS) in this cholestasis. In primary cultured hepatocytes, pretreatment with specific inhibitors of ERK1/2 and p38MAPK impeded E17G-induced ROS production; contrarily, NOX inhibition did not affect ERK1/2 and p38MAPK phosphorylation. Both, knockdown of p47phox by siRNA and preincubation with apocynin in sandwich-cultured rat hepatocytes significantly prevented E17G-induced internalization of Mrp2, suggesting a crucial role for NOX in this phenomenon. Concluding, E17G-induced cholestasis is partially mediated by NOX-generated ROS through internalization of canalicular transporters like Mrp2, being ERK1/2 and p38MAPK necessary for NOX activation.
Assuntos
Estradiol , Hepatócitos , NADPH Oxidases , Espécies Reativas de Oxigênio , Animais , NADPH Oxidases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Ratos , Hepatócitos/metabolismo , Hepatócitos/efeitos dos fármacos , Estradiol/farmacologia , Estradiol/metabolismo , Estradiol/análogos & derivados , Feminino , Colestase/induzido quimicamente , Colestase/metabolismo , Colestase/patologia , Ratos Wistar , Acetofenonas/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Acetilcisteína/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Células Cultivadas , Antioxidantes/farmacologia , Antioxidantes/metabolismo , Colestase Intra-Hepática , Complicações na Gravidez , Transportadores de Cassetes de Ligação de ATPRESUMO
Oxidative stress (OS) is a key factor in the development of gastrointestinal disorders, in which the intestinal barrier is altered. However, the Multidrug resistance-associated protein 2 (Mrp2) status, an essential component of the intestinal transcellular barrier exhibiting pharmaco-toxicological relevance by limiting the orally ingested toxicants and drugs absorption, has not been investigated. We here evaluated the short-term effect of OS on Mrp2 by treatment of isolated rat intestinal sacs with tert-butyl hydroperoxide (TBH) for 30 min. OS induction by TBH (250 and 500 µM) was confirmed by increased lipid peroxidation end products, decreased reduced glutathione (GSH) content and altered antioxidant enzyme activities. Under this condition, assessment of Mrp2 distribution between brush border (BBM) and intracellular (IM) membrane fractions, showed that Mrp2 protein decreased in BBM and increased in IM, consistent with an internalization process. This was associated with decreased efflux activity and, consequently, impaired barrier function. Subsequent incubation with N-Acetyl-L-Cysteine (NAC, 1 mM) reestablished GSH content and reverted concomitantly the alteration in Mrp2 localization and function induced by TBH. Cotreatment with a specific inhibitor of classic calcium-dependent Protein Kinase C (cPKC) implicated this kinase in TBH-effects. In conclusion, we demonstrated a negative posttranslational regulation of rat intestinal Mrp2 after short-term exposition to OS, a process likely mediated by cPKC and dependent on intracellular GSH content. The concomitant impairment of the Mrp2 barrier function may have implications in xenobiotic absorption and toxicity in a variety of human diseases linked to OS, with notable consequences on the toxicity/safety of therapeutic agents.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Mucosa Intestinal/metabolismo , Jejuno/metabolismo , Microvilosidades/metabolismo , Estresse Oxidativo/fisiologia , Processamento de Proteína Pós-Traducional/fisiologia , Animais , Relação Dose-Resposta a Droga , Mucosa Intestinal/efeitos dos fármacos , Jejuno/efeitos dos fármacos , Masculino , Microvilosidades/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Ratos , Ratos Wistar , terc-Butil Hidroperóxido/toxicidadeRESUMO
The extensive intestinal surface offers an advantage regarding nutrient, ion and water absorptive capacity but also brings along a high exposition to xenobiotics, including drugs of therapeutic use and food contaminants. After absorption of these compounds by the enterocytes, apical ABC transporters play a key role in secreting them back to the intestinal lumen, hence acting as a transcellular barrier. Rapid and reversible modulation of their activity is a subject of increasing interest for pharmacologists. On the one hand, a decrease in transporter activity may result in increased absorption of therapeutic agents given orally. On the other hand, an increase in transporter activity would decrease their absorption and therapeutic efficacy. Although of less relevance, apical ABC transporters also contribute to disposition of drugs systemically administered. This review article summarizes the present knowledge on the mechanisms aimed to rapidly regulate the activity of the main apical ABC transporters of the gut: multidrug resistance protein 1 (MDR1), multidrug resistance-associated protein 2 (MRP2) and breast cancer resistance protein (BCRP). Regulation of these mechanisms by drugs, drug delivery systems, drug excipients and nutritional components are particularly considered. This information could provide the basis for controlled regulation of bioavailability of therapeutic agents and at the same time would help to prevent potential drug-drug interactions.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Trato Gastrointestinal/metabolismo , Preparações Farmacêuticas/metabolismo , Animais , Disponibilidade Biológica , HumanosRESUMO
Mercury is a widespread pollutant. Mercuric ions uptake into tubular cells is supported by the Organic anion transporter 1 (Oat1) and 3 (Oat3) and its elimination into urine is through the Multidrug resistance-associated protein 2 (Mrp2). We investigated the effect of recombinant human erythropoietin (Epo) on renal function and on renal expression of Oat1, Oat3, and Mrp2 in a model of mercuric chloride (HgCl2)-induced renal damage. Four experimental groups of adult male Wistar rats were used: Control, Epo, HgCl2, and Epo + HgCl2. Epo (3000 IU/kg, b.w., i.p.) was administered 24 h before HgCl2 (4 mg/kg, b.w., i.p.). Experiments were performed 18 h after the HgCl2 dose. Parameters of renal function and structure were evaluated. The protein expression of Oat1, Oat3 and Mrp2 in renal tissue was assessed by immunoblotting techniques. Mercury levels were determined by cold vapor atomic absorption spectrometry. Pretreatment with Epo ameliorated the HgCl2-induced tubular injury as assessed by histopathology and urinary biomarkers. Immunoblotting showed that pretreatment with Epo regulated the renal expression of mercury transporters in a way to decrease mercury content in the kidney. Epo pretreatment ameliorates HgCl2-induced renal tubular injury by modulation of mercury transporters expression in the kidneys.
Assuntos
Eritropoetina/uso terapêutico , Nefropatias/tratamento farmacológico , Cloreto de Mercúrio/toxicidade , Substâncias Protetoras/uso terapêutico , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Eritropoetina/genética , Eritropoetina/farmacologia , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Nefropatias/induzido quimicamente , Nefropatias/metabolismo , Nefropatias/patologia , Masculino , Mercúrio/sangue , Mercúrio/metabolismo , Mercúrio/urina , Proteína 1 Transportadora de Ânions Orgânicos/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Substâncias Protetoras/farmacologia , Ratos Wistar , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Ureia/sangueRESUMO
AIM: MRP2 is an intestinal ABC transporter that prevents the absorption of dietary xenobiotics. The aims of this work were: (1) to evaluate whether a short-term regulation of intestinal MRP2 barrier function takes place in vivo after luminal incorporation of nutrients and (2) to explore the underlying mechanism. METHODS: MRP2 activity and localization were assessed in an in vivo rat model with preserved irrigation and innervation. Nutrients were administered into distal jejunum. After 30-minutes treatments, MRP2 activity was assessed in proximal jejunum by quantifying the transport of the model substrate 2,4-dinitrophenyl-S-glutathione. MRP2 localization was determined by quantitative confocal microscopy. Participation of extracellular mediators was evaluated using selective inhibitors and by immunoneutralization. Intracellular pathways were explored in differentiated Caco-2 cells. RESULTS: Oleic acid, administered intraluminally at dietary levels, acutely stimulated MRP2 insertion into brush border membrane. This was associated with increased efflux activity and, consequently, enhanced barrier function. Immunoneutralization of the gut hormone glucagon-like peptide-2 (GLP-2) prevented oleic acid effect on MRP2, demonstrating the participation of this trophic factor as a main mediator. Further experiments using selective inhibitors demonstrated that extracellular adenosine synthesis and its subsequent binding to enterocytic A2B adenosine receptor (A2BAR) take place downstream GLP-2. Finally, studies in intestinal Caco-2 cells revealed the participation of A2BAR/cAMP/PKA intracellular pathway, ultimately leading to increased MRP2 localization in apical domains. CONCLUSION: These findings reveal an on-demand, acute regulation of MRP2-associated barrier function, constituting a novel physiological mechanism of protection against the absorption of dietary xenobiotics in response to food intake.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Peptídeo 2 Semelhante ao Glucagon , Animais , Células CACO-2 , Humanos , Mucosa Intestinal , Nutrientes , Ratos , Ratos WistarRESUMO
Multidrug resistance-associated protein 2 (Mrp2), expressed at the brush border membrane (BBM) of the enterocyte, is an ABC transporter with relevant intestinal barrier function. Its toxicological relevance lies in preventing absorption and tissue accumulation of dietary contaminants, drugs, and potentially harmful endogenous metabolites. Expression and activity of intestinal Mrp2 is downregulated in LPS-induced endotoxemia. In addition, confocal microscopy studies demonstrated internalization of the transporter to endocytic vesicles. Since IL-1ß plays an important role as early mediator of LPS-inflammatory responses, we evaluated whether IL-1ß mediates LPS-induced impairment of Mrp2 function. Two protocols were used: I) In vivo administration of LPS (5 mg/kg b.wt., i.p., single dose) to rats in simultaneous with administration of anti-IL-1ß (25 µg/kg b.wt., i.p., 4 doses), followed by studies of Mrp2 expression, localization and activity, 24 h after LPS administration; II) In vitro incubation of isolated intestinal sacs with IL-1ß (10 ng/mL) for 30 min, followed by analysis of Mrp2 activity and localization. We found that in vivo immunoneutralization of IL-1ß partially prevented the decrease of Mrp2 protein expression and activity as well as its internalization to intracellular domains induced by LPS. Involvement of IL-1ß in the alteration of Mrp2 localization and activity was more directly demonstrated in isolated intestinal sacs, as incubation with IL-1ß resulted in detection of Mrp2 in intracellular regions of the enterocyte in simultaneous with alteration of transport activity. In conclusion, IL-1ß induces early internalization of intestinal Mrp2, which could partially explain loss of expression at the BBM under conditions of experimental endotoxemia. Concomitant impairment of Mrp2-dependent barrier function may have pathophysiological relevance since IL-1ß mediates the effect of many local and systemic inflammatory processes.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Endotoxemia/metabolismo , Interleucina-1beta/metabolismo , Mucosa Intestinal/metabolismo , Animais , Western Blotting , Endotoxemia/patologia , Feminino , Mucosa Intestinal/ultraestrutura , Microscopia Confocal , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Taurolithocholate (TLC) is a cholestatic bile salt that induces disinsertion of the canalicular transporter Abcc2 (Mrp2, multidrug resistance-associated protein 2). This internalization is mediated by different intracellular signaling proteins such as PI3K, PKCε and MARCK but the initial receptor of TLC remains unknown. A few G protein-coupled receptors interact with bile salts in hepatocytes. Among them, sphingosine-1 phosphate receptor 2 (S1PR2) represents a potential initial receptor for TLC. The aim of this study was to evaluate the role of this receptor and its downstream effectors in the impairment of Abcc2 function induced by TLC. In vitro, S1PR2 inhibition by JTE-013 or its knockdown by small interfering RNA partially prevented the decrease in Abcc2 activity induced by TLC. Moreover, adenylyl cyclase (AC)/PKA and PI3K/Akt inhibition partially prevented TLC effect on canalicular transporter function. TLC produced PKA and Akt activation, which were blocked by JTE-013 and AC inhibitors, connecting S1PR2/AC/PKA and PI3K/Akt in a same pathway. In isolated perfused rat liver, injection of TLC triggered endocytosis of Abcc2 that was accompanied by a sustained decrease in the bile flow and the biliary excretion of the Abcc2 substrate dinitrophenyl-glutathione until the end of the perfusion period. S1PR2 or AC inhibition did not prevent the initial decay, but they accelerated the recovery of these parameters and the reinsertion of Abcc2 into the canalicular membrane. In conclusion, S1PR2 and the subsequent activation of AC, PKA, PI3K and Akt is partially responsible for the cholestatic effects of TLC through sustained internalization of Abcc2.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Adenilil Ciclases/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Receptores de Esfingosina-1-Fosfato/metabolismo , Ácido Taurolitocólico/farmacologia , Animais , Células Cultivadas , Feminino , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Redes e Vias Metabólicas/efeitos dos fármacos , Técnicas de Cultura de Órgãos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirazóis/farmacologia , Piridinas/farmacologia , Ratos Wistar , Receptores de Esfingosina-1-Fosfato/antagonistas & inibidores , Receptores de Esfingosina-1-Fosfato/genética , Ácido Taurolitocólico/metabolismoRESUMO
Intestinal multidrug resistance-associated protein 2 is an ABC transporter that limits the absorption of xenobiotics ingested orally, thus acting as essential component of the intestinal biochemical barrier. Metabolic Syndrome (MetS) is a pathological condition characterized by dyslipidemia, hyperinsulinemia, insulin resistance, chronic inflammation, and oxidative stress (OS). In a previous study we demonstrated that MetS-like conditions induced by fructose in drinking water (10% v/v, during 21 days), significantly reduced the expression and activity of intestinal Mrp2 in rats. We here evaluated the potential beneficial effect of geraniol or vitamin C supplementation, natural compounds with anti-inflammatory and anti-oxidant properties, in reverse fructose-induced Mrp2 alterations. After MetS-like conditions were induced (21 days), animals were cotreated with geraniol or vitamin C or vehicle for another 14 days. Decreased expression of Mrp2 protein and mRNA due to fructose administration was reversed by geraniol and by vitamin C, consistent with restoration of Mrp2 activity evaluated in everted intestinal sacs. Concomitantly, increased intestinal IL-1ß and IL-6 levels induced by fructose were totally and partially counterbalanced, respectively, by geraniol administration. The intestinal redox unbalance generated by fructose was improved by geraniol and vitamin C, as evidenced by decreasing lipid peroxidation products and activity of Superoxide Dismutase and by normalizing glutathione reduced/oxidized glutathione ratio. The restoration effects exhibited by geraniol and vitamin C suggest that local inflammatory response and OS generated under MetS-like conditions represent important mediators of the intestinal Mrp2 down-regulation. Additionally, both agents could be considered of potential therapeutic value to preserve Mrp2 function under MetS conditions.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Monoterpenos Acíclicos/farmacologia , Ácido Ascórbico/farmacologia , Frutose/efeitos adversos , Mucosa Intestinal/efeitos dos fármacos , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Antioxidantes/farmacologia , Peso Corporal/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Ingestão de Alimentos/efeitos dos fármacos , Glucose/metabolismo , Inflamação , Resistência à Insulina , Mucosa Intestinal/metabolismo , Masculino , Estresse Oxidativo/efeitos dos fármacos , Ratos Wistar , Triglicerídeos/sangueRESUMO
TNFα is a cytokine whose levels are increased in inflammatory pathologies that are associated with cholestasis. Endocytic internalization of Abcc2 (multidrug resistance-associated protein 2), a canalicular transporter of organic anions that is implicated in the clearance of clinically important drugs, is a phenomenon that occurs in inflammatory liver diseases, and it has been established that cytokines act as mediators. However, the intracellular mechanism involved in this effect remains unknown. The aim of the present work was to characterize the internalization of Abcc2 induced by TNFα and to study the role of ERK1/2 and reactive oxygen species as signaling mediators of transporter internalization. Using rat hepatocyte couplets, we found that TNFα (6.25â¯pg/ml) induced a decrease in Abcc2 activity estimated by the accumulation of the Abcc2 substrate glutathione methylfluorescein in the canalicular vacuole that was accompanied by internalization of Abcc2 from the canalicular membrane. Inhibition of MEK1/2 (upstream of ERK1/2) partially prevented TNFα effects on Abcc2 internalization and activity impairment. Reactive oxygen species (ROS) scavengers such as vitamin C and mannitol partially prevented both TNFα-induced decrease in Abcc2 activity and ERK1/2 phosphorylation. Apocynin, a NADPH oxidase inhibitor, prevented the increase in ROS and the phosphorylation of ERK1/2 produced by TNFα. Taken together, these results indicate that TNFα activates a pathway involving NADPH oxidase, ROS and MEK1/2-ERK1/2 that is partially responsible for the internalization of Abcc2. This internalization leads to an altered transport activity of Abcc2 that could impair drug disposal, enhancing drug toxicity in patients suffering from inflammatory liver diseases.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Hepatócitos/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Fator de Necrose Tumoral alfa/farmacologia , Animais , Relação Dose-Resposta a Droga , Feminino , Hepatócitos/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Ratos , Ratos WistarRESUMO
Multidrug resistance-associated proteins (MRP) 1 and 2 belong to the ABC (ATP-Binding Cassette) transporters. These transport proteins are involved in the removal of various drugs and xenobiotics, as well as in multiple physiological, pathological, and pharmacological processes. There is a strong correlation between different polymorphisms and their clinical implication in resistance to antiepileptic drugs, anticancer, and anti-infective agents. In our study, we evaluated exon regions of MRP1 (ABCC1)/MRP2 (ABCC2) in a Colombian cohort of healthy subjects to determine single nucleotide polymorphisms (SNPs) and to determine the allelic and genomic frequency. Results showed there are SNPs in our population that have been previously reported for both MRP1/ABCC1 (rs200647436, rs200624910, rs150214567) and MRP2/ABCC2 (rs2273697, rs3740066, rs142573385, rs17216212). Additionally, 13 new SNPs were identified. Evidence also shows a significant clinical correlation for polymorphisms rs3740066 and rs2273697 in the transport of multiple drugs, which suggests a genetic variability in regards to that reported in other populations.
RESUMO
OBJECTIVE: To clarify the clinical, pathologic, and genetic features of neonatal Dubin-Johnson syndrome. STUDY DESIGN: Ten patients with neonatal Dubin-Johnson syndrome were recruited from 6 pediatric centers in Japan between September 2013 and October 2016. Clinical and laboratory course, macroscopic and microscopic liver findings, and molecular genetic findings concerning ATP-binding cassette subfamily C member 2 (ABCC2) were retrospectively and prospectively examined. RESULTS: All neonates exhibited cholestasis, evident as prolonged jaundice with or without acholic stools and elevations of serum direct bilirubin as well as γ-glutamyltransferase or total bile acids. Only 38% (3 of 8) of patients who underwent liver biopsy showed a grossly black liver or melanin-like pigment deposits in hepatocytes; their biopsies were performed in early infancy. Immunohistochemically, all liver specimens showed no expression of multidrug resistance-associated protein 2 but increased expression of the bile salt export pump protein. Homozygous or compound heterozygous pathogenic variants of ABCC2 were identified in all patients, representing 11 distinct pathogenic variants including 2 not previously reported. CONCLUSIONS: Immunohistochemical staining of the liver for multidrug resistance-associated protein 2 and molecular genetic analysis of ABCC2 are crucial for accurate diagnosis of neonatal Dubin-Johnson syndrome.
Assuntos
Icterícia Idiopática Crônica/diagnóstico , Icterícia Idiopática Crônica/genética , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/metabolismo , Ácidos e Sais Biliares/metabolismo , Bilirrubina/metabolismo , China , Feminino , Hepatócitos/metabolismo , Humanos , Recém-Nascido , Doenças do Recém-Nascido , Japão , Icterícia , Icterícia Idiopática Crônica/patologia , Icterícia Idiopática Crônica/cirurgia , Fígado/metabolismo , Fígado/patologia , Masculino , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Mutação , Estudos Prospectivos , Estudos RetrospectivosRESUMO
The accumulation of mercury in the liver causes hepatotoxicity. The organic anion transporter 3 (Oat3) and the multidrug-resistance associated protein 2 (Mrp2) are involved in the hepatic excretion of toxins and drugs and in the hepatic handling of mercury. The aim of this work was to study if there are gender-related differences in mercuric chloride (HgCl2)-induced hepatotoxicity in rats. Total mercury levels and protein expressions of Oat3 and Mrp2 in liver samples were also assessed to clarify the mechanisms underlying mercury-induced liver damage in male and female rats. Control and HgCl2-treated male and female Wistar rats were used. Hepatotoxicity was evaluated by plasma activity of transaminases and alkaline phosphatase, as well as by histopathological analysis. Oat3 and Mrp2 expression was assessed by immunoblotting. Female rats displayed a higher HgCl2-induced hepatotoxicity than male rats as demonstrated by the higher alterations in the plasma markers of liver damage and in the histopathology. The sex-related differences observed in the hepatic damage can be explained by the higher accumulation of mercury in liver from female rats. In this connection, after mercury treatment the expression of Mrp2 decreased in both sexes and the expression of Oat3 decreased only in males. The decreased in Oat3 abundance in the hepatocytes membranes in mercury-treated males would limit the uptake of mercuric ions into the liver protecting them from mercury hepatotoxicity.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/patologia , Fígado/patologia , Mercúrio/toxicidade , Animais , Biomarcadores/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Feminino , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Ratos , Ratos Wistar , Fatores SexuaisRESUMO
Multidrug resistance-associated protein 2 (MRP2) is an ATP-dependent transporter expressed at the brush border membrane of the enterocyte that confers protection against absorption of toxicants from foods or bile. Acute, short-term regulation of intestinal MRP2 activity involving changes in its apical membrane localization was poorly explored. We evaluated the effects of dibutyryl-cAMP (db-cAMP), a permeable analog of cAMP, and estradiol-17ß-D-glucuronide (E217G), an endogenous derivative of estradiol, on MRP2 localization and activity using isolated rat intestinal sacs and Caco-2 cells, a model of human intestinal epithelium. Changes in MRP2 localization were studied by Western blotting of plasma membrane (PM) vs. intracellular membrane (IM) fractions in both experimental models, and additionally, by confocal microscopy in Caco-2 cells. After 30 min of exposure, db-cAMP-stimulated sorting of MRP2 from IM to PM both in rat jejunum and Caco-2 cells at 10 and 100 µM concentrations, respectively, with increased excretion of the model substrate 2,4-dinitrophenyl-S-glutathione. In contrast, E217G (400 µM) induced internalization of MRP2 together with impairment of transport activity. Confocal microscopy analysis performed in Caco-2 cells confirmed Western blot results. In the particular case of E217G, MRP2 exhibited an unusual pattern of staining compatible with endocytic vesiculation. Use of selective inhibitors demonstrated the participation of cAMP-dependent protein kinase and classic calcium-dependent protein kinase C in db-cAMP and E217G effects, respectively. We conclude that localization of MRP2 in intestine may be subjected to a dynamic equilibrium between plasma membrane and intracellular domains, thus allowing for rapid regulation of MRP2 function.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Bucladesina/farmacologia , Estradiol/análogos & derivados , Mucosa Intestinal/efeitos dos fármacos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Animais , Células CACO-2 , Membrana Celular/metabolismo , AMP Cíclico , Estradiol/farmacologia , Humanos , Mucosa Intestinal/metabolismo , Masculino , Proteína 2 Associada à Farmacorresistência Múltipla , Ratos , Ratos WistarRESUMO
Multidrug resistance-associated protein 2 (Mrp2, ABCC2) and P-glycoprotein (P-gp, ABCB1) constitute essential components of the intestinal biochemical barrier that prevent incorporation of food contaminants, drugs or toxic metabolites into the blood stream. Endotoxemia induced in rats by administration of bacterial lipopolysaccharide (LPS) results in elevated intestinal permeability and toxicity of xenobiotics in part associated with down-regulation of expression and activity of Mrp2 and P-gp. We evaluated the protective effect of glucagon-like peptide 2 (GLP-2), a peptide hormone with enterotrophic properties, on Mrp2 and P-gp alterations induced by single i.p. injection of LPS (5mg/kg b.wt.) to rats. Two different protocols of GLP-2 administration, namely prevention and reversion, were examined. The prevention protocol consisted of 7s.c. injections of GLP-2 (125µg/kg b.wt.) administered every 12h, starting 60h before LPS administration. The reversion protocol consisted of 2 doses of GLP-2, starting 3h after LPS injection. Intestinal samples were collected 24h after LPS administration and expression (protein and mRNA) and activity of Mrp2 were evaluated in proximal jejunum whereas those of P-gp were studied in ileum. GLP-2 completely neutralized down-regulation of expression of Mrp2 and P-gp and loss of their respective activities induced by LPS under prevention protocol. GLP-2 was also able to prevent internalization of both transporters from the apical membrane of the enterocyte to intracellular compartments, as detected by confocal microscopy. LPS induced an increase in IL-1ß and oxidized glutathione tissue levels, which were also counterbalanced by GLP-2 administration. In contrast, the reversion protocol failed to attenuate Mrp2 and P-gp down-regulation induced by LPS. We conclude that GLP-2 can prevent down-regulation of intestinal expression and activity of Mrp2 and P-gp in endotoxemic rats and that IL-1ß and oxidative stress constitute potential targets of GLP-2 protective effects.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Endotoxemia/prevenção & controle , Peptídeo 2 Semelhante ao Glucagon/administração & dosagem , Jejuno/metabolismo , 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 , Animais , Antioxidantes/metabolismo , Modelos Animais de Doenças , Regulação para Baixo , Esquema de Medicação , Endotoxemia/induzido quimicamente , Endotoxemia/metabolismo , Feminino , Glutationa/metabolismo , Injeções Subcutâneas , Interleucina-1beta/metabolismo , Absorção Intestinal , Lipopolissacarídeos , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Permeabilidade , Ratos Wistar , Fatores de TempoRESUMO
AIM: Furosemide is a loop diuretic. Different authors demonstrated that continuous administration of furosemide modulates the expression of organic anion transporters. This study was undertaken to simultaneously evaluate the effects of furosemide pretreatment on organic anion transporter 1 (Oat1) and multidrug resistance protein 2 (Mrp2) renal expressions, on p-aminohippurate (PAH) pharmacokinetics and on renal and urinary PAH levels in rats. METHODS: Male Wistar rats were treated with furosemide (6 mg/100 g body weight per day, subcutaneously, 4 days) (treated group) or saline (control group). On the fifth day, PAH was administered as a bolus infusion in the femoral vein, and plasma samples were obtained from femoral artery at different time points. PAH levels in renal tissue and urine were also assessed. Renal Oat1 and Mrp2 expressions were evaluated by western blotting. RESULTS: Furosemide pretreatment increased both the expression of Oat1 and Mrp2. PAH plasma concentrations decreased following a biexponential function. The furosemide-treated group showed higher PAH plasma levels, a lower systemic clearance and elimination rate constant from the peripheral compartment, indicating that PAH renal elimination was decreased. PAH levels in renal tissue were significantly elevated and in urine appeared to be significantly lower as compared with control animals. CONCLUSIONS: Furosemide pretreatment caused a significant decrease of PAH renal elimination, despite Oat1 and Mrp2 augmented renal expression. The goal of the present study is the addition of important information in the wide gap of knowledge that exists about drug-drug interactions. Because of furosemide worldwide use, the data obtained are interesting and useful in terms of translation to clinical practice.
Assuntos
Furosemida/farmacologia , Rim/efeitos dos fármacos , Proteína 1 Transportadora de Ânions Orgânicos/efeitos dos fármacos , Inibidores de Simportadores de Cloreto de Sódio e Potássio/farmacologia , Ácido p-Aminoipúrico/farmacocinética , Transportadores de Cassetes de Ligação de ATP/efeitos dos fármacos , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Interações Medicamentosas , Furosemida/administração & dosagem , Injeções Intravenosas , Injeções Subcutâneas , Rim/metabolismo , Masculino , Taxa de Depuração Metabólica , Modelos Biológicos , Proteína 1 Transportadora de Ânions Orgânicos/metabolismo , Ratos Wistar , Eliminação Renal/efeitos dos fármacos , Inibidores de Simportadores de Cloreto de Sódio e Potássio/administração & dosagem , Regulação para Cima , Ácido p-Aminoipúrico/administração & dosagem , Ácido p-Aminoipúrico/sangue , Ácido p-Aminoipúrico/urinaRESUMO
Expression and activity of jejunal multidrug resistance-associated protein 2 (Mrp2) and glutathione-S-transferase (GST) were examined in fructose fed Wistar rats, an experimental model of metabolic syndrome. Animals were fed on (a) control diet or (b) control diet plus 10% w/vol fructose in the drinking water. Mrp2 and the α class of GST proteins as well as their corresponding mRNAs were decreased, suggesting a transcriptional regulation by fructose. Confocal microscopy studies reaffirmed down-regulation of Mrp2. Everted intestinal sacs were incubated with 1-chloro-2,4-dinitrobenzene in the mucosal compartment, and the glutathione-conjugated derivative, dinitrophenyl- S-glutathione (DNP-SG; model Mrp2 substrate), was measured in the same compartment to estimate Mrp2 activity. Excretion of DNP-SG was substantially decreased by fructose treatment, consistent with simultaneous down-regulation of Mrp2 and GST. In addition, the effect of fructose on intestinal barrier function exerted by Mrp2 was evaluated in vivo using valsartan, a recognized Mrp2 substrate of therapeutic use. After intraduodenal administration as a bolus, intestinal absorption of valsartan was increased in fructose-drinking animals. Fructose administration also induced oxidative stress in intestinal tissue as demonstrated by significant increases of intestinal lipid peroxidation end products and activity of the antioxidant enzyme superoxide dismutase, by a decreased GSH/GSSG ratio. Moreover, fructose treatment conduced to increased intestinal levels of the proinflammatory cytokines IL-ß1 and IL-6. Collectively, our results demonstrate that metabolic syndrome-like conditions, induced by a fructose-rich diet, result in down-regulation of intestinal Mrp2 expression and activity and consequently in an impairment of its barrier function.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Frutose/efeitos adversos , Intestinos/efeitos dos fármacos , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Antioxidantes/metabolismo , Peso Corporal/efeitos dos fármacos , Citocinas/metabolismo , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Glutationa Transferase/metabolismo , Mucosa Intestinal/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Síndrome Metabólica/induzido quimicamente , Ratos Wistar , Superóxido Dismutase/metabolismoRESUMO
The gastrointestinal epithelium functions as a selective barrier to absorb nutrients, electrolytes and water, but at the same time restricts the passage into the systemic circulation of intraluminal potentially toxic compounds. This epithelium maintains its selective barrier function through the presence of very selective and complex intercellular junctions and the ability of the absorptive cells to reject those compounds. Accordingly, the enterocytes metabolize orally incorporated xenobiotics and secrete the hydrophilic metabolites back into the intestinal lumen through specific transporters localized apically. In the recent decades, there has been increasing recognition of the existence of the intestinal cellular barrier. In the present review we focus on the role of the multidrug resistance-associated protein 2 (MRP2, ABCC2) in the apical membrane of the enterocytes, as an important component of this intestinal barrier, as well as on its regulation. We provide a detailed compilation of significant contributions demonstrating that MRP2 expression and function vary under relevant physiological and pathophysiological conditions. Because MRP2 activity modulates the availability and pharmacokinetics of many therapeutic drugs administered orally, their therapeutic efficacy and safety may vary as well.
Assuntos
Intestinos/fisiologia , Intestinos/fisiopatologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/fisiologia , Animais , Humanos , Proteína 2 Associada à Farmacorresistência MúltiplaRESUMO
Estradiol-17ß-D-glucuronide (E17G) induces acute endocytic internalization of canalicular transporters, including multidrug resistance-associated protein 2 (Abcc2) in rat, generating cholestasis. Several proteins organized in at least two different signaling pathways are involved in E17G cholestasis: one pathway involves estrogen receptor alpha (ERα), Ca(2+)-dependent protein kinase C and p38-mitogen activated protein kinase, and the other pathway involves GPR30, PKA, phosphoinositide 3-kinase/AKT and extracellular signal-related kinase 1/2. EGF receptor (EGFR) can potentially participate in both pathways since it interacts with GPR30 and ERα. Hence, the aim of this study was to analyze the potential role of this receptor and its downstream effectors, members of the Src family kinases in E17G-induced cholestasis. In vitro, EGFR inhibition by Tyrphostin (Tyr), Cl-387785 or its knockdown with siRNA strongly prevented E17G-induced impairment of Abcc2 function and localization. Activation of EGFR was necessary but not sufficient to impair the canalicular transporter function, whereas the simultaneous activation of EGFR and GPR30 could impair Abcc2 transport. The protection of Tyr was not additive to that produced by the ERα inhibitor ICI neither with that produced by Src kinase inhibitors, suggesting that EGFR shared the signaling pathway of ERα and Src. Further analysis of ERα, EGFR and Src activations induced by E17G, demonstrated that ERα activation precedes that of EGFR and EGFR activation precedes that of Src. In conclusion, activation of EGFR is a key factor in the alteration of canalicular transporter function and localization induced by E17G and it occurs before that of Src and after that of ERα.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Receptores ErbB/metabolismo , Estradiol/análogos & derivados , Receptor alfa de Estrogênio/metabolismo , Hepatócitos/metabolismo , Animais , Canalículos Biliares/efeitos dos fármacos , Canalículos Biliares/metabolismo , Canalículos Biliares/fisiopatologia , Células Cultivadas , Colestase/induzido quimicamente , Colestase/metabolismo , Receptores ErbB/genética , Estradiol/metabolismo , Estradiol/farmacologia , Antagonistas do Receptor de Estrogênio/farmacologia , Feminino , Fulvestranto , Técnicas de Silenciamento de Genes , Hepatócitos/efeitos dos fármacos , Quinazolinas/farmacologia , Ratos , Ratos Wistar , Tirfostinas/farmacologia , Quinases da Família src/metabolismoRESUMO
ABC transporters including MRP2, MDR1 and BCRP play a major role in tissue defense. Epidemiological and experimental studies suggest a cytoprotective role of estrogens in intestine, though the mechanism remains poorly understood. We evaluated whether pharmacologic concentrations of ethynylestradiol (EE, 0.05pM to 5nM), or concentrations of genistein (GNT) associated with soy ingestion (0.1-10µM), affect the expression and activity of multidrug resistance proteins MRP2, MDR1 and BCRP using Caco-2 cells, an in vitro model of intestinal epithelium. We found that incubation with 5pM EE and 1µM GNT for 48h increased expression and activity of both MRP2 and MDR1. Estrogens did not affect expression of BCRP protein at any concentration studied. Irrespective of the estrogen tested, up-regulation of MDR1 and MRP2 protein was accompanied by increased levels of MDR1 mRNA, whereas MRP2 mRNA remained unchanged. Cytotoxicity assays demonstrated association of MRP2 and MDR1 up-regulation with increased resistance to cell death induced by 1-chloro-2,4-dinitrobenzene, an MRP2 substrate precursor, and by paraquat, an MDR1 substrate. Experiments using an estrogen receptor (ER) antagonist implicate ER participation in MRP2 and MDR1 regulation. GNT but not EE increased the expression of ERß, the most abundant form in human intestine and in Caco-2 cells, which could lead in turn to increased sensitivity to estrogens. We conclude that specific concentrations of estrogens can confer resistance against cytotoxicity in Caco-2 cells, due in part to positive modulation of ABC transporters involved in extrusion of their toxic substrates. Although extrapolation of these results to the in vivo situation must be cautiously done, the data could explain tentatively the cytoprotective role of estrogens against chemical injury in intestine.