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1.
Drug Des Devel Ther ; 15: 3241-3254, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34349501

RESUMO

PURPOSE: Berberine (BBR) is an active component of Phellodendri Cortex (PC), which is a traditional Chinese medicine that has been prescribed clinically for hyperuricemia (HUA) for hundreds of years. Many studies reported the anti-inflammatory and nephroprotective properties of BBR and PC; however, the therapeutic effects of BBR on HUA have not been explored. This study aims to investigate the efficacy and mechanism of BBR for treating HUA. METHODS: The mechanism of BBR in the treatment of HUA were predicted by network pharmacology. A mouse model of HUA established by potassium oxonate and hypoxanthine was used to verify the prediction. The levels of serum uric acid (UA), urea nitrogen (BUN) and creatinine (CRE) were determined by biochemical test kits. Hematoxylin and eosin staining of kidney tissues was used to observe the kidney damage. ELISA kits were applied to detect the levels of interleukin (IL)-1ß and IL-18 in serum and kidney tissues. Quantitative real-time PCR and Western blotting were adopted to analyze the expression of NLRP3, ASC, Caspase1, IL-1ß and URAT1. The expressions of URAT1 in the kidney tubules were visualized by immunohistochemical staining. Molecular docking was used to assess the interaction between URAT1 and BBR. RESULTS: The network pharmacology screened out 82 genes and several inflammation-related signaling pathways related to the anti-hyperuricemia effect of BBR. In the in vivo experiment, BBR substantially decreased the level of UA, BUN and CRE, and alleviated the kidney damage in mice with HUA. BBR reduced IL-1ß and IL-18, and downregulated expressions of NLRP3, ASC, Caspase1 and IL-1ß. BBR also inhibited expression of URAT1 and exhibited strong affinity with this target in silico docking. CONCLUSION: BBR exerts anti-HUA and nephroprotective effects via inhibiting activation of NLRP3 inflammasome and correcting the aberrant expression of URAT1 in kidney. BBR might be a novel therapeutic agent for treating HUA.


Assuntos
Berberina/uso terapêutico , Hiperuricemia/tratamento farmacológico , Nefropatias/tratamento farmacológico , Farmacologia em Rede , Animais , Berberina/farmacologia , Modelos Animais de Doenças , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Masculino , Camundongos , Simulação de Acoplamento Molecular , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Transportadores de Ânions Orgânicos/análise , Transportadores de Ânions Orgânicos/antagonistas & inibidores , Ácido Úrico/sangue
2.
Drug Metab Dispos ; 48(4): 245-254, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31959703

RESUMO

The levels of drug-metabolizing enzymes (DMEs) and transporter proteins in the human intestine are pertinent to determine oral drug bioavailability. Despite the paucity of reports on such measurements, it is well recognized that these values are essential for translating in vitro data on drug metabolism and transport to predict drug disposition in gut wall. In the current study, clinically relevant DMEs [cytochrome P450 (P450) and uridine 5'-diphospho-glucuronosyltransferase (UGT)] and drug transporters were quantified in total mucosal protein preparations from the human jejunum (n = 4) and ileum (n = 12) using quantification concatemer-based targeted proteomics. In contrast to previous reports, UGT2B15 and organic anion-transporting polypeptide 1 (OATP1A2) were quantifiable in all our samples. Overall, no significant disparities in protein expression were observed between jejunum and ileum. Relative mRNA expression for drug transporters did not correlate with the abundance of their cognate protein, except for P-glycoprotein 1 (P-gp) and organic solute transporter subunit alpha (OST-α), highlighting the limitations of RNA as a surrogate for protein expression in dynamic tissues with high turnover. Intercorrelations were found within P450 [2C9-2C19 (P = 0.002, R 2 = 0.63), 2C9-2J2 (P = 0.004, R 2 = 0.40), 2D6-2J2 (P = 0.002, R 2 = 0.50)] and UGT [1A1-2B7 (P = 0.02, R 2 = 0.87)] family of enzymes. There were also correlations between P-gp and several other proteins [OST-α (P < 0.0001, R 2 = 0.77), UGT1A6 (P = 0.009, R 2 = 0.38), and CYP3A4 (P = 0.007, R 2 = 0.30)]. Incorporating such correlations into building virtual populations is crucial for obtaining plausible characteristics of simulated individuals. SIGNIFICANCE STATEMENT: A number of drug transporters were quantified for the first time in this study. Several intercorrelations of protein abundance were reported. mRNA expression levels proved to be a poor reflection of differences between individuals regarding the level of protein expression in gut. The reported abundance of drug-metabolizing enzymes and transporters and their intercorrelations will contribute to better predictions of oral drug bioavailability and drug-drug interactions by linking in vitro observations to potential outcomes through physiologically based pharmacokinetic models.


Assuntos
Sistema Enzimático do Citocromo P-450/análise , Glucuronosiltransferase/análise , Jejuno/enzimologia , Transportadores de Ânions Orgânicos/análise , Administração Oral , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Disponibilidade Biológica , Sistema Enzimático do Citocromo P-450/metabolismo , Interações Medicamentosas , Feminino , Humanos , Jejuno/cirurgia , Masculino , Taxa de Depuração Metabólica , Pessoa de Meia-Idade , Modelos Biológicos , Transportadores de Ânions Orgânicos/metabolismo , Proteômica/métodos
3.
FEBS J ; 287(12): 2468-2485, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-31770475

RESUMO

Organic anion-transporting polypeptide 1A2 (OATP1A2), expressed in the human blood-brain barrier, promotes drug uptake from the blood and hence can be exploited for central nervous system-targeted drug delivery. The thyroid transporter OATP1C1, expressed in the choroid plexus and in astrocytes, is also a potential pharmacological target. Based on their established pharmacological relevance, screening the drug interaction profile of OATP1A2 and OATP1C1 is highly desirable. However, drug interaction screens require suitable model systems and functional assays. In the current study, uptake of a set of cell-impermeable fluorescent dyes was screened in HEK-293 and A431 cell lines overexpressing OATP1A2 and OATP1C1. Based on the uptake of fluorescent dye substrates, a functional assay was developed, which was used to characterize OATP inhibitors/substrates. We identify Live/Dead Green (LDG), Live-or-Dye 488, and sulforhodamines 101, G, and B as novel fluorescent substrates of OATP1A2 and OATP1C1. We show that LDG uptake is proportional to OATP1A2/1C1 expression, allowing the isolation of cells expressing high transporter levels. Additionally, dye uptake can be used to characterize the drug interaction pattern of OATP1A2 and OATP1C1. We demonstrate that third-generation P-glycoprotein inhibitors elacridar, tariquidar, and zosuquidar inhibit OATP1A2 function. Increased toxicity of elacridar in OATP1A2-expressing cells suggests that OATP1A2 may modulate the distribution of this compound. The fluorescence-based assays developed in the current study are a good alternative of radioligand-based tests and pave the way toward high-throughput screens for OATP1A2/1C1 drug interaction studies.


Assuntos
Acridinas/farmacologia , Dibenzocicloeptenos/farmacologia , Corantes Fluorescentes/química , Transportadores de Ânions Orgânicos/antagonistas & inibidores , Quinolinas/farmacologia , Tetra-Hidroisoquinolinas/farmacologia , Acridinas/química , Linhagem Celular , Dibenzocicloeptenos/química , Humanos , Transportadores de Ânions Orgânicos/análise , Transportadores de Ânions Orgânicos/metabolismo , Quinolinas/química , Tetra-Hidroisoquinolinas/química
4.
Mol Med Rep ; 20(2): 1645-1654, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31257523

RESUMO

Chronic gouty arthritis, caused by a persistent increase in, and the deposition of, soluble uric acid (sUA), can induce pathological chondrocyte destruction; however, the effects of urate transport and intracellular sUA on chondrocyte functionality and viability are yet to be fully determined. Thus, the aim of the present study was to investigate the presence and functionality of a urate transport system in chondrocytes. The expression profiles of two primary urate reabsorptive transporters, glucose transporter 9 (GLUT9) and urate transporter 1 (URAT1), in human articular cartilage and chondrocyte cell lines were examined via western blotting, reverse transcription­quantitative PCR, immunohistochemistry and immunofluorescence. Then, chondrocytes were incubated with exogenous sUA at increasing concentrations. Negative control assays were conducted via the specific knockdown of GLUT9 and URAT1 with lentiviral short hairpin (sh)RNAs, and by pretreatment with benzbromarone, a known inhibitor of the two transporters. Intracellular UA concentrations were measured using colorimetric assays. The expression levels of GLUT9 and URAT1 were determined in cartilage tissues and chondrocyte cell lines. Incubation of chondrocytes with sUA led to a concentration­dependent increase in intracellular urate concentrations, which was inhibited by GLUT9 or URAT1 knockdown, or by benzbromarone pretreatment (27.13±2.70, 44.22±2.34 and 58.46±2.32% reduction, respectively). In particular, benzbromarone further decreased the already­reduced intracellular UA concentrations in HC­shGLUT9 and HC­shURAT1 cells by 46.79±2.46 and 39.79±2.22%, respectively. Cells overexpressing GLUT9 and URAT1 were used as the positive cell control, which showed increased intracellular UA concentrations that could be reversed by treatment with benzbromarone. In conclusion, chondrocytes may possess an active UA transport system. GLUT9 and URAT1 functioned synergistically to transport UA into the chondrocyte cytoplasm, which was inhibited by specific gene knockdowns and drug­induced inhibition. These results may be fundamental in the further investigation of the pathological changes to chondrocytes induced by sUA during gouty arthritis, and identified UA transport processes as potential targets for the early control of chronic gouty arthritis.


Assuntos
Condrócitos/metabolismo , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Ácido Úrico/metabolismo , Transporte Biológico , Cartilagem Articular/metabolismo , Linhagem Celular , Proteínas Facilitadoras de Transporte de Glucose/análise , Células HEK293 , Humanos , Transportadores de Ânions Orgânicos/análise , Proteínas de Transporte de Cátions Orgânicos/análise
5.
Int J Mol Sci ; 19(9)2018 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-30231490

RESUMO

Aluminum-activated malate transporters (ALMTs) play an important role in aluminum tolerance, stomatal opening, and fruit acidity in plants. However, the evolutionary pattern of the ALMT gene family in apples remains relatively unknown. In this study, a total of 25 MdALMT genes were identified from the apple reference genome of the "Golden Delicious" doubled-haploid tree (GDDH13). The physiological and biochemical properties, gene structure, and conserved motifs of MdALMT genes were examined. Chromosome location and gene-duplication analysis indicated that whole-genome duplication/segmental duplication played an important role in the expansion of the MdALMT gene family. The Ka/Ks ratio of duplicated MdALMT genes showed that members of this family have undergone strong purifying selection. Through exploration of the phylogenetic relationships, seven subgroups were classified, and higher old gene duplication frequency and significantly different evolutionary rates of the ALMT gene families were detected. In addition, the functional divergence of ALMT genes occurred during the evolutionary process of Rosaceae species. Furthermore, the functional divergence of MdALMT genes was confirmed by expression discrepancy and different subcellular localizations. This study provides the foundation to better understand the molecular evolution of MdALMT genes and further facilitate functional analysis to unravel their exact role in apples.


Assuntos
Alumínio/metabolismo , Evolução Molecular , Malatos/metabolismo , Malus/genética , Transportadores de Ânions Orgânicos/genética , Filogenia , Proteínas de Plantas/genética , Duplicação Gênica , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Malus/metabolismo , Transportadores de Ânions Orgânicos/análise , Transportadores de Ânions Orgânicos/metabolismo , Proteínas de Plantas/análise , Proteínas de Plantas/metabolismo
6.
Gastroenterology ; 155(5): 1578-1592.e16, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30063921

RESUMO

BACKGROUND & AIMS: Bile acid transporters maintain bile acid homeostasis. Little is known about the functions of some transporters in cholestasis or their regulatory mechanism. We investigated the hepatic expression of solute carrier organic anion transporter family member 3A1 (SLCO3A1, also called OATP3A1) and assessed its functions during development of cholestasis. METHODS: We measured levels of OATP3A1 protein and messenger RNA and localized the protein in liver tissues from 22 patients with cholestasis and 21 patients without cholestasis, using real-time quantitative polymerase chain reaction, immunoblot, and immunofluorescence analyses. We performed experiments with Slco3a1-knockout and C57BL/6J (control) mice. Mice and Sprague-Dawley rats underwent bile duct ligation (BDL) or a sham operation. Some mice were placed on a 1% cholic acid (CA) diet to induce cholestasis or on a control diet. Serum and liver tissues were collected and analyzed; hepatic levels of bile acids and 7-α-C4 were measured using liquid chromatography/mass spectrometry. Human primary hepatocytes and hepatoma (PLC/PRF/5) cell lines were used to study mechanisms that regulate OATP3A1 expression and transport. RESULTS: Hepatic levels of OATP3A1 messenger RNA and protein were significantly increased in liver tissues from patients with cholestasis and from rodents with BDL or 1% CA diet-induced cholestasis. Levels of fibroblast growth factor 19 (FGF19, FGF15 in rodents) were also increased in liver tissues from patients and rodents with cholestasis. FGF19 signaling activated the Sp1 transcription factor and nuclear factor κB to increase expression of OATP3A1 in hepatocytes; we found binding sites for these factors in the SLCO3A1 promoter. Slco3a1-knockout mice had shorter survival times and increased hepatic levels of bile acid, and they developed more liver injury after the 1% CA diet or BDL than control mice. In hepatoma cell lines, we found OATP3A1 to take prostaglandin E2 and thyroxine into cells and efflux bile acids. CONCLUSIONS: We found levels of OATP3A1 to be increased in cholestatic liver tissues from patients and rodents compared with healthy liver tissues. We show that OATP3A1 functions as a bile acid efflux transporter that is up-regulated as an adaptive response to cholestasis.


Assuntos
Ácidos e Sais Biliares/metabolismo , Colestase/metabolismo , Transportadores de Ânions Orgânicos/fisiologia , Animais , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Fatores de Crescimento de Fibroblastos/análise , Fatores de Crescimento de Fibroblastos/fisiologia , Humanos , Fígado/química , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transportadores de Ânions Orgânicos/análise , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Fator de Transcrição Sp1/fisiologia , Fator de Transcrição RelA/fisiologia
7.
Anticancer Res ; 37(10): 5693-5700, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28982888

RESUMO

BACKGROUND/AIM: We assessed the utility of dynamic magnetic resonance imaging (MRI) with gadoxetate-ethoxybenzyl-diethylenetriamine penta-aceticpenta-acetic acid (Gd-EOB-DTPA) (EOB-MRI) for estimating functional liver volume compared to 99mTc-galactosyl albumin single-photon-emission computed tomography (99mTc-GSA SPECT). PATIENTS AND METHODS: Regional functional liver volume (left lateral, medial, right anterior, right posterior) of 58 hepatectomized patients was assessed using EOB-MRI and 99mTc-GSA SPECT, and compared to the actual liver volume with MDCT-3D volumetry. RESULTS: 99mTc-GSA SPECT found a significantly lower functional volume of the left lateral section than the actual volume found by MDCT-3D volumetry (p=0.003) and EOB-MRI (p<0.001). Functional liver volume of right anterior section found with 99mTc-GSA SPECT was significantly higher than that found by MDCT-3D volumetry (p=0.04), despite no differences in asialoglycoprotein receptor 1 (ASGR1) or ATP-dependent organic anion transporting polypeptide 1 (OATP) expression between the left lateral and right anterior sections. CONCLUSION: 99mTc-GSA SPECT might underestimate the function of the left lobe and overestimate that of the right lobe. Therefore, EOB-MRI could be better for estimating the true regional functional liver reserve.


Assuntos
Meios de Contraste/administração & dosagem , Gadolínio DTPA/administração & dosagem , Fígado/diagnóstico por imagem , Imageamento por Ressonância Magnética/métodos , Tomografia Computadorizada Multidetectores , Compostos Radiofarmacêuticos/administração & dosagem , Tomografia Computadorizada com Tomografia Computadorizada de Emissão de Fóton Único/métodos , Agregado de Albumina Marcado com Tecnécio Tc 99m/administração & dosagem , Pentetato de Tecnécio Tc 99m/administração & dosagem , Adulto , Idoso , Idoso de 80 Anos ou mais , Receptor de Asialoglicoproteína/análise , Biomarcadores/análise , Biópsia , Feminino , Hepatectomia , Humanos , Imuno-Histoquímica , Fígado/química , Fígado/cirurgia , Masculino , Pessoa de Meia-Idade , Tamanho do Órgão , Transportadores de Ânions Orgânicos/análise , Valor Preditivo dos Testes , Reprodutibilidade dos Testes
8.
Neurosci Lett ; 659: 99-103, 2017 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-28870626

RESUMO

It has been suggested that urate plays a protective role in neurons, while hyperuricemia is correlated with atherosclerosis and cardiovascular disease. However, whether there is a system that directly transports urate into the brain remains to be clarified. In this study, the localization of glucose transporter 9 (GLUT9) and urate transporter 1 (URAT1), which are known to be representative reabsorptive urate transporters, was immunohistochemically examined in autopsied human brains. Immunoreactivity of GLUT9 was observed on the apical side of the cytoplasm of epithelial cells in the choroid plexus and in the cilia of ependymal cells of the human brain. Immunoreactivity of URAT1 was observed on the basolateral side of the cytoplasm of epithelial cells in the choroid plexus. In addition, immunoreactivity of GLUT9 and URAT1 was not observed in microvessels of the human brains. The choroid plexus and renal proximal tubule were similar in having a polarized distribution of these two transporters with the two transporters on opposite membranes, but the two transporters' distribution differs between the choroid plexus and the kidney in terms of which membrane (apical/basal) expresses which transporter. These findings support the hypothesis of the direct transport of intravascular urate into the central nervous system through the choroid plexus.


Assuntos
Encéfalo/imunologia , Plexo Corióideo/imunologia , Células Epiteliais/imunologia , Proteínas Facilitadoras de Transporte de Glucose/análise , Proteínas Facilitadoras de Transporte de Glucose/imunologia , Transportadores de Ânions Orgânicos/análise , Transportadores de Ânions Orgânicos/imunologia , Proteínas de Transporte de Cátions Orgânicos/análise , Proteínas de Transporte de Cátions Orgânicos/imunologia , Encéfalo/citologia , Encéfalo/metabolismo , Plexo Corióideo/citologia , Plexo Corióideo/metabolismo , Epêndima/imunologia , Células Epiteliais/metabolismo , Humanos , Imuno-Histoquímica , Túbulos Renais Proximais/imunologia
10.
Histochem Cell Biol ; 148(4): 345-357, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28493059

RESUMO

Organic anion-transporting polypeptides (OATPs) are membrane proteins that mediate cellular uptake of structurally diverse endogenous and exogenous compounds, including bile salts, thyroid and sex hormones, pharmacological agents, and toxins. Roles of OATPs in human liver are well established. Our recent report suggested the presence of the hepatic transporter OATP1B3 in human ß cells. The aim of this study was to better characterize cellular localization and interindividual variation in OATP1B3 expression in human adult islets as a function of age, sex, and pancreatic disease, and to assess the expression of other OATPs. High transcript levels of OATP1B3, OATP2B1, OATP1A2, but not OATP1B1 were observed in isolated human adult islets. While OATP1B3 protein expression was variable, the carrier co-localized more frequently with glucagon-positive α cells than insulin-positive ß cells in islets of normal pancreatic tissues from ten subjects using dual immunostaining. Moreover, OATP1B3 co-staining with endocrine cells was two- to three-fold higher in older (≥60 years) than younger (<60 years) subjects. In comparison, in a subset of three individuals, OATP2B1 was primarily found in ß cells, suggesting a distinct expression pattern for OATP1B3 and OATP2B1 in islets. Abundant OATP1B3 staining was also observed in islet as well as ductal cells of diseased tissues of patients with pancreatitis or pancreatic adenocarcinoma. Considering the abundance of key OATP carriers in ß and α cells, potential implications of OATP transport in islet cell function may be suggested. Future studies are needed to gain insights into their specific endocrine roles as well as pharmacological relevance.


Assuntos
Ilhotas Pancreáticas/metabolismo , Transportadores de Ânions Orgânicos/genética , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de Soluto/genética , Adulto , Humanos , Ilhotas Pancreáticas/química , Ilhotas Pancreáticas/citologia , Transportadores de Ânions Orgânicos/análise , Transportadores de Ânions Orgânicos/metabolismo , RNA Mensageiro/genética , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de Soluto/análise , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de Soluto/metabolismo
11.
Pharm Biol ; 54(12): 3151-3155, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27563755

RESUMO

CONTEXT: Hyperuricaemia is known as an abnormally increased uric acid level in the blood. Although it was observed many years ago, since uric acid excretion via the intestine pathway accounted for approximately one-third of total elimination of uric acid, the molecular mechanism of 'extra-renal excretion' was poorly understood until the finding of uric acid transporters. OBJECTIVE: The objective of this study was to gather all information related to uric acid transporters in the intestine and present this information as a comprehensive and systematic review article. METHODS: A literature search was performed from various databases (e.g., Medline, Science Direct, Springer Link, etc.). The key terms included uric acid, transporter and intestine. The period for the search is from the 1950s to the present. The bibliographies of papers relating to the review subject were also searched for further relevant references. RESULTS: The uric acid transporters identified in the intestine are discussed in this review. The solute carrier (SLC) transporters include GLUT9, MCT9, NPT4, NPT homolog (NPT5) and OAT10. The ATP binding cassette (ABC) transporters include ABCG2 (BCRP), MRP2 and MRP4. Bacterial transporter YgfU is a low-affinity and high-capacity transporter for uric acid. CONCLUSION: The present review may be helpful for further our understanding of hyperuricaemia and be of value in designing future studies on novel therapeutic pathways.


Assuntos
Mucosa Intestinal/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Proteínas Carreadoras de Solutos/metabolismo , Ácido Úrico/metabolismo , Animais , Humanos , Intestinos/química , Transportadores de Ânions Orgânicos/análise , Proteínas Carreadoras de Solutos/análise , Ácido Úrico/análise
12.
Mol Med Rep ; 14(4): 3243-50, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27510522

RESUMO

The genotoxicity and cytotoxicity of aristolochic acids is well documented, and the Aristolochiaceae plant family has been widely used in China and India for medical purposes. However, the mechanisms of aristolochic acid I (AAI) in treatment and toxicity remain to be fully elucidated. According to the theory of traditional Chinese medicine (TCM), the spleen is responsible for transportation and transformation, in which a substance is transformed, absorbed and distributed in the body. In the present study, rats were randomized into a blank group without spleen deficiency and a spleen deficiency group to investigate the metabolism of AAI. The results showed that the concentration of AAI was higher in the spleen deficiency group, compared with that of the blank group. To further elucidate this process, the expression of organic anion transporting peptide (oatp)2a1 in the rats of the two groups were examined following oral administration of AAI. It was observed that the mRNA level of oatp2a1 in the small intestine of the blank+AAI 60 min group was downregulated, compared with that in the blank group. Compared with the mRNA level of oatp2a1 in the spleen deficiency group, the expression levels in the lung and liver were downregulated in the spleen deficiency+AAI 5 min group, whereas expression levels in the kidney in the spleen deficiency+AAI 60 min group were upregulated. Based on the above results, it was hypothesized that the expression of oatp2a1 may be one of the mechanisms of AAI metabolism in rats. In TCM, the spleen and certain functions of the small intestine, are important in AAI metabolism, and affect the toxicity of AAI. In addition, the lung, liver and kidney may also be involved in spleen deficiency syndrome in rats.


Assuntos
Ácidos Aristolóquicos/metabolismo , Medicamentos de Ervas Chinesas/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Baço/metabolismo , Animais , Ácidos Aristolóquicos/administração & dosagem , Ácidos Aristolóquicos/química , Ácidos Aristolóquicos/farmacocinética , Asarum/química , Medicamentos de Ervas Chinesas/administração & dosagem , Medicamentos de Ervas Chinesas/química , Medicamentos de Ervas Chinesas/farmacocinética , Masculino , Medicina Tradicional Chinesa , Transportadores de Ânions Orgânicos/análise , Transportadores de Ânions Orgânicos/genética , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley
13.
Methods ; 82: 38-46, 2015 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-25916617

RESUMO

Flow cytometry is a powerful tool for the quantitation of fluorescence and is proven to be able to correlate the fluorescence intensity to the number of protein on cells surface. Mass spectroscopy can also be used to determine the number of proteins per cell. Here we have developed two methods, using flow cytometry and mass spectroscopy to quantify number of transporters in human cells. These two approaches were then used to analyse the same samples so that a direct comparison could be made. Transporters have a major impact on the behaviour of a diverse number of drugs in human systems. While active uptake studies by transmembrane protein transporters using model substrates are routinely undertaken in human cell lines and hepatocytes as part of drug discovery and development, the interpretation of these results is currently limited by the inability to quantify the number of transporters present in the test samples. Here we provide a flow cytometric method for accurate quantification of transporter levels both on the cell surface and within the cell, and compare this to a quantitative mass spectrometric approach. Two transporters were selected for the study: OATP1B1 (also known as SLCO1B1, LST-1, OATP-C, OATP2) due to its important role in hepatic drug uptake and elimination; P-gp (also known as P-glycoprotein, MDR1, ABCB1) as a well characterised system and due to its potential impact on oral bioavailability, biliary and renal clearance, and brain penetration of drugs that are substrates for this transporter. In all cases the mass spectrometric method gave higher levels than the flow cytometry method. However, the two methods showed very similar trends in the relative ratios of both transporters in the hepatocyte samples investigated. The P-gp antibody allowed quantitative discrimination between externally facing transporters located in the cytoplasmic membrane and the total number of transporters on and in the cell. The proportion of externally facing transporter varied considerably in the four hepatocyte samples analysed, ranging from only 6% to 35% of intact and viable cells. The sample with only 6% externally facing transporter was further analysed by confocal microscopy which qualitatively confirmed the low level of transporter in the membrane and the large internal population. Here we prove that flow cytometry is an important tool for future protein analysis as it can not only quantify the number of proteins that a cell express but also identify the number of proteins on the surface and it is easy to apply for routine assays.


Assuntos
Citometria de Fluxo , Hepatócitos/metabolismo , Espectrometria de Massas , Transportadores de Ânions Orgânicos/análise , Subfamília B de Transportador de Cassetes de Ligação de ATP/análise , Linhagem Celular , Hepatócitos/química , Humanos , Transportador 1 de Ânion Orgânico Específico do Fígado
14.
PLoS One ; 10(4): e0123895, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25923111

RESUMO

Prostaglandin (PG) E2 exhibits an anti-fibrotic effect in the lung in response to inflammatory reactions and is a high-affinity substrate of PG transporter (SLCO2A1). The present study aimed to evaluate the pathophysiological relevance of SLCO2A1 to bleomycin (BLM)-induced pulmonary fibrosis in mice. Immunohistochemical analysis indicated that Slco2a1 protein was expressed in airway and alveolar type I (ATI) and II (ATII) epithelial cells, and electron-microscopic immunohistochemistry further demonstrated cell surface expression of Slco2a1 in ATI cells in wild type (WT) C57BL/6 mice. PGE2 uptake activity was abrogated in ATI-like cells from Slco2a1-deficient (Slco2a1-/-) mice, which was clearly observed in the cells from WT mice. Furthermore, the PGE2 concentrations in lung tissues were lower in Slco2a1-/- than in WT mice. The pathological relevance of SLCO2A1 was further studied in mouse BLM-induced pulmonary fibrosis models. BLM (1 mg/kg) or vehicle (phosphate buffered saline) was intratracheally injected into WT and Slco2a1-/- mice, and BLM-induced fibrosis was evaluated on day 14. BLM induced more severe fibrosis in Slco2a1-/- than in WT mice, as indicated by thickened interstitial connective tissue and enhanced collagen deposition. PGE2 levels were higher in bronchoalveolar lavage fluid, but lower in lung tissues of Slco2a1-/- mice. Transcriptional upregulation of TGF-ß1 was associated with enhanced gene transcriptions of downstream targets including plasminogen activator inhitor-1. Furthermore, Western blot analysis demonstrated a significant activation of protein kinase C (PKC) δ along with a modest activation of Smad3 in lung from Slco2a1-/- mice, suggesting a role of PKCδ associated with TGF-ß signaling in aggravated fibrosis in BLM-treated Slco2a1-/- mice. In conclusion, pulmonary PGE2 disposition is largely regulated by SLCO2A1, demonstrating that SLCO2A1 plays a critical role in protecting the lung from BLM-induced fibrosis.


Assuntos
Bleomicina , Dinoprostona/imunologia , Pulmão/patologia , Transportadores de Ânions Orgânicos/imunologia , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/imunologia , Animais , Líquido da Lavagem Broncoalveolar , Células Cultivadas , Dinoprostona/análise , Deleção de Genes , Regulação da Expressão Gênica , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Masculino , Camundongos Endogâmicos C57BL , Transportadores de Ânions Orgânicos/análise , Transportadores de Ânions Orgânicos/genética , Fibrose Pulmonar/genética , Fibrose Pulmonar/patologia , RNA Mensageiro/genética , Ratos Wistar
15.
BMC Pregnancy Childbirth ; 14: 241, 2014 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-25048443

RESUMO

BACKGROUND: Elucidation of the biochemical pathways involved in activation of preterm and term human labour would facilitate the development of effective management and inform judgements regarding the necessity for preterm tocolysis and post-term induction. Prostaglandins act at all stages of human reproduction, and are potentially activators of labour. METHODS: Expression of 15 genes involved in prostaglandin synthesis, transport and degradation was measured by qPCR using tissue samples from human placenta, amnion and choriodecidua at preterm and full-term vaginal and caesarean delivery. Cellular localisation of eight prostaglandin pathway proteins was determined by immunohistochemistry. RESULTS: Expression of prostaglandin pathway genes was differentially affected by factors including gestational age at delivery, and the incidence and duration of labour. Chorioamnionitis/deciduitis was associated with upregulation of PTGS2 (prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxygenase)), along with the inflammatory genes IL8 (interleukin 8), S100A8 (S100 calcium binding protein A8) and TLR2 (toll-like receptor 2), in amnion and choriodecidua, and with downregulation of CBR1 (carbonyl reductase 1) and HPGD (hydroxyprostaglandin dehydrogenase 15-(NAD)) in choriodecidua. Protein localisation differed greatly between the various maternal and fetal cell types. CONCLUSIONS: Preterm and term labour are associated with distinct prostaglandin pathway expression profiles; inflammation provokes specific changes, unrelated to the presence of labour; spontaneous and induced term labour are indistinguishable.


Assuntos
Expressão Gênica , Trabalho de Parto/genética , Trabalho de Parto Prematuro/genética , Prostaglandinas/análise , Prostaglandinas/genética , Transdução de Sinais/genética , 3-Hidroxiesteroide Desidrogenases/análise , 3-Hidroxiesteroide Desidrogenases/genética , Adulto , Oxirredutases do Álcool/análise , Oxirredutases do Álcool/genética , Aldeído Redutase/análise , Aldeído Redutase/genética , Membro C3 da Família 1 de alfa-Ceto Redutase , Âmnio/química , Calgranulina A/análise , Calgranulina A/genética , Corioamnionite/genética , Córion/química , Ciclo-Oxigenase 2/análise , Ciclo-Oxigenase 2/genética , Sistema Enzimático do Citocromo P-450/análise , Sistema Enzimático do Citocromo P-450/genética , Decídua/química , Regulação para Baixo , Feminino , Idade Gestacional , Humanos , Hidroxiprostaglandina Desidrogenases/análise , Hidroxiprostaglandina Desidrogenases/genética , Interleucina-1/análise , Interleucina-1/genética , Oxirredutases Intramoleculares/análise , Oxirredutases Intramoleculares/genética , Trabalho de Parto/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/análise , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Trabalho de Parto Prematuro/metabolismo , Transportadores de Ânions Orgânicos/análise , Transportadores de Ânions Orgânicos/genética , Placenta/química , Gravidez , Prostaglandina-E Sintases , Prostaglandinas/metabolismo , Receptor 2 Toll-Like/análise , Receptor 2 Toll-Like/genética , Regulação para Cima , Adulto Jovem
16.
J Endod ; 40(8): 1112-7, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25069917

RESUMO

INTRODUCTION: The prostaglandin transporter (Pgt) and multidrug resistance-associated protein (Mrp) 4 are membrane transport proteins that play crucial roles in the transmembrane uptake and/or efflux of prostaglandins (PGs). This study attempted to analyze the protein expression of Pgt and Mrp4 and their involvement in PGE2 efflux transport in lipopolysaccharide (LPS)-inflamed rat incisor pulp tissue. METHODS: Pulpitis was induced in the upper incisors of Wistar rats by treating them with LPS for 24 hours. The protein expression levels of Pgt, Mrp4, and microsomal PGE synthase (mPGES) were analyzed with immunofluorescent staining. The amount of PGE2 released from the inflamed pulp tissue in the presence or absence of dipyridamole (an Mrp4 inhibitor) was assessed by using an enzyme-linked immunosorbent assay. RESULTS: Double immunofluorescence staining revealed that the Pgt, Mrp4, and mPGES immunoreactivity co-localized in CD31-expressing endothelial cells. Moreover, the Mrp4 inhibitor caused a significant decrease in the amount of PGE2 released from the LPS-inflamed pulp (P < .01 at 24 hours). CONCLUSION: Pgt, Mrp4, and mPGES expression was detected in the endothelial cells of normal and LPS-inflamed rat incisor pulp tissue, suggesting that these cells are associated with the biosynthesis and transmembrane transport of PGE2. The significant decrease in PGE2 release induced by the Mrp4 inhibitor suggests that Mrp4 contributes to the transport of PGE2 in the transmembrane efflux pathway.


Assuntos
Dinoprostona/metabolismo , Lipopolissacarídeos/efeitos adversos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Pulpite/metabolismo , Animais , Antígenos CD/análise , Antígenos de Diferenciação Mielomonocítica/análise , Polpa Dentária/metabolismo , Polpa Dentária/patologia , Dinoprostona/análise , Dipiridamol/farmacologia , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Escherichia coli , Oxirredutases Intramoleculares/análise , Macrófagos/patologia , Masculino , Proteínas Associadas à Resistência a Múltiplos Medicamentos/análise , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Neutrófilos/patologia , Transportadores de Ânions Orgânicos/análise , Molécula-1 de Adesão Celular Endotelial a Plaquetas/análise , Prostaglandina-E Sintases , Pulpite/patologia , Ratos , Ratos Wistar , Organismos Livres de Patógenos Específicos
17.
J Biomed Opt ; 18(10): 101306, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23812606

RESUMO

The liver is important in the biotransformation of various drugs, where hepatic transporters facilitate uptake and excretion. Ischemia-reperfusion (I/R) injury is a common occurrence in liver surgery, and the developing oxidative stress can lead to graft failure. We used intravital multiphoton tomography, with fluorescence lifetime imaging, to characterize metabolic damage associated with hepatic I/R injury and to model the distribution of fluorescein as a measure of liver function. In addition to measuring a significant increase in serum alanine transaminase levels, characteristic of hepatic I/R injury, a decrease in the averaged weighted lifetime of reduced nicotinamide adenine dinucleotide phosphate was observed, which can be attributed to a changed metabolic redox state of the hepatocytes. I/R injury was associated with delayed uptake and excretion of fluorescein and elevated area-under-the-curve within the hepatocytes compared to sham (i.e., untreated control) as visualized and modeled using images recorded by intravital multiphoton tomography. High-performance liquid chromatography analysis showed no differences in plasma or bile concentrations of fluorescein. Finally, altered fluorescein distribution was associated with acute changes in the expression of liver transport proteins. In summary, multiphoton intravital imaging is an effective approach to measure liver function and is more sensitive in contrasting the impact of I/R injury than measuring plasma and bile concentrations of fluorescein.


Assuntos
Fluoresceína/farmacocinética , Fígado/irrigação sanguínea , Fígado/metabolismo , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Traumatismo por Reperfusão/metabolismo , Transportadores de Cassetes de Ligação de ATP/análise , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Alanina Transaminase/sangue , Proteínas Angiogênicas/análise , Proteínas Angiogênicas/genética , Proteínas Angiogênicas/metabolismo , Animais , Western Blotting , Cromatografia Líquida de Alta Pressão , Fluoresceína/análise , Fluoresceína/química , Histocitoquímica , Processamento de Imagem Assistida por Computador/métodos , Fígado/química , Masculino , Transportadores de Ânions Orgânicos/análise , Transportadores de Ânions Orgânicos/genética , Transportadores de Ânions Orgânicos/metabolismo , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real
18.
Clin Pharmacol Ther ; 93(3): 275-82, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23361102

RESUMO

Individual variability in expression and function of organic anion-transporting polypeptide 1B1 (OATP1B1), multidrug resistance protein 1 (MDR1), and/or cytochrome P450 3A4 (CYP3A4) may impact the clinical response of many drugs. We investigated the correlation between expression of these proteins and pharmacokinetics of atorvastatin, a substrate of all three, in 21 obese patients with paired biopsies from liver and intestinal segments. The patients were also screened for the SLCO1B1 c.521T→C variant alleles. Approximately 30% (r(2) = 0.28) of the variation in oral clearance (CL/F) of atorvastatin was explained by hepatic OATP1B1 protein expression (P = 0.041). Patients carrying the SLCO1B1 c.521C variant allele (homozygous, n = 4; heterozygous, n = 2) exhibited 45% lower CL/F of atorvastatin than the c.521TT carriers (P = 0.067). No association between hepatic and intestinal expression of MDR1 or CYP3A4 and atorvastatin pharmacokinetics was found (P > 0.149). In conclusion, this study suggests that OATP1B1 phenotype is more important than CYP3A4 and MDR1 phenotypes for the individual pharmacokinetic variability of atorvastatin.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/fisiologia , Citocromo P-450 CYP3A/fisiologia , Ácidos Heptanoicos/farmacocinética , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacocinética , Mucosa Intestinal/metabolismo , Fígado/metabolismo , Obesidade/metabolismo , Transportadores de Ânions Orgânicos/fisiologia , Pirróis/farmacocinética , Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/análise , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Adulto , Atorvastatina , Índice de Massa Corporal , Citocromo P-450 CYP3A/análise , Citocromo P-450 CYP3A/genética , Feminino , Humanos , Transportador 1 de Ânion Orgânico Específico do Fígado , Masculino , Pessoa de Meia-Idade , Transportadores de Ânions Orgânicos/análise , Transportadores de Ânions Orgânicos/genética
19.
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
20.
Bioorg Med Chem ; 20(12): 3703-9, 2012 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-22607882

RESUMO

Drug transporters mediate the uptake and elimination of drugs in various organs; therefore, having knowledge of how a transporter functions in the body would play a key role in ensuring drug efficacy in in vivo systems. In this context, we designed and synthesized [(11)C]dehydropravastatin, a novel PET probe that would be potentially useful for evaluation of the functions of the OATP1B1 and MRP2 transporters, based on the use of palladium(0)-mediated rapid C-[(11)C]methylation (viz., the rapid cross-coupling between [(11)C]methyl iodide and a boron intermediate).


Assuntos
Transportadores de Cassetes de Ligação de ATP/análise , Fígado/química , Transportadores de Ânions Orgânicos/análise , Tomografia por Emissão de Pósitrons , Pravastatina/análogos & derivados , Compostos Radiofarmacêuticos/síntese química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Fígado/metabolismo , Transportador 1 de Ânion Orgânico Específico do Fígado , Masculino , Transportadores de Ânions Orgânicos/metabolismo , Pravastatina/síntese química , Pravastatina/química , Compostos Radiofarmacêuticos/química , Ratos , Ratos Sprague-Dawley
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