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1.
Drug Metab Pharmacokinet ; 36: 100363, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33189558

RESUMO

Organic anion transporter (OAT) 4, which is localized at the apical membrane of human renal proximal tubules, transports olmesartan, an angiotensin II receptor blocker (ARB). Many ARBs, including olmesartan, undergo partial tubular secretion as active forms, and inhibit OAT4-mediated uptake activity. Here, we examined the substrate recognition of various ARBs by OAT4 in order to assess whether OAT4 might be involved in the renal handling of ARBs. Concentration-dependent OAT4-mediated uptake of azilsartan, candesartan, carboxylosartan, losartan, and valsartan was observed with Km values of 6.6, 31, 7.2, 13, and 1.7 µM, respectively, in the absence of extracellular Cl-. In the presence of extracellular Cl-, OAT4-mediated uptake of dianionic ARBs (azilsartan, candesartan, carboxylosartan, and valsartan) was lower and reached a steady state faster than in the absence of extracellular Cl-. Thus, OAT4 is proposed to use extracellular Cl- as a counterpart for anion efflux. Our results suggest that OAT4 may play a role in the excretion of azilsartan, candesartan, carboxylosartan, and valsartan, as well as olmesartan. In contrast, OAT4-mediated uptake of losartan, a monoanionic ARB, was little affected by extracellular Cl-, suggesting that only OAT4-mediated dianion transport is Cl--sensitive.


Assuntos
Antagonistas de Receptores de Angiotensina/metabolismo , Rim/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Espectrometria de Massas em Tandem/métodos , Antagonistas de Receptores de Angiotensina/análise , Antagonistas de Receptores de Angiotensina/farmacologia , Cromatografia Líquida/métodos , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Rim/efeitos dos fármacos , Espectrometria de Massas/métodos , Transportadores de Ânions Orgânicos Sódio-Independentes/análise , Especificidade por Substrato/fisiologia
2.
J Pharmacol Exp Ther ; 373(2): 261-268, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32127372

RESUMO

In vitro approaches for predicting drug-drug interactions (DDIs) caused by alterations in transporter protein regulation are not well established. However, reports of transporter regulation via nuclear receptor (NR) modulation by drugs are increasing. This study examined alterations in transporter protein levels in sandwich-cultured human hepatocytes (SCHH; n = 3 donors) measured by liquid chromatography-tandem mass spectrometry-based proteomic analysis after treatment with N-[4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)phenyl]-N-(2,2,2-trifluoroethyl)benzenesulfonamide (T0901317), the first described synthetic liver X receptor agonist. T0901317 treatment (10 µM, 48 hours) decreased the levels of organic cation transporter (OCT) 1 (0.22-, 0.43-, and 0.71-fold of control) and organic anion transporter (OAT) 2 (0.38-, 0.38-, and 0.53-fold of control) and increased multidrug resistance protein (MDR) 1 (1.37-, 1.48-, and 1.59-fold of control). The induction of NR downstream gene expression supports the hypothesis that T0901317 off-target effects on farnesoid X receptor and pregnane X receptor activation are responsible for the unexpected changes in OCT1, OAT2, and MDR1. Uptake of the OCT1 substrate metformin in SCHH was decreased by T0901317 treatment. Effects of decreased OCT1 levels on metformin were simulated using a physiologically-based pharmacokinetic (PBPK) model. Simulations showed a clear decrease in metformin hepatic exposure resulting in a decreased pharmacodynamic effect. This DDI would not be predicted by the modest changes in simulated metformin plasma concentrations. Altogether, the current study demonstrated that an approach combining SCHH, proteomic analysis, and PBPK modeling is useful for revealing tissue concentration-based DDIs caused by unexpected regulation of hepatic transporters by NR modulators. SIGNIFICANCE STATEMENT: This study utilized an approach combining sandwich-cultured human hepatocytes, proteomic analysis, and physiologically based pharmacokinetic modeling to evaluate alterations in pharmacokinetics (PK) and pharmacodynamics (PD) caused by transporter regulation by nuclear receptor modulators. The importance of this approach from a mechanistic and clinically relevant perspective is that it can reveal drug-drug interactions (DDIs) caused by unexpected regulation of hepatic transporters and enable prediction of altered PK and PD changes, especially for tissue concentration-based DDIs.


Assuntos
Hepatócitos/efeitos dos fármacos , Hidrocarbonetos Fluorados/farmacologia , Receptores X do Fígado/agonistas , Proteômica/métodos , Sulfonamidas/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/análise , Adulto , Células Cultivadas , Interações Medicamentosas , Feminino , Hepatócitos/metabolismo , Humanos , Hidrocarbonetos Fluorados/farmacocinética , Pessoa de Meia-Idade , Modelos Biológicos , Fator 1 de Transcrição de Octâmero/análise , Transportadores de Ânions Orgânicos Sódio-Independentes/análise , Sulfonamidas/farmacocinética
3.
Mol Pharm ; 15(8): 3227-3235, 2018 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-29906129

RESUMO

Organic anion transporter (OAT) 2 and OAT7 were recently shown to be involved in the hepatic uptake of drugs; however, there is limited understanding of the population variability in the expression of these transporters in liver. There is also a need to derive relative expression-based scaling factors (REFs) that can be used to bridge in vitro functional data to the in vivo drug disposition. To this end, we quantified OAT2 and OAT7 surrogate peptide abundance in a large number of human liver tissue samples ( n = 52), as well as several single-donor cryopreserved human hepatocyte lots ( n = 30) by a novel, validated liquid chromatography tandem mass spectrometry (LC-MS/MS) method. The average surrogate peptide expression of OAT2 and OAT7 in the liver samples was 1.52 ± 0.57 and 4.63 ± 1.58 fmol/µg membrane protein, respectively. While we noted statistically significant differences ( p < 0.05) in hepatocyte and liver tissue abundances for both OAT2 and OAT7, the differences were relatively small (1.8- and 1.5-fold difference in median values, respectively). Large interindividual variability was noted in the hepatic expression of OAT2 (16-fold in liver tissue and 23-fold in hepatocytes). OAT7, on the other hand, showed less interindividual variability (4-fold) in the livers, but high variability for the hepatocyte lots (27-fold). A significant positive correlation in OAT2 and OAT7 expression was observed, but expression levels were neither associated with age nor sex. In conclusion, our data suggest marked interindividual variability in the hepatic expression of OAT2/7, which may contribute to the pharmacokinetic variability of their substrates. Because both transporters were less abundant in hepatocytes than livers, a REF-based approach is recommended when scaling in vitro hepatocyte transport data to predict hepatic drug clearance and liver exposure of OAT2/7 substrates.


Assuntos
Hepatócitos/metabolismo , Fígado/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Proteômica/métodos , Adulto , Fatores Etários , Idoso , Variação Biológica da População , Criança , Cromatografia Líquida , Feminino , Células HEK293 , Humanos , Fígado/citologia , Masculino , Pessoa de Meia-Idade , Transportadores de Ânions Orgânicos Sódio-Independentes/análise , Cultura Primária de Células , Fatores Sexuais , Espectrometria de Massas em Tandem , Adulto Jovem
4.
Toxicol Lett ; 263: 58-67, 2016 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-27765674

RESUMO

Disruption of the vectorial bile acid transport in the liver is a key feature of cholestatic drugs, although many causal and mechanistic aspects are still unknown. The aim of the present study was to explore if cholestatic drugs can repress or induce the expression of hepatic transporters. To this end, sandwich-cultured rat hepatocytes were treated with cholestatic and non-cholestatic (steatotic, non-hepatotoxic, etc.) drugs and the mRNA expression of 10 uptake and efflux biliary transporters was measured. Results evidenced that all cholestatic drugs cause extensive alterations in the mRNA expression of most biliary transporters. Surprisingly, nearly all steatotic drugs also affected the expression of these genes. The most frequent alterations triggered by both types of drugs were the repression of OATP1A1, NTCP and BSEP, and the induction of MRP2/3/4, MDR2 and ABCG5/8. The majority of these alterations were also observed in vivo, in the livers of treated rats. The common signature of cholestatic and steatotic drugs was the repression of OATP1A1. Indeed, ROC curve analysis indicated that OATP1A1 mRNA is a very sensitive marker to identify drugs with cholestatic or steatotic potential, with a maximal sensitivity and specificity of 0.917 and 0.941, respectively. We conclude that alteration of expression of hepatobiliary transporters is a hallmark of both cholestatic and steatotic drugs, lending support to a connection between these two mechanisms of hepatotoxicity. Moreover, OATP1A1 mRNA is proposed as a very simple and useful screening biomarker for the prediction of new cholestatic or steatotic drugs in early drug development.


Assuntos
Bile/metabolismo , Biomarcadores/análise , Proteínas de Transporte/metabolismo , Colestase/induzido quimicamente , Avaliação Pré-Clínica de Medicamentos/métodos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Fígado Gorduroso/induzido quimicamente , Hepatócitos/efeitos dos fármacos , RNA Mensageiro/biossíntese , Animais , Bile/efeitos dos fármacos , Proteínas de Transporte/biossíntese , Células Cultivadas , Masculino , Transportadores de Ânions Orgânicos Sódio-Independentes/análise , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Valor Preditivo dos Testes , Ratos , Ratos Sprague-Dawley , Tetraciclinas/toxicidade
5.
Mol Pharm ; 13(9): 3130-40, 2016 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-27467266

RESUMO

One of the characteristics of chronic kidney disease (CKD) is the accumulation of uremic solutes in the plasma. Less is known about the effects of uremic solutes on transporters that may play critical roles in pharmacokinetics. We evaluated the effect of 72 uremic solutes on organic anion transporter 1 and 3 (OAT1 and OAT3) using a fluorescent probe substrate, 6-carboxyfluorescein. A total of 12 and 13 solutes were identified as inhibitors of OAT1 and OAT3, respectively. Several of them inhibited OAT1 or OAT3 at clinically relevant concentrations and reduced the transport of other OAT1/3 substrates in vitro. Review of clinical studies showed that the active secretion of most drugs that are known substrates of OAT1/3 deteriorated faster than the renal filtration in CKD. Collectively, these data suggest that through inhibition of OAT1 and OAT3, uremic solutes contribute to the decline in renal drug clearance in patients with CKD.


Assuntos
Proteína 1 Transportadora de Ânions Orgânicos/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Insuficiência Renal Crônica/metabolismo , Animais , Fluoresceínas/metabolismo , Humanos , Modelos Biológicos , Proteína 1 Transportadora de Ânions Orgânicos/análise , Transportadores de Ânions Orgânicos Sódio-Independentes/análise
6.
J Hepatol ; 64(3): 708-16, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26632635

RESUMO

The incidence of hepatocellular carcinomas (HCCs) has increased worldwide in line with an improved screening by high-resolution imaging of cirrhotic livers. Besides abdominal ultrasonography and computerised tomography, magnetic resonance imaging (MRI) is an important tool to detect HCCs. With commercialisation of MR hepatobiliary contrast agents that cross membrane transporters in hepatocytes or tumour cells, MRI adds new information to detect and characterise HCCs. When tumour cells lose organic anion transporting polypeptides (OATP1B1/B3) in cell membranes facing sinusoidal blood, tumours appear hypointense (decreased contrast agent concentrations) in comparison to surrounding normal or cirrhotic liver that retains OATP1B1/B3 expression. However, expression, regulation, and prognostic significance of transporter evolution along carcinogenesis are not completely known. Moreover, understanding signal intensities in focal lesions also relies on transport functions of cellular efflux transporters. This manuscript reviews all the publications that associate liver imaging with hepatobiliary contrast agents and expression of transporters. The regulation of transporters along carcinogenesis to anticipate the prognosis of focal lesions is also included.


Assuntos
Carcinoma Hepatocelular/diagnóstico por imagem , Neoplasias Hepáticas/diagnóstico por imagem , Fígado/diagnóstico por imagem , Imageamento por Ressonância Magnética/métodos , Carcinoma Hepatocelular/patologia , Meios de Contraste , Humanos , Neoplasias Hepáticas/patologia , Transportador 1 de Ânion Orgânico Específico do Fígado/análise , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/análise , Transportadores de Ânions Orgânicos Sódio-Independentes/análise , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de Soluto , beta Catenina/fisiologia
7.
Magn Reson Med Sci ; 15(1): 111-20, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26346403

RESUMO

PURPOSE: We attempted to clarify the relationship between the signal intensity (SI) in the hepatobiliary phase of gadoxetic acid-enhanced magnetic resonance (MR) imaging and the efficacy of hepatic arterial infusion chemotherapy (HAIC) in hepatocellular carcinomas (HCCs). METHODS: We enrolled 14 patients with HCCs who underwent gadoxetic acid-enhanced MR imaging prior to HAIC using cisplatin and 5-fluorouracil. In the hepatobiliary phase, we calculated the SI of the HCCs and the background liver. In cases with multiple HCCs, we calculated the SI of the largest lesion. Patients were classified into high (n = 7) and low intensity (n = 7) groups based on the median value of the SI ratio (SI of the tumor/SI of the background liver). We analyzed progression-free survival using the Kaplan-Meier method and the log-rank test. In the 5 patients with a history of HCC surgery, we compared the expression of immunohistochemical organic anion-transporting polypeptide (OATP) 8 between the high and low intensity groups by chi-square test. RESULTS: The SI ratios were 0.568 ± 0.093 (mean ± standard deviation) in the high intensity group and 0.251 ± 0.086 in the low intensity group. Compared to the group with low signal intensity, the group with high signal intensity demonstrated significantly lower serum levels of alpha fetoprotein (AFP) (P = 0.0350), significantly higher progression-free survival (P = 0.0108), better differentiation of tumor grade at histologic examination (P = 0.0253), and significantly higher OATP8 expression (P = 0.0253). CONCLUSION: Patients with HCCs of high SI ratio in the hepatobiliary phase of gadoxetic acid-enhanced MR imaging can respond better to HAIC.


Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Meios de Contraste , Gadolínio DTPA , Aumento da Imagem/métodos , Neoplasias Hepáticas/tratamento farmacológico , Imageamento por Ressonância Magnética/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Antimetabólitos Antineoplásicos/administração & dosagem , Antineoplásicos/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Cisplatino/administração & dosagem , Intervalo Livre de Doença , Feminino , Fluoruracila/administração & dosagem , Previsões , Artéria Hepática/patologia , Humanos , Processamento de Imagem Assistida por Computador/métodos , Bombas de Infusão Implantáveis , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Transportadores de Ânions Orgânicos Sódio-Independentes/análise , Transportadores de Ânions Orgânicos Sódio-Independentes/efeitos dos fármacos , Estudos Retrospectivos , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de Soluto , Resultado do Tratamento , alfa-Fetoproteínas/análise , alfa-Fetoproteínas/efeitos dos fármacos
8.
Bioconjug Chem ; 27(1): 87-101, 2016 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-26684581

RESUMO

Luminogenic probes were designed and synthesized for the detection of uptake transporter activity in a lytic cell-based assay. These probes rely on a self-cleavable trimethyl lock quinone-cyanobenzothiazole (TMQ-CNBT) or trimethyl lock quinone-luciferin (TMQ-Luc) linked to the anion transporter substrate fluorescein. Upon cellular transport, the TMQ is reduced by viable cells, resulting in the facile intramolecular lactonization and rapid release of the bioluminescent reporter molecule. The uptake transporter activity can then be detected without removing and washing off the extracellular substrates. Six probes were tested with OATP1B1*1a and OATP1B3 overexpressing HEK293 cells, and all compounds showed up to 10.2-fold enhancement in uptake when compared to control cells. Uptake of TMQ-luciferin compounds 2, 4, and 6 increased linearly over time up to 30 min at a concentration ranging from 40 nM to 20 µM. The apparent Km values obtained at different time intervals up to 30 min were nearly identical for a given compound, which validates the 30 min window as appropriate for uptake transporter assays. The average apparent Km values ranged from 0.3 to 0.8 µM and 0.2 to 1.3 µM for OATP1B1*1a and OATP1B3, respectively, indicating good affinities to these anion transporters. Furthermore, uptake of compound 2 was inhibited by two inhibitors of OATP1B1*1a and OATP1B3: rifampicin and ritonavir. The preliminary results obtained from compound 2 exhibited a time-dependent, saturatable, and inhibitable nature of uptake, indicating the feasibility of using the probe for the detection of a transporter-mediated process. This add-and-read homogeneous assay may provide a convenient, rapid, and facile way to detect changes in transporter activity in a high-throughput format, and this assay design strategy could create a new platform for a general cell uptake assay for biomaterials in the future.


Assuntos
Transportador 1 de Ânion Orgânico Específico do Fígado/metabolismo , Substâncias Luminescentes/química , Imagem Molecular/métodos , Sondas Moleculares/química , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Benzotiazóis/química , Transporte Biológico/efeitos dos fármacos , Técnicas de Química Sintética , Luciferina de Vaga-Lumes/análogos & derivados , Luciferina de Vaga-Lumes/química , Luciferina de Vaga-Lumes/farmacocinética , Fluoresceína/química , Fluoresceínas/química , Fluoresceínas/farmacocinética , Células HEK293 , Humanos , Cinética , Transportador 1 de Ânion Orgânico Específico do Fígado/análise , Transportador 1 de Ânion Orgânico Específico do Fígado/genética , Medições Luminescentes/métodos , Sondas Moleculares/síntese química , Sondas Moleculares/farmacocinética , Nitrilas/química , Transportadores de Ânions Orgânicos Sódio-Independentes/análise , Transportadores de Ânions Orgânicos Sódio-Independentes/genética , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de Soluto
9.
Oncology ; 86(1): 53-62, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24401597

RESUMO

OBJECTIVES: Recent reports indicated that reduced SLC22A7 (a gene-encoding organic anion transporter 2) expression in noncancerous liver tissue predicts hepatocellular carcinoma (HCC) recurrence after curative resection. Our study aimed to elucidate the association between SLC22A7 expression and HCC development in chronic hepatitis C patients. METHODS: HCC recurrence after local ablation therapy and SLC22A7 expression in noncancerous liver tissue were analyzed in 20 patients. Subsequently, the association between de novo HCC development and SLC22A7 expression was examined at baseline in 38 hepatitis C patients without HCC who subsequently developed HCC as well as in 76 hepatitis C patients who did not develop HCC and were matched for age, gender and stage of fibrosis. RESULTS: In the patients whose HCC had been cured, reduced SLC22A7 expression in noncancerous liver tissue was significantly associated with a high incidence of multifocal HCC recurrence. In patients without HCC at baseline, cumulative incidence of de novo HCC development was significantly higher with a reduced SLC22A7 expression than with a normal expression (p = 0.01). This difference remained significant among patients without known risk factors for HCC like age and advanced fibrosis. CONCLUSION: Reduced SLC22A7 expression in the liver indicates a significant risk for HCC development in chronic hepatitis C, independently of other risk factors.


Assuntos
Carcinoma Hepatocelular/etiologia , Hepatite C Crônica/complicações , Neoplasias Hepáticas/etiologia , Transportadores de Ânions Orgânicos Sódio-Independentes/análise , Pontuação de Propensão , Idoso , Carcinoma Hepatocelular/química , Carcinoma Hepatocelular/cirurgia , Ablação por Cateter , Feminino , Humanos , Neoplasias Hepáticas/química , Neoplasias Hepáticas/cirurgia , Masculino , Pessoa de Meia-Idade , Modelos de Riscos Proporcionais , Fatores de Risco
10.
J Pharm Sci ; 102(9): 3302-8, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23630107

RESUMO

Organic anion transporters (OATs) and organic cation transporters (OCT) play pivotal roles in the uptake of drugs into epithelial cells at the basolateral membranes, and multidrug and toxin extrusion (MATE) mediates drug secretion into urine at the brush-border membranes. In this study, the expression and distribution of apical MATE1 and MATE2-K, and basolateral OAT1, OAT3, and OCT2 were compared using serial sections of human kidney cortex. First, mRNA expression in the proximal tubules was evaluated using laser microdissection. Levels of OAT, OCT2, and MATE mRNA in the proximal tubules were greatly higher compared with glomerulus. The results quantitatively indicated that these transporters were localized to proximal tubules in the renal cortex. Second, MATE1 and MATE2-K protein were detected in proximal epithelial cells in which OCT2 protein was expressed at the basolateral membranes. In addition, MATE1 was expressed at the brush-border membranes of tubular epithelial cells in which OAT1 and OAT3 were expressed. The results confirmed that OAT1, OAT3, OCT2, MATE1, and MATE2-K were coexpressed in tubular epithelial cells. The cooperation among OAT, OCT, and MATE in renal drug secretion was consistent with their distribution.


Assuntos
Rim/metabolismo , Proteína 1 Transportadora de Ânions Orgânicos/análise , Transportadores de Ânions Orgânicos Sódio-Independentes/análise , Proteínas de Transporte de Cátions Orgânicos/análise , Idoso , Expressão Gênica , Humanos , Imuno-Histoquímica , Rim/ultraestrutura , Masculino , Pessoa de Meia-Idade , Proteína 1 Transportadora de Ânions Orgânicos/genética , Transportadores de Ânions Orgânicos Sódio-Independentes/genética , Proteínas de Transporte de Cátions Orgânicos/genética , Transportador 2 de Cátion Orgânico , RNA Mensageiro/análise , RNA Mensageiro/genética
11.
Pharm Res ; 30(9): 2260-9, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23307416

RESUMO

PURPOSE: Organic Anion Transporting Polypeptides (OATPs) are expressed in various epithelial tissues in the body. Because they can be expressed in cancers and because they can transport anticancer drugs, OATPs could be potential targets for cancer therapy. Therefore we examined their expression in human pancreatic ductal adenocarcinomas. METHODS: Expression of all 11 human OATPs was measured at the mRNA level and OATPs with highest expression were characterized at the protein level. RESULTS: Transcripts of SLCO1B3, SLCO2A1, SLCO3A1 and SLCO4A1 were detected in all the tested pancreatic tissues. OATP1B3, OATP2A1, OATP3A1 and OATP4A1 protein expression was confirmed in these tissues and expression of all four transporters increased in pancreatic adenocarcinoma compared to normal pancreas. OATP1B3 expression was highest in pancreatic hyperplasia and stage one adenocarcinomas compared to stage two and three adenocarcinomas. CONCLUSION: OATP1B3, OATP2A1, OATP3A1 and OATP4A1 are up-regulated in pancreatic adenocarcinoma and could potentially be used to target anticancer drugs to pancreatic cancer. Additionally, because expression of OATP1B3 is highest in pancreatitis and stage one adenocarcinoma, which leads to pancreatic cancer, OATP1B3 is a potential marker to diagnose patients with early stage pancreatic adenocarcinomas.


Assuntos
Adenocarcinoma/genética , Regulação Neoplásica da Expressão Gênica , Transportadores de Ânions Orgânicos/genética , Pâncreas/patologia , Neoplasias Pancreáticas/genética , Adenocarcinoma/patologia , Doença Crônica , Humanos , Transportadores de Ânions Orgânicos/análise , Transportadores de Ânions Orgânicos Sódio-Independentes/análise , Transportadores de Ânions Orgânicos Sódio-Independentes/genética , Pâncreas/metabolismo , Neoplasias Pancreáticas/patologia , Pancreatite/genética , Pancreatite/patologia , RNA Mensageiro/análise , RNA Mensageiro/genética , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de Soluto
12.
Jpn J Radiol ; 30(9): 777-82, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22911100

RESUMO

We report a male case of beta-catenin-activated hepatocellular adenoma (HCA) focusing on findings of gadoxetic-acid-enhanced magnetic resonance imaging (EOB-MRI) and discussing the molecular background and possible clinical significance. The patient was a 31-year-old man in whom computed tomography (CT) showed a large nodule of 14 cm in diameter in the right liver lobe. On dynamic contrast-enhanced CT, heterogeneous and slight to moderate enhancement was observed during the early phase, with washout in the late phase. Focal fat deposits and a scar-like portion in the lesion were also seen. Most of the lesion was slightly hyperintense compared with the background liver on the hepatobiliary phase of EOB-MRI. After operation, this patient was confirmed pathologically as having beta-catenin-activated HCA with a portion suggestive of malignant transformation. In addition, intense organic anion transporter polypeptide 8 expression was observed throughout the tumor by immunohistochemical staining.


Assuntos
Adenoma de Células Hepáticas/química , Adenoma de Células Hepáticas/patologia , Neoplasias Hepáticas/química , Neoplasias Hepáticas/patologia , Imageamento por Ressonância Magnética , Transportadores de Ânions Orgânicos Sódio-Independentes/análise , beta Catenina/fisiologia , Adulto , Gadolínio DTPA , Humanos , Imuno-Histoquímica , Masculino , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de Soluto , Tomografia Computadorizada por Raios X
13.
J Endod ; 38(5): 648-52, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22515894

RESUMO

INTRODUCTION: Membrane transport proteins (transporters) play a crucial role in the transmembrane uptake and/or efflux of various compounds such as inorganic ions, endogenous bioactive substances such as prostaglandins (PGs), and drugs such as nonsteroidal anti-inflammatory drugs. This study aimed to analyze mRNA expression of selected transporters related to drug disposition and PG transport in normal and lipopolysaccharide (LPS)-inflamed rat incisor pulp. METHODS: Pulp tissues were subjected to reverse transcription-polymerase chain reaction (PCR) detection for transporter isoforms belonging to organic anion transporting polypeptide (Oatp), organic anion transporter (Oat), organic cation transporter (Oct), multidrug resistance-associated protein (Mrp), and multidrug resistance protein (Mdr) families. The levels of mRNA expression for PG transporters (Oatp1a5, Oatp1b2, Oatp2a1, Oatp2b1, and Oatp3a1) were compared in normal and LPS-inflamed pulps by using real-time PCR. RESULTS: The pulp tissue expressed mRNAs for various transporters belonging to the Oatp, Oat, Oct, Mrp, and Mdr families. LPS inflammation caused significant up-regulation of Oatp2a1 (P < .01) and significant down-regulation of Oatp1a5, Oatp2b1 (P < .01), and Oatp3a1 (P < .05). CONCLUSIONS: Rat incisor dental pulp expressed mRNAs for various transporter isoforms. The levels of mRNA expression for PG transporters were significantly up-regulated or down-regulated in LPS-inflamed dental pulp.


Assuntos
Polpa Dentária/metabolismo , Lipopolissacarídeos/efeitos adversos , Proteínas de Membrana Transportadoras/genética , Pulpite/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/análise , Animais , Anti-Inflamatórios não Esteroides/metabolismo , Proteínas da Membrana Plasmática de Transporte de Catecolaminas/análise , Perfilação da Expressão Gênica , Masculino , Proteínas Associadas à Resistência a Múltiplos Medicamentos/análise , Transportadores de Ânions Orgânicos/análise , Transportadores de Ânions Orgânicos Sódio-Independentes/análise , Proteínas de Transporte de Cátions Orgânicos/análise , Prostaglandinas/metabolismo , Isoformas de Proteínas/análise , Pulpite/induzido quimicamente , RNA Mensageiro/análise , Ratos , Ratos Wistar , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de Soluto , Organismos Livres de Patógenos Específicos , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATP
14.
Anal Chim Acta ; 717: 67-76, 2012 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-22304817

RESUMO

OATP1B1, OATP1B3 and OATP2B1 are important members of the organic anion transporting polypeptides (OATP) family and are implicated in the hepatic disposition of endobiotics and xenobiotics. Quantitating the expression levels of human OATP1B1, OATP1B3 and OATP2B1 in in vitro systems and tissue samples could significantly improve attempts to scale up in vitro data and result in more effective in vitro-in vivo correlation of transporter-mediated effects on drug disposition, such as hepatic clearance. In the present study, a quantification method was developed, characterized, and implemented for simultaneous determination of human OATP1B1, OATP1B3 and OATP2B1 in HEK cells transfected with OATP-expressing plasmid vectors (SLCO1B1, SLCO1B3, and SLCO2B1, respectively), human hepatocytes, human brain capillary endothelial cells, and humanized mouse liver tissue using UPLC-MRM MS. Purified membrane protein standards prepared and characterized as previously reported (Protein Expr. Purif. 2008, 57, 163-71) were first used as standards for absolute quantification of the expression levels of the three human OATP membrane proteins. The specificity of the optimized MRM transitions were characterized by analyzing the tryptic digests of the membrane protein fraction of wild type HEK cells and control mouse liver tissue using the herein reported UPLC-MRM MS method. The linearity of the calibration curve spanned from 0.2 µg mL(-1) (0.040 µg mg(-1)) to 20 µg mL(-1) (4.0 µg mg(-1)), with accuracy (% RE) within 15% at all concentrations examined for all three OATPs of interest in this study. The intra- and inter-day assay accuracy (% RE) and coefficient of variations (% CV) of triplicates are all within 15% for all levels of quality control samples prepared by mixing the membrane fraction of control mouse liver tissue with the required amount of purified human OATP1B1, OATP1B3 and OATP2B1.


Assuntos
Cromatografia Líquida de Alta Pressão , Espectrometria de Massas , Transportadores de Ânions Orgânicos Sódio-Independentes/análise , Transportadores de Ânions Orgânicos/análise , Animais , Encéfalo/irrigação sanguínea , Capilares/citologia , Linhagem Celular , Cromatografia Líquida de Alta Pressão/métodos , Células Endoteliais/química , Hepatócitos/química , Humanos , Transportador 1 de Ânion Orgânico Específico do Fígado , Espectrometria de Massas/métodos , Camundongos , Transportadores de Ânions Orgânicos/genética , Transportadores de Ânions Orgânicos Sódio-Independentes/genética , Sensibilidade e Especificidade , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de Soluto , Transfecção
15.
Drug Metab Pharmacokinet ; 25(3): 274-82, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20610886

RESUMO

The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor pravastatin has been reported to have a beneficial effect on reducing the new onset of diabetes as well as lowering plasma lipids. Because pravastatin is a water-soluble organic anion, it cannot easily penetrate the lipid bilayer of the cell membrane. As the precise mechanisms of the effect of pravastatin on glucose metabolism and diabetes have not been clarified, we examined the roles of the organic anion transporter family on pravastatin-treated islet and adipocyte functions. Rat oatp1/slco1a1, oatp2/slco1a4 and oatp3/slco1a5 were expressed in the pancreas, and rat oatp3/slco1a5 was also detected in rat insulinoma cell line INS-1e. Pravastatin was transported not only by oatp1/slco1a1 and oatp2/slco1a4, but also by rat oatp3/slco1a5. Pravastatin uptake into INS-1e cells was detected and this transport was inhibited by sulfobromophthalein and rifampicin, both of which are known to inhibit oatp family-mediated uptake. In addition, pravastatin enhanced the glucose-stimulated insulin secretion from INS-1e cells. When fat-loaded db/db mice were treated with pravastatin, glucose intolerance and insulin resistance were prevented. In addition, insulin secretion from isolated islets was enhanced by pravastatin. These data suggest that pravastatin has pleiotropic effects on islets through membrane transport under high fat/glucose conditions.


Assuntos
Insulina/metabolismo , Ilhotas Pancreáticas/citologia , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Pravastatina/farmacologia , Pravastatina/farmacocinética , Adiponectina/sangue , Animais , Transporte Biológico/efeitos dos fármacos , Linhagem Celular Tumoral , Diabetes Mellitus/tratamento farmacológico , Hidroximetilglutaril-CoA Redutases , Imuno-Histoquímica , Insulina/agonistas , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos , Modelos Animais , Transportadores de Ânions Orgânicos Sódio-Independentes/análise , Pâncreas/citologia , Pâncreas/efeitos dos fármacos , Ratos , Rifampina/farmacologia , Sulfobromoftaleína/farmacologia
16.
Pharm Res ; 27(4): 589-96, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20140636

RESUMO

PURPOSE: Human organic anion transporter 4 (hOAT4) belongs to a family of organic anion transporters that play critical roles in the body disposition of clinically important drugs. We have previously shown that the activity of hOAT4 was down-regulated by activation of PKC and up-regulated by PDZ protein NHERF-1. Here, we investigated the mechanisms underlying such regulations. METHODS: COS-7 cells expressing hOAT4 were treated with PKC activator phorbol 12-myristate 13-acetate (PMA) or transfected with dominant negative mutants of dynamin-2 or Eps15 or transfected with NHERF-1. The internalization and the function of hOAT4 were then determined. RESULTS: We showed that hOAT4 constitutively internalized from and recycled back to plasma membrane. Transfection of dominant negative mutants of dynamin-2 or Eps15 into the cells, all of which block clathrin-dependent endocytotic pathway, significantly blocked hOAT4 internalization. Treatment of cells with PMA accelerated hOAT4 internalization, whereas transfection of cells with NHERF-1 attenuated hOAT4 internalization. CONCLUSION: Our studies demonstrated that i) hOAT4 undergoes constitutive trafficking between cell surface and intracellular compartments, ii) hOAT4 internalization partly occurs through clathrin-dependent pathway, iii) the down-regulation of hOAT4 activity by activation of PKC and the up-regulation of hOAT4 activity by NHERF-1 are mediated through alteration of hOAT4 internalization.


Assuntos
Endocitose , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Fosfoproteínas/metabolismo , Proteína Quinase C/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Animais , Células COS , Chlorocebus aethiops , Clatrina/metabolismo , Humanos , Transportadores de Ânions Orgânicos Sódio-Independentes/análise , Transporte Proteico , Proteínas de Transporte Vesicular/análise
17.
Reprod Toxicol ; 26(3-4): 220-30, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18926897

RESUMO

Knowledge of the ontogeny of the various systems involved in distribution and elimination of drugs is important for adequate interpretation of the findings during safety studies in juvenile animals. The present study was designed to collect information on plasma concentrations of total protein and albumin, enzyme activity and mRNA expression of cytochrome P450 isoenzymes (CYP1A1/2, CYP2B1/2, CYP2E1, CYP3A1/2, and CYP4A1), carboxylesterase and thyroxin glucuronidation (T4-GT) activity in liver microsomes, and mRNA expression of transporters (Mdr1a/b, Mrp1-3 and 6, Bsep and Bcrp, Oct1-2, Oat1-3 and Oatp1a4) in liver, kidney and brain tissue during development in Sprague-Dawley rats. Enzyme activities were determined by measuring the metabolism of marker substrates; expression of mRNAs was assessed using RTq-PCR. There were considerable differences in the ontogeny of the individual cytochrome P450 isoenzymes. In addition, ontogeny patterns of enzyme activity did not always parallel ontogeny patterns of mRNA expression. Ontogeny of the transporters depended on the transporter and the organ studied. Changes in mRNA expression of the various transporters during development are likely to result in altered elimination and/or tissue distribution of substrates, with concomitant changes in hepatic metabolism, renal excretion and passage through the blood-brain barrier. Consideration of the ontogeny of metabolizing enzymes and transporters may improve the design and interpretation of results of toxicity studies in juvenile animals.


Assuntos
Transportadores de Cassetes de Ligação de ATP/análise , Proteínas Sanguíneas/análise , Sistema Enzimático do Citocromo P-450/análise , Transportadores de Cassetes de Ligação de ATP/genética , Fatores Etários , Animais , Animais Recém-Nascidos , Proteínas da Membrana Plasmática de Transporte de Catecolaminas/análise , Proteínas da Membrana Plasmática de Transporte de Catecolaminas/genética , Sistema Enzimático do Citocromo P-450/genética , Feminino , Masculino , Microssomos Hepáticos/enzimologia , Proteína 1 Transportadora de Ânions Orgânicos/análise , Proteína 1 Transportadora de Ânions Orgânicos/genética , Transportadores de Ânions Orgânicos Sódio-Independentes/análise , Transportadores de Ânions Orgânicos Sódio-Independentes/genética , Ratos , Ratos Sprague-Dawley
18.
Am J Nephrol ; 28(5): 772-83, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18441523

RESUMO

BACKGROUND/AIMS: Renal organic anion transporters (OAT1 and OAT3) localized in the basolateral membrane mediate the uptake of organic anions from the blood into proximal tubules. This study aimed to examine the effects of renal ischemia/reperfusion injury (IRI) on the expression of cortical renal OAT1 and OAT3 and the functional impact. METHODS: Male rats underwent a right nephrectomy and clamping of the left renal pedicle for 50 min or sham operation, followed by reperfusion for 1, 2, 4 and 6 days. The expression of OAT1 and OAT3 was detected by RT-PCR, immunohistochemistry and Western blot analysis. Na(+)-K(+)-ATPase activity was also estimated. RESULTS: The renal clearance of para-aminohippurate was significantly decreased on day 1 in IRI rats compared with sham-operated rats and returned to normal when the tubular injury recovered. There were significant increases in the mRNA and protein levels of OAT1 and OAT3 in renal cortex homogenates and basolateral membranes on day 1 after IRI, while on days 2 and 4 after IRI, the renal expression of OAT1 and OAT3 decreased gradually but was still significantly higher than that of the sham-operated rats. The Na(+)-K(+)-ATPase activity in renal cortex homogenates decreased significantly on day 1 after IRI but gradually increased on days 2, 4 and 6. CONCLUSIONS: Renal para-aminohippurate clearance was depressed in response to IRI; however, the expressions of renal cortex OAT1 and OAT3 were significantly elevated in the early stage of IRI which may have substantial impact on renal excretion of some drugs and toxic compounds.


Assuntos
Córtex Renal/química , Proteína 1 Transportadora de Ânions Orgânicos/análise , Transportadores de Ânions Orgânicos Sódio-Independentes/análise , Traumatismo por Reperfusão/metabolismo , Regulação para Cima , Animais , Western Blotting , Imuno-Histoquímica , Masculino , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ácido p-Aminoipúrico/metabolismo
19.
J Pharmacol Sci ; 103(1): 48-55, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17220594

RESUMO

Renal organic anion transporters play an important role in the handling of a number of endogenous and exogenous anionic substances in the kidney. In this study, we investigated prostaglandin E(2) (PGE(2)) transport properties and intrarenal localization of mouse organic anion transporter 3 (mOat3). When expressed in Xenopus oocytes, mOat3 mediated the time- and concentration-dependent transport of PGE(2) (K(m): 1.48 microM). PGE(2) transport mediated by mOat3 was trans-stimulated by intracellular glutarate injected into the oocytes. PGE(2) efflux via mOat3 was also trans-stimulated by extracellular glutarate. Thus, mOat3 was shown to mediate the bidirectional transport of PGE(2), partly coupled to the dicarboxylate exchange mechanism. Immunohistochemical study revealed that mOat3 protein was localized at the basolateral membrane of renal proximal and distal tubules. Furthermore, diffuse expression of mOat3, including expression in the basolateral membrane in macula densa (MD) cells, was observed. These results indicate that mOat3 plays an important role as a basolateral transport pathway of PGE(2) in the distal nephron including MD cells that may constitute one of the indispensable steps for renin release and regulation of the tubuloglomerular feedback mechanism.


Assuntos
Dinoprostona/metabolismo , Rim/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/fisiologia , Animais , Transporte Biológico , Feminino , Masculino , Camundongos , Camundongos Endogâmicos ICR , Transportadores de Ânions Orgânicos Sódio-Independentes/análise , Coelhos , Xenopus laevis
20.
J Pharmacol Exp Ther ; 319(2): 809-17, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16895978

RESUMO

Contrast-enhanced magnetic resonance imaging (CE-MRI) is a valuable technique for the diagnosis of liver diseases. As gadocoletic acid trisodium salt (B22956/1), a new contrast agent showing high biliary excretion, may be potentially advantageous in hepatobiliary imaging, the aim of the study was to investigate the molecular mechanisms of hepatic transport of the B22956 ion in a cellular model of hepatic tumor. B22956 ion uptake was measured in tumoral (HepG2) and nontumoral (Chang liver) hepatic cell lines. Absolute quantitative real-time reverse transcriptase (RT)-polymerase chain reaction (PCR) analyses, using cloned PCR products as standards, were performed on total RNA of both cell lines and normal liver to evaluate the transcription of 12 transport genes: SLCO1A2, SLCO2B1, SLCO1B1, SLCO3A1, SLCO4A1, SLCO1B3, SLC22A7, SLC22A8, SLC22A1, SLC10A1, SLC15A1, and SLC15A2. B22956 transport was more efficient in Chang liver than in HepG2 cells and was inhibited by cholecystokinin-8, a specific substrate of OATP1B3. Real-time RT-PCR analyses revealed different transcription profiles in the tumoral and nontumoral cell lines. Compared with normal liver, the expression of SLCO1B1, SLCO3A1, and SLCO1B3 was greatly repressed in HepG2 cells, whereas SLCO2B1, SLC22A7, and SLC22A8 expression was either maintained or increased. On the contrary, in Chang liver cells, SLC22A7 and SLC22A8 genes were undetectable, whereas the expression of SLCO3A1, SLCO4A1, and SLCO1B3 was similar to normal liver. Transport studies and gene expression analyses indicated that B22956 ion is a good substrate to the liver-specific OATP1B3, reported to be poorly expressed or absent in human liver tumors. Therefore, B22956 may be helpful in detecting hepatic neoplastic lesions by CE-MRI.


Assuntos
Meios de Contraste/farmacocinética , Fígado/metabolismo , Compostos Organometálicos/farmacocinética , Transporte Biológico , Western Blotting , Linhagem Celular , Humanos , Neoplasias Hepáticas/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de Soluto , Ácido Taurocólico/farmacocinética
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