RESUMEN
Our recent study revealed that SLC49A4, known as disrupted in renal carcinoma 2, is a H+-coupled lysosomal exporter for pyridoxine (vitamin B6), a cationic compound, and involved in the regulation of its lysosomal and cellular levels. We here examined a possibility that this transporter might also transport cationic amphiphilic drugs (CADs) that are known to undergo lysosomal trapping, using pyrilamine, an H1-antagonist, as a model CAD and the COS-7 cell line as a model cell system for transient introduction of human SLC49A4 and a recombinant SLC49A4 protein (SLC49A4-AA), in which the N-terminal dileucine motif involved in lysosomal localization was removed by replacing with dialanine for redirected localization to the plasma membrane. The introduction of SLC49A4 into COS-7 cells induced a significant decrease in the accumulation of pyrilamine in the intracellular compartments in the cells treated with digitonin for permeabilization of plasma membranes, suggesting its operation for lysosomal pyrilamine export. Accordingly, functional analysis using the SLC49A4-AA mutant, which operates for cellular uptake at the plasma membrane, in transiently transfected COS-7 cells demonstrated its H+-coupled operation for pyrilamine transport, which was saturable with a Michaelis constant of 132 µM at pH 5.5. In addition, many CADs that may potentially undergo lysosomal trapping, which include imipramine, propranolol, verapamil, and some others, were found to inhibit SLC49A4-AA-mediated pyrilamine transport, suggesting their affinity for SLC49A4. These results suggest that SLC49A4 is involved in the lysosomal trapping of pyrilamine, operating for its exit. The CADs that inhibited SLC49A4-AA-mediated pyrilamine transport could also be SLC49A4 substrate candidates. Significance Statement SLC49A4 mediates the transport of pyrilamine in a H+-coupled manner at the lysosomal membrane. This could be a newly identified mechanism for lysosomal export involved in its lysosomal trapping.
RESUMEN
Disrupted in renal carcinoma 2 (DIRC2) has gained interest because of its association with the development of renal cancer and cosegregation with a chromosomal translocation. It is a member of the SLC49 family (SLC49A4) and is considered to be an electrogenic lysosomal metabolite transporter; however, its molecular function has not been fully defined. To perform a detailed functional analysis of human DIRC2, we used a recombinant DIRC2 protein (DIRC2-AA), in which the N-terminal dileucine motif involved in its lysosomal localization was removed by replacing with dialanine for redirected localization to the plasma membrane, exposing intralysosomal segments to the extracellular space. The DIRC2-AA mutant induced the cellular uptake of pyridoxine (vitamin B6) under acidic conditions when expressed transiently in COS-7 cells. In addition, uptake was markedly inhibited by protonophores, indicating its function through an H+-coupled mechanism. In separate experiments, the transient overexpression of unmodified DIRC2 (tagged with HA) in human embryonic kidney 293 cells reduced cellular pyridoxine accumulation induced by transiently introduced human thiamine transporter 2/SLC19A3 (tagged with FLAG), a plasma membrane thiamine transporter that also transports pyridoxine. The cellular accumulation of pyridoxine in Caco-2 cells as a cell model was increased by the knockdown of endogenous DIRC2. Overall, the results indicate that DIRC2 is an H+-driven lysosomal pyridoxine exporter. Its overexpression leads to a reduction in cellular pyridoxine accumulation associated with reduced lysosomal accumulation and, conversely, its suppression results in an increase in lysosomal and cellular pyridoxine accumulation.
Asunto(s)
Carcinoma de Células Renales , Neoplasias Renales , Proteínas de Transporte de Membrana , Humanos , Células CACO-2 , Carcinoma de Células Renales/genética , Neoplasias Renales/genética , Lisosomas , Proteínas de Transporte de Membrana/genética , Piridoxina , TiaminaRESUMEN
Recent studies have shown that human solute carrier SLC19A3 (hSLC19A3) can transport pyridoxine (vitamin B6) in addition to thiamine (vitamin B1), its originally identified substrate, whereas rat and mouse orthologs of hSLC19A3 can transport thiamine but not pyridoxine. This finding implies that some amino acid residues required for pyridoxine transport, but not for thiamine transport, are specific to hSLC19A3. Here, we sought to identify these residues to help clarify the unique operational mechanism of SLC19A3 through analyses comparing hSLC19A3 and mouse Slc19a3 (mSlc19a3). For our analyses, hSLC19A3 mutants were prepared by replacing selected amino acid residues with their counterparts in mSlc19a3, and mSlc19a3 mutants were prepared by substituting selected residues with their hSLC19A3 counterparts. We assessed pyridoxine and thiamine transport by these mutants in transiently transfected human embryonic kidney 293 cells. Our analyses indicated that the hSLC19A3-specific amino acid residues of Gln86, Gly87, Ile91, Thr93, Trp94, Ser168, and Asn173 are critical for pyridoxine transport. These seven amino acid residues were found to be mostly conserved in the SLC19A3 orthologs that can transport pyridoxine but not in orthologs that are unable to transport pyridoxine. In addition, these residues were also found to be conserved in several SLC19A2 orthologs, including rat, mouse, and human orthologs, which were all found to effectively transport both pyridoxine and thiamine, exhibiting no species-dependent differences. Together, these findings provide a molecular basis for the unique functional characteristics of SLC19A3 and also of SLC19A2.
Asunto(s)
Aminoácidos , Proteínas de Transporte de Membrana/metabolismo , Aminoácidos/metabolismo , Animales , Transporte Biológico , Células Epiteliales/metabolismo , Humanos , Ratones , Ratas , Tiamina/genética , Tiamina/metabolismoRESUMEN
The thiamine transporters, SLC19A2 and SLC19A3, have recently been shown to transport pyridoxine in addition to thiamine, the originally identified substrate, in our study on human orthologs. Based on these results, we characterized the rat and mouse orthologs for pyridoxine transport function. Through the assessment of pyridoxine uptake in human embryonic kidney 293 cells transiently expressing the SLC19A2/3 orthologs, we found that both rat and mouse Slc19a2 can transport pyridoxine, but rat or mouse Slc19a3 cannot. However, all SLC19A2/3 orthologs were capable of thiamine transport. We subsequently demonstrated in the rat small intestine that a carrier-mediated mechanism exists for thiamine uptake, but not for pyridoxine uptake. This is supported by the finding that rat Slc19a3, for which the human ortholog operates for the intestinal uptake of both pyridoxine and thiamine, lacks the pyridoxine transport function. Thus, SLC19A3s from different animal species exhibit differences in pyridoxine transport. Rats and mice, in which Slc19a3 lacks this function, are not suitable model animals for studies involving pyridoxine disposition and related issues.
Asunto(s)
Intestino Delgado , Proteínas de Transporte de Membrana , Piridoxina , Tiamina , Animales , Transporte Biológico , Intestino Delgado/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Ratones , Piridoxina/metabolismo , Ratas , Especificidad de la Especie , Tiamina/metabolismoRESUMEN
Orotate, a nutritional compound typically utilized as an intermediate in pyrimidine synthesis, has been suggested to undergo renal reabsorption. However, the detailed mechanisms involved in the process remain unclear, with only urate transporter 1 (URAT1/SLC22A12) being indicated as a transporter involved in its tubular uptake. As an attempt to identify transporters involved in that to help clarify the mechanisms, we examined a possibility that organic anion transporter 10 (OAT10/SLC22A13), which is present at the brush border membrane in renal tubular epithelial cells, could transport orotate. The operation of human OAT10 for orotate transport was demonstrated indeed and analyzed in detail in Madin-Darby canine kidney II cells introduced with this transporter by stable transfection. Orotate transport by OAT10 was found to be kinetically saturable with a biphasic characteristic and dependent on Cl-. These are unique characteristics previously unknown in its operation for the other substrates. Orotate transport by OAT10 was, on the other hand, inhibited by several anionic compounds known as OAT10 inhibitors. Finally, the rat ortholog of OAT10 was found not to be able to transport orotate, indicating animal species differences in that function. Thus, human OAT10 has been demonstrated to operate for orotate transport with unique characteristics.
Asunto(s)
Transportadores de Anión Orgánico , Animales , Transporte Biológico , Perros , Humanos , Riñón/metabolismo , Células de Riñón Canino Madin Darby , Microvellosidades/metabolismo , Transportadores de Anión Orgánico/genética , Transportadores de Anión Orgánico/metabolismo , Proteínas de Transporte de Catión Orgánico/metabolismo , RatasRESUMEN
Thiamin (vitamin B1) plays critical roles in normal metabolism and function of all mammalian cells. Pancreatic acinar cells (PACs) import thiamin from circulation via specific carrier-mediated uptake that involves thiamin transporter-1 and -2 (THTR-1 and -2; products of SLC19A2 and SLC19A3, respectively). Our aim in this study was to investigate the effect(s) of proinflammatory cytokines on thiamin uptake by PACs. We used human primary (h)PACs, PAC 266-6 cells, and mice in vivo as models in the investigations. First, we examined the level of expression of THTR-1 and -2 mRNA in pancreatic tissues of patients with chronic pancreatitis and observed severe reduction in their expression compared with normal control subjects. Exposing hPACs and PAC 266-6 to proinflammatory cytokines (hyper IL-6, TNF-α, and IL-1ß) was found to lead to a significant inhibition in thiamin uptake. Focusing on hyper-IL-6 (which also inhibited thiamin uptake by primary mouse PACs), the inhibition in thiamin uptake was found to be associated with significant reduction in THTR-1 and -2 proteins and mRNA expression as well as in activity of the SLC19A2 and SLC19A3 promoters; it was also associated with reduction in level of expression of the transcription factor Sp1 (which is required for activity of these promoters). Finally, blocking the intracellular Stat3 signaling pathway was found to lead to a significant reversal in the inhibitory effect of hyper IL-6 on thiamin uptake by PAC 266-6. These results show that exposure of PACs to proinflammatory cytokines negatively impacts thiamin uptake via (at least in part) transcriptional mechanism(s).NEW & NOTEWORTHY Findings of the current study demonstrate, for the first time, that exposure of pancreatic acinar cells to proinflammatory cytokines (including hyper IL-6) cause significant inhibition in vitamin B1 (thiamin; a micronutrient that is essential for normal cellular energy metabolism) and that this effect is mediated at the level of transcription of the thiamin transporter genes SLC19A2 and SLC19A3.
Asunto(s)
Células Acinares/efectos de los fármacos , Transporte Biológico/efectos de los fármacos , Citocinas/farmacología , Células Epiteliales/efectos de los fármacos , Células Acinares/metabolismo , Animales , Citocinas/metabolismo , Células Epiteliales/metabolismo , Humanos , Proteínas de Transporte de Membrana/efectos de los fármacos , Proteínas de Transporte de Membrana/genética , Ratones , Páncreas/efectos de los fármacos , Páncreas/metabolismo , Páncreas Exocrino/efectos de los fármacos , Páncreas Exocrino/metabolismo , Regiones Promotoras Genéticas/efectos de los fármacos , ARN Mensajero/metabolismoRESUMEN
SLC19A2 and SLC19A3, also known as thiamine transporters (THTR) 1 and 2, respectively, transport the positively charged thiamine (vitamin B1) into cells to enable its efficient utilization. SLC19A2 and SLC19A3 are also known to transport structurally unrelated cationic drugs, such as metformin, but whether this charge selectivity extends to other molecules, such as pyridoxine (vitamin B6), is unknown. We tested this possibility using Madin-Darby canine kidney II (MDCKII) cells and human embryonic kidney 293 (HEK293) cells for transfection experiments, and also using Caco-2 cells as human intestinal epithelial model cells. The stable expression of SLC19A2 and SLC19A3 in MDCKII cells (as well as their transient expression in HEK293 cells) led to a significant induction in pyridoxine uptake at pH 5.5 compared with control cells. The induced uptake was pH-dependent, favoring acidic conditions over neutral to basic conditions, and protonophore-sensitive. It was saturable as a function of pyridoxine concentration, with an apparent Km of 37.8 and 18.5 µm, for SLC19A2 and SLC19A3, respectively, and inhibited by the pyridoxine analogs pyridoxal and pyridoxamine as well as thiamine. We also found that silencing the endogenous SLC19A3, but not SLC19A2, of Caco-2 cells with gene-specific siRNAs lead to a significant reduction in carrier-mediated pyridoxine uptake. These results show that SLC19A2 and SLC19A3 are capable of recognizing/transporting pyridoxine, favoring acidic conditions for operation, and suggest a possible role for these transporters in pyridoxine transport mainly in tissues with an acidic environment like the small intestine, which has an acidic surface microclimate.
Asunto(s)
Ácidos/metabolismo , Células Epiteliales/metabolismo , Intestino Delgado/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Microclima , Animales , Transporte Biológico , Perros , Humanos , Concentración de Iones de Hidrógeno , Células de Riñón Canino Madin Darby , Tiamina/metabolismoRESUMEN
It has long been suggested that a Na+-dependent carrier-mediated transport system is involved in the absorption of nucleobases and analogs, including some drugs currently in therapeutic use, for their uptake at the brush border membrane of epithelial cells in the small intestine, mainly based on studies in non-primate experimental animals. The presence of this transport system was indeed proved by the recent identification of sodium-dependent nucleobase transporter 1 (SNBT1/Slc23a4) as its molecular entity in rats. However, this transporter has been found to be genetically deficient in humans and higher primates. Aware of this deficiency, we need to revisit the issue of the absorption of these compounds in the human small intestine so that we can understand the mechanisms and gain information to assure the more rational use and development of drugs analogous to nucleobases. Here, we review the current understanding of the intestinal absorption of nucleobases and analogs. This includes recent knowledge about the efflux transport of those compounds across the basolateral membrane when exiting epithelial cells, following brush border uptake, in order to complete the overall absorption process; the facilitative transporters of equilibrative nucleoside transporter 1 (ENT1/SLC29A1) and equilibrative nucleobase transporter 1 (ENBT1/SLC43A3) may be involved in that in many animal species, including human and rat, without any major species differences.
Asunto(s)
Sistemas de Transporte de Aminoácidos/metabolismo , Tranportador Equilibrativo 1 de Nucleósido/metabolismo , Absorción Intestinal/genética , Purinas/farmacocinética , Pirimidinas/farmacocinética , Sistemas de Transporte de Aminoácidos/genética , Animales , Membrana Celular , Tranportador Equilibrativo 1 de Nucleósido/genética , Humanos , Mucosa Intestinal/metabolismo , Intestino Delgado/metabolismo , Proteínas de Transporte de Nucleobases/metabolismo , Ratas , Especificidad de la EspecieRESUMEN
The water-soluble vitamin B1 (thiamin) plays essential roles in normal metabolism and function of all human/mammalian cells, including the pancreatic acinar cells (PACs). PACs obtain thiamin from their surrounding circulation via transport across the plasma membrane, a process that is mediated by thiamin transporter (THTR)-1 and THTR-2. We have previously characterized different aspects of thiamin uptake by mouse and human primary PACs, but little is known about posttranscriptional regulation of the uptake event. We addressed this by focusing on the predominant thiamin transporter THTR-1 (encoded by SLC19A2 gene) in PACs. Transfecting pmirGLO-SLC19A2 3'-untranslated region (UTR) into mouse-derived PAC 266-6 cells leads to a significant reduction in luciferase activity compared with cells transfected with empty vector. Subjecting the SLC19A2 3'-UTR to different in silico algorithms identified multiple putative microRNA binding sites in this region. Focusing on miR-200a-3p (since it is highly expressed in mouse and human pancreas), we found that transfecting PAC 266-6 and human primary PACs (hPACs) with mimic miR-200a-3p leads to a significant inhibition of THTR-1 expression (both protein and mRNA levels) and in thiamin uptake. In contrast, transfection by miR-200a-3p inhibitor leads to an increase in THTR-1 expression and thiamin uptake. Additionally, truncating the region carrying miR-200a-3p binding site in SLC19A2 3'-UTR and mutating the binding site lead to abrogation in the inhibitory effect of this microRNA on luciferase activity in PAC 266-6. These results demonstrate that expression of THTR-1 and thiamin uptake in PACs is subject to posttranscriptional regulation by microRNAs.NEW & NOTEWORTHY The findings of this study show, for the first time, that the membrane transporter of vitamin B1, i.e., thiamin transporter-1 (THTR-1), is subject to regulation by microRNAs (specifically miR-200a-3p) in mouse and human primary pancreatic acinar cells (PACs). The results also show that this posttranscriptional regulation has functional consequences on the ability of PACs to take in the essential micronutrient thiamin.
Asunto(s)
Células Acinares/metabolismo , Proteínas de Transporte de Membrana/genética , MicroARNs/genética , Páncreas/metabolismo , Procesamiento Postranscripcional del ARN/genética , Regiones no Traducidas 3'/genética , Animales , Humanos , Ratones , Mutación , Cultivo Primario de Células , Tiamina/metabolismoRESUMEN
To develop a novel intestinal drug absorption system using intestinal epithelial cells derived from human induced pluripotent stem (iPS) cells, the cells must possess sufficient pharmacokinetic functions. However, the CYP3A4/5 activities of human iPS cell-derived small intestinal epithelial cells prepared using conventional differentiation methods is low. Further, studies of the CYP3A4/5 activities of human iPS-derived and primary small intestinal cells are not available. To fill this gap in our knowledge, here we used forskolin to develop a new differentiation protocol that activates adenosine monophosphate signaling. mRNA expressions of human iPS cell-derived small intestinal epithelial cells, such as small intestine markers, drug-metabolizing enzymes, and drug transporters, were comparable to or greater than those of the adult small intestine. The activities of CYP3A4/5 in the differentiated cells were equal to those of human primary small intestinal cells. The differentiated cells had P-glycoprotein and PEPT1 activities equivalent to those of Caco-2 cells. Differentiated cells were superior to Caco-2 cells for predicting the membrane permeability of drugs that were absorbed through a paracellular pathway and via drug transporters. In summary, here we produced human iPS cell-derived small intestinal epithelial cells with CYP3A4/5 activities equivalent to those of human primary small intestinal cells.
Asunto(s)
Células Epiteliales/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Intestino Delgado/metabolismo , Ácidos Alcanesulfónicos/farmacocinética , Células CACO-2 , Células Cultivadas , Ciclosporinas/farmacocinética , Digoxina/farmacocinética , Dipéptidos/farmacocinética , Humanos , Ibuprofeno/farmacocinética , Intestino Delgado/citología , Morfolinas/farmacocinéticaRESUMEN
Equilibrative nucleobase transporter 1 (ENBT1/SLC43A3) has recently been identified as a purine-selective nucleobase transporter. Although it is highly expressed in the liver, its role in nucleobase transport has not been confirmed yet in hepatocytes or any relevant cell models. We, therefore, examined its role in adenine transport in the HepG2 cell line as a human hepatocyte model. The uptake of [3H]adenine in HepG2 cells was highly saturable, indicating the involvement of carrier-mediated transport. The carrier-mediated transport component, for which the Michaelis constant was estimated to be 0.268 µM, was sensitive to decynium-22, an ENBT1 inhibitor, with the half maximal inhibitory concentration of 2.59 µM, which was comparable to that of 2.30 µM for [3H]adenine uptake by ENBT1 in its transient transfectant human embryonic kidney 293 cells. Although equilibrative nucleoside transporter 1 (ENT1/SLC29A1) and ENT2/SLC29A2 are also known to be able to transport adenine, [3H]adenine uptake in HepG2 cells was not inhibited by the ENT1/2-specific inhibitor of either dipyridamole or nitrobenzylthioinosine. Finally, [3H]adenine uptake was extensively reduced by silencing of ENBT1 by RNA interference in the hepatocyte model. All these results, taken together, suggest the predominant role of ENBT1 in the uptake of adenine in HepG2 cells.
Asunto(s)
Tranportador Equilibrativo 1 de Nucleósido , Transportador Equilibrativo 2 de Nucleósido , Adenina , Sistemas de Transporte de Aminoácidos/metabolismo , Transporte Biológico , Tranportador Equilibrativo 1 de Nucleósido/genética , Tranportador Equilibrativo 1 de Nucleósido/metabolismo , Transportador Equilibrativo 2 de Nucleósido/genética , Transportador Equilibrativo 2 de Nucleósido/metabolismo , Células Hep G2 , HumanosRESUMEN
Human proton-coupled folate transporter (hPCFT/SLC46A1) has recently been found to be inhibited by myricetin by a sustained mechanism, raising a concern that the inhibition might lead to malabsorption of folates in the intestine, where hPCFT works for their epithelial uptake. However, rat PCFT (rPCFT) has more recently been found not to be inhibited by myricetin. Prompted by this finding, we attempted to determine the amino acid residue involved in that by analyses comparing between hPCFT and rPCFT. In the initial analysis, chimeric constructs prepared from hPCFT and rPCFT were examined for myricetin sensitivity to determine the hPCFT segment involved in the sensitivity. Focusing on the thereby determined segment from 83rd to 186th amino acid residue, hPCFT mutants having a designated amino acid residue replaced with its counterpart in rPCFT were prepared for the subsequent analysis. Among them, only G158N-substituted hPCFT was found to be transformed to be insensitive to myricetin and, accordingly, oppositely N158G-substituted rPCFT was transformed to be sensitive to myricetin. These results indicate the critical role of Gly158 in the myricetin sensitivity of hPCFT. This finding would help advance the elucidation of the mechanism of the myricetin-induced inhibition of hPCFT and manage the potential risk arising from that.
Asunto(s)
Flavonoides/farmacología , Glicina/genética , Transportador de Folato Acoplado a Protón/genética , Sustitución de Aminoácidos , Ácido Fólico/farmacocinética , Células HEK293 , Humanos , Absorción Intestinal/efectos de los fármacos , Absorción Intestinal/fisiología , Mutagénesis Sitio-Dirigida , Transportador de Folato Acoplado a Protón/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Licorice-induced pseudoaldosteronism is a common adverse effect in traditional Japanese Kampo medicine, and 3-monoglucuronyl glycyrrhetinic acid (3MGA) was considered as a causative agent of it. Previously, we found 22α-hydroxy-18ß-glycyrrhetyl-3-O-sulfate-30-glucuronide (1), one of the metabolites of glycyrrhizin (GL) in the urine of Eisai hyperbilirubinuria rats (EHBRs) treated with glycyrrhetinic acid (GA), and suggested that it is also a possible causative agent of pseudoaldosteronism. The discovery of 1 also suggested that there might be other metabolites of GA as causal candidates. In this study, we found 22α-hydroxy-18ß-glycyrrhetyl-3-O-sulfate (2) and 18ß-glycyrrhetyl-3-O-sulfate (3) in EHBRs' urine. 2 and 3 more strongly inhibited rat type 2 11ß-hydroxysteroid dehydrogenase than 1 did in vitro. When EHBRs were orally treated with GA, GA and 1-3 in plasma and 1-3 in urine were detected; the levels of 3MGA were quite low. 2 and 3 were shown to be the substrates of organic anion transporter (OAT) 1 and OAT3. In the plasma of a patient suffering from pseudoaldosteronism with rhabdomyolysis due to licorice, we found 8.6 µM of 3, 1.3 µM of GA, and 87 nM of 2, but 1, GL, and 3MGA were not detected. These findings suggest that 18ß-glycyrrhetyl-3-O-sulfate (3) is an alternative causative agent of pseudoaldosteronism, rather than 3MGA and 1.
Asunto(s)
Glycyrrhiza/efectos adversos , Ácido Glicirrínico/efectos adversos , Síndrome de Liddle/etiología , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 2/antagonistas & inhibidores , Animales , Relación Dosis-Respuesta a Droga , Femenino , Glycyrrhiza/química , Ácido Glicirrínico/química , Ácido Glicirrínico/aislamiento & purificación , Ácido Glicirrínico/orina , Túbulos Renales/efectos de los fármacos , Túbulos Renales/metabolismo , Síndrome de Liddle/diagnóstico , Espectroscopía de Resonancia Magnética , Estructura Molecular , RatasRESUMEN
Pseudoaldosteronism is a common adverse effect associated with traditional Japanese Kampo medicines. The pathogenesis is mainly caused by 3-monoglucuronyl glycyrrhetinic acid (3MGA), one of the metabolites of glycyrrhizin (GL) contained in licorice. We developed an anti-3MGA monoclonal antibody (MAb) and an ELISA system to easily detect 3MGA in the plasma and urine of the patients. However, we found that some metabolites of GL cross-reacted with this MAb. Mrp2-deficient Eisai Hyperbilirubinemia rats (EHBRs) were administered glycyrrhetinic acid (GA), and we isolated 22α-hydroxy-18ß-glycyrrhetyl-3-O-sulfate-30-glucuronide (1) from the pooled urine with the guidance of positive immunostaining of eastern blot as the new metabolite of GL. The IC50 of 1 for type 2 11ß-hydroxysteroid dehydrogenase (11ß-HSD2) was 2.0 µM. Similar plasma concentrations of 1 and GA were observed 12 h after oral administration of GA to EHBR. Compound 1 was eliminated via urine, whereas GA was not. In Sprague-Dawley (SD) rats orally treated with GA, compound 1 was absent from both the plasma and the urine. Compound 1 was actively transported into cells via OAT1 and OAT3, whereas GA was not. Compound 1, when produced in Mrp2-deficiency, represents a potential causative agent of pseudoaldosteronism, and might be used as a biomarker to prevent the adverse effect.
Asunto(s)
Ácido Glicirretínico/análogos & derivados , Ácido Glicirrínico/análogos & derivados , Síndrome de Liddle/etiología , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Perros , Femenino , Ácido Glicirretínico/farmacocinética , Ácido Glicirretínico/toxicidad , Humanos , Células de Riñón Canino Madin Darby , Masculino , Ratones , Ratones Endogámicos BALB C , Proteína 1 de Transporte de Anión Orgánico/metabolismo , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Eliminación RenalRESUMEN
Sodium-dependent nucleobase transporter 1 (SNBT1) is a nucleobase-specific transporter identified in our recent study. In an attempt to search for its potential substrates other than nucleobases in this study, we could successfully find urate, a metabolic derivative of purine nucleobases, as a novel substrate, as indicated by its specific Na+ -dependent and saturable transport, with a Michaelis constant of 433 µmol/L, by rat SNBT1 (rSNBT1) stably expressed in Madin-Darby canine kidney II cells. However, urate uptake was observed only barely in the everted tissue sacs of the rat small intestine, in which rSNBT1 operates for nucleobase uptake. These findings suggested that urate undergoes a futile cycle, in which urate transported into epithelial cells is immediately effluxed back by urate efflux transporters, in the small intestine. In subsequent attempts to examine that possibility, such a futile urate cycle was demonstrated in the human embryonic kidney 293 cell line as a model cell system, where urate uptake induced by transiently introduced rSNBT1 was extensively reduced by the co-introduction of rat breast cancer resistance protein (rBCRP), a urate efflux transporter present in the small intestine. However, urate uptake was not raised in the presence of Ko143, a BCRP inhibitor, in the everted intestinal tissue sacs, suggesting that some other transporter might also be involved in urate efflux. The newly found urate transport function of SNBT1, together with the suggested futile urate cycle in the small intestine, should be of interest for its evolutional and biological implications, although SNBT1 is genetically deficient in humans.
Asunto(s)
Proteínas de Transporte de Nucleobases/metabolismo , Ácido Úrico/metabolismo , Animales , Transporte Biológico , Perros , Células HEK293 , Humanos , Intestino Delgado/metabolismo , Células de Riñón Canino Madin Darby , Masculino , Ratas WistarRESUMEN
Bioluminescence (BL) imaging based on d-luciferin (d-luc)-luciferase reaction allows noninvasive and real-time monitoring of luciferase-expressing cells. Because BL intensity depends on photons generated through the d-luc-luciferase reaction, an approach to increase intracellular levels of d-luc could improve the detection sensitivity. In the present study, we showed that organic anion transporter 1 (OAT1) is useful, as a d-luc transporter, in boosting the BL intensity in luciferase-expressing cells. Functional screening of several transporters showed that the expression of OAT1 in HEK293â¯cells stably expressing Pyrearinus termitilluminans luciferase (HEK293/eLuc) markedly enhanced BL intensity in the presence of d-luc. When OAT1 was transiently expressed in HEK293â¯cells, intracellular accumulation of d-luc was higher than that in control cells, and the specific d-luc uptake mediated by OAT1 was saturable with a Michaelis constant (Km) of 0.23⯵M. The interaction between OAT1 and d-luc was verified using 6-carboxyfluorescein, a typical substrate of OAT1, which showed that d-luc inhibited the uptake of 6-carboxyfluorescein mediated by OAT1. BL intensity was concentration-dependent at steady states in HEK293/eLuc cells stably expressing OAT1, and followed Michaelis-Menten kinetics with an apparent Km of 0.36⯵M. In addition, the enhanced BL was significantly inhibited by OAT1-specific inhibitors. Thus, OAT1-mediated transport of d-luc could be a rate-limiting step in the d-luc-luciferase reaction. Furthermore, we found that expressing OAT1 in HEK293/eLuc cells implanted subcutaneously in mice also significantly increased the BL after intraperitoneal injection of d-luc. Our findings suggest that because OAT1 is capable of transporting d-luc, it can also be used to improve visualization and monitoring of luciferase-expressing cells.
Asunto(s)
Benzotiazoles/metabolismo , Aumento de la Imagen/métodos , Luciferasas/metabolismo , Mediciones Luminiscentes/métodos , Proteína 1 de Transporte de Anión Orgánico/metabolismo , Genes Reporteros/genética , Células HEK293 , Humanos , Luciferasas/genética , Imagen Molecular/métodos , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Myricetin is a flavonoid that inhibits human proton-coupled folate transporter (hPCFT) in a transient manner, in which inhibition is manifested in its presence, and also in a sustained manner, in which inhibition induced in its presence persists after its removal. In an effort to elucidate the mechanisms involved in those, we examined if myricetin might or might not act similarly on some other transporters. Transporters examined for that, in comparison with hPCFT, were its rat ortholog (rPCFT) and human riboflavin transporter 3 (hRFVT3). Experiments were conducted, using human embryonic kidney 293 cells transiently expressing the transporter to be examined, to assess the effects of myricetin (100 µM) on the uptake of folate by the PCFTs and riboflavin by hRFVT3. For hPCFT, myricetin was confirmed to induce a transient inhibition and also a sustained inhibition. However, myricetin induced neither transient nor sustained type of rPCFT inhibition. hRFVT3 was inhibited by myricetin in a transient manner, but not in a sustained manner. These results suggest the involvement of a hPCFT-specific mechanism in the sustained inhibition. The transient inhibition may be induced by a mechanism specific to hPCFT and also hRFVT3.
Asunto(s)
Flavonoides/farmacología , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Transportador de Folato Acoplado a Protón/antagonistas & inhibidores , Animales , Relación Dosis-Respuesta a Droga , Flavonoides/metabolismo , Ácido Fólico/farmacocinética , Células HEK293 , Humanos , Proteínas de Transporte de Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Transportador de Folato Acoplado a Protón/metabolismo , Ratas , Receptores Acoplados a Proteínas G , Riboflavina/farmacocinética , Relación Estructura-ActividadRESUMEN
The intestinal absorption of atenolol has recently been reported to be reduced by simultaneous ingestion of fruit juices, such as apple juice. This finding implies a possibility that an unidentified carrier-mediated transport system, which could be interfered by some components of those juices, might be involved in atenolol absorption. In an attempt to explore that possibility, we successfully identified plasma membrane monoamine transporter (PMAT/SLC29A4) as a transporter that can operate for cellular atenolol uptake in the intestine, using Madin-Darby canine kidney II cells stably expressing PMAT. The specific uptake of atenolol by PMAT was greatest at around pH 6.0 and decreased with an increase in pH. At pH 6.0, the PMAT-specific uptake of atenolol was saturable with a Michaelis constant of 0.907 mM. Moreover, PMAT-specific atenolol uptake was extensively inhibited by phloretin and quercetin, which are the major flavonoids contained in apple juice, with the half maximal inhibitory concentrations of 33.3 and 116.3 µM, respectively. PMAT-specific atenolol uptake was also inhibited by several ß-blockers, suggesting that they may also be recognized and transported by PMAT. These results suggest that PMAT is an atenolol transporter that may be involved in intestinal atenolol absorption and sensitive to flavonoids contained in apple juice.
Asunto(s)
Atenolol/metabolismo , Proteínas de Transporte de Nucleósido Equilibrativas/metabolismo , Flavonoides/metabolismo , Jugos de Frutas y Vegetales/análisis , Malus/química , Animales , Atenolol/química , Transporte Biológico , Línea Celular , Membrana Celular/metabolismo , Perros , Flavonoides/química , Expresión Génica , Células HEK293 , Humanos , Absorción Intestinal/fisiología , Riñón/metabolismo , Especificidad por Sustrato , TermodinámicaRESUMEN
A suicide gene therapy using herpes simplex virus thymidine kinase (HSV-TK) with ganciclovir (GCV) has been under development as a tumor-targeted therapy; however, the mechanism of cellular GCV uptake, which is prerequisite in the therapy, has not been clarified. In an attempt to resolve this situation and gain information to optimize HSV-TK/GCV system for cancer therapy, we found that human equilibrative nucleobase transporter 1 (ENBT1) can transport GCV with a Michaelis constant of 2.75 mM in Madin-Darby canine kidney II (MDCKII) cells stably transfected with this transporter. In subsequent experiments using green fluorescent protein (GFP)-tagged ENBT1 (GFP-ENBT1) and HSV-TK, the uptake of GCV (30 µM), which was minimal in MDCKII cells and unchanged by their transfection with HSV-TK alone, was increased extensively by their transfection with GFP-ENBT1, together with HSV-TK. Accordingly, cytotoxicity, which was assessed by the WST-8 cell viability assay after the treatment of those cells with GCV (30 µM) for 72 hours, was induced in those transfected with GFP-ENBT1, together with HSV-TK but not in those transfected with HSV-TK alone. These results suggest that ENBT1 could facilitate GCV uptake and thereby enhance cytotoxicity in HSV-TK/GCV system. We also identified Helacyton gartleri (HeLa) and HepG2 as cancer cell lines that are rich with ENBT1 and A549, HCT-15 and MCF-7 as those poor with ENBT1. Accordingly, the HSV-TK/GCV system was effective in inducing cytotoxicity in the former but not in the latter. Thus, ENBT1 was found to be a GCV transporter that could enhance the performance of HSV-TK/GCV suicide gene therapy.
Asunto(s)
Sistemas de Transporte de Aminoácidos/metabolismo , Apoptosis/genética , Ganciclovir/metabolismo , Ganciclovir/farmacología , Terapia Genética , Simplexvirus/enzimología , Timidina Quinasa/genética , Animales , Transporte Biológico , Línea Celular , Perros , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Simplexvirus/genéticaRESUMEN
OBJECTIVE: A genome-wide association study (GWAS) of gout and its subtypes was performed to identify novel gout loci, including those that are subtype-specific. METHODS: Putative causal association signals from a GWAS of 945 clinically defined gout cases and 1213 controls from Japanese males were replicated with 1396 cases and 1268 controls using a custom chip of 1961 single nucleotide polymorphisms (SNPs). We also first conducted GWASs of gout subtypes. Replication with Caucasian and New Zealand Polynesian samples was done to further validate the loci identified in this study. RESULTS: In addition to the five loci we reported previously, further susceptibility loci were identified at a genome-wide significance level (p<5.0×10-8): urate transporter genes (SLC22A12 and SLC17A1) and HIST1H2BF-HIST1H4E for all gout cases, and NIPAL1 and FAM35A for the renal underexcretion gout subtype. While NIPAL1 encodes a magnesium transporter, functional analysis did not detect urate transport via NIPAL1, suggesting an indirect association with urate handling. Localisation analysis in the human kidney revealed expression of NIPAL1 and FAM35A mainly in the distal tubules, which suggests the involvement of the distal nephron in urate handling in humans. Clinically ascertained male patients with gout and controls of Caucasian and Polynesian ancestries were also genotyped, and FAM35A was associated with gout in all cases. A meta-analysis of the three populations revealed FAM35A to be associated with gout at a genome-wide level of significance (p meta =3.58×10-8). CONCLUSIONS: Our findings including novel gout risk loci provide further understanding of the molecular pathogenesis of gout and lead to a novel concept for the therapeutic target of gout/hyperuricaemia.