Biomedical subjects

Tomohiro Terada

Publications and source records attributed to Tomohiro Terada.

At least 19 recordsLinked to original sources

PubMed2006

Gene expression and regulation of drug transporters in the intestine and kidney.

Intestinal absorption and renal secretion of ionic drugs are controlled by a number of drug transporters expressed at the brush-border and basolateral membranes of epithelial cells. Over the last several years, considerable progress has been made regarding the molecular identification and functional characterization of drug transporters. Under some physiological and pathophysiological conditions, the expression and transport activity of drug transporters are changed, affecting the pharmacokinetics of substrate drugs. The regulation of transport activity in response to endogenous and exogenous signals can occur at various levels such as transcription, mRNA stability, translation, and posttranslational modification. Transcriptional regulation is of particular interest, because changes in transport activity are dynamically regulated by increases or decreases in levels of mRNA expression. The tissue-specific expression of drug transporters is also under transcriptional control, and recent studies using clinical samples from human tissues have revealed the expression profiles of drug transporters in the human body. The purpose of this research updates is to review the recent progress in the study of the gene expression and regulation of intestinal and renal drug transporters.

Animals↗

PubMed2006

Oppositely directed H+ gradient functions as a driving force of rat H+/organic cation antiporter MATE1.

Recently, we have isolated the rat (r) H(+)/organic cation antiporter multidrug and toxin extrusion 1 (MATE1) and reported its tissue distribution and transport characteristics. Functional characterization suggested that an oppositely directed H(+) gradient serves as a driving force for the transport of a prototypical organic cation, tetraethylammonium, by MATE1, but there is no direct evidence to prove this. In the present study, therefore, we elucidated the driving force of tetraethylammonium transport via rMATE1 using plasma membrane vesicles isolated from HEK293 cells stably expressing rMATE1 (HEK-rMATE1 cells). A 70-kDa rMATE1 protein was confirmed to exist in HEK-rMATE1 cells, and the transport of various organic cations including [(14)C]tetraethylammonium was stimulated in intracellular acidified HEK-rMATE1 cells but not mock cells. The transport of [(14)C]tetraethylammonium in membrane vesicles from HEK-rMATE1 cells exhibited the overshoot phenomenon only when there was an outwardly directed H(+) gradient, as observed in rat renal brush-border membrane vesicles. The overshoot phenomenon was not observed in the vesicles from mock cells. The stimulated [(14)C]tetraethylammonium uptake by an H(+) gradient [intravesicular H(+) concentration ([H(+)](in)) > extravesicular H(+) concentration ([H(+)](out))] was significantly reduced in the presence of a protonophore, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). [(14)C]tetraethylammonium uptake was not changed in the presence of valinomycin-induced membrane potential. These findings definitively indicate that an oppositely directed H(+) gradient serves as a driving force of tetraethylammonium transport via rMATE1, and this is the first demonstration to identify the driving force of the MATE family. The present experimental strategy is very useful in identifying the driving force of cloned transporters whose driving force has not been evaluated.

Animals↗

PubMed2006

Human organic anion transporter 3 gene is regulated constitutively and inducibly via a cAMP-response element.

Human organic anion transporter (OAT) 3 (SLC22A8) is localized to the basolateral membranes of renal tubular epithelial cells and plays a critical role in the excretion of anionic compounds. We previously reported that interindividual variation in the OAT3 mRNA level corresponded to interindividual differences in the rate of renal excretion of cefazolin. However, there is little information available on the molecular mechanisms regulating the gene expression of OAT3. Therefore, in the present study, we examined the transcriptional regulation of human OAT3. A deletion analysis of the OAT3 promoter suggested that the region spanning -214 to -77 base pairs was essential for basal transcriptional activity. This region contained a perfectly conserved cAMP-response element (CRE), and a mutation here led to a reduction in promoter activity. Electrophoretic mobility shift assays showed that CRE-binding protein (CREB)-1 and activating transcription factor (ATF)-1 bound to CRE. The activity of the OAT3 promoter was increased through the phosphorylation of CREB-1 and ATF-1 by treatment with 8-bromo-cAMP. This paper reports the first characterization of the human OAT3 promoter and shows that CREB-1 and ATF-1 function as constitutive and inducible transcriptional regulators of the human OAT3 gene via CRE.

Base Sequence↗

PubMed2006

Identification and functional characterization of a new human kidney-specific H+/organic cation antiporter, kidney-specific multidrug and toxin extrusion 2.

A cDNA coding a new H+/organic cation antiporter, human kidney-specific multidrug and toxin extrusion 2 (hMATE2-K), has been isolated from the human kidney. The hMATE2-K cDNA had an open reading frame that encodes a 566-amino acid protein, which shows 94, 82, 52, and 52% identity with the hMATE2, hMATE2-B, hMATE1, and rat MATE1, respectively. Reverse transcriptase-PCR revealed that hMATE2-K mRNA but not hMATE2 was expressed predominantly in the kidney, and hMATE2-B was ubiquitously found in all tissues examined except the kidney. The immunohistochemical analyses revealed that the hMATE2-K as well as the hMATE1 was localized at the brush border membranes of the proximal tubules. HEK293 cells that were transiently transfected with the hMATE2-K cDNA but not hMATE2-B exhibited the H+ gradient-dependent antiport of tetraethylammonium (TEA). Transfection of hMATE2-B had no affect on the hMATE2-K-mediated transport of TEA. hMATE2-K also transported cimetidine, 1-methyl-4-phenylpyridinium (MPP), procainamide, metformin, and N1-methylnicotinamide. Kinetic analyses demonstrated that the Michaelis-Menten constants for the hMATE2-K-mediated transport of TEA, MPP, cimetidine, metformin, and procainamide were 0.83 mM, 93.5 microM, 0.37 mM, 1.05 mM, and 4.10 mM, respectively. Ammonium chloride-induced intracellular acidification significantly stimulated the hMATE2-K-dependent transport of organic cations such as TEA, MPP, procainamide, metformin, N1-methylnicotinamide, creatinine, guanidine, quinidine, quinine, thiamine, and verapamil. These results indicate that hMATE2-K is a new human kidney-specific H+/organic cation antiporter that is responsible for the tubular secretion of cationic drugs across the brush border membranes.

Alternative Splicing↗

PubMed2006

Induction of intestinal peptide transporter 1 expression during fasting is mediated via peroxisome proliferator-activated receptor alpha.

We previously demonstrated that starvation markedly increased the amount of mRNA and protein levels of the intestinal H+/peptide cotransporter (PEPT1) in rats, leading to altered pharmacokinetics of the PEPT1 substrates. In the present study, the mechanism underlying this augmentation was investigated. We focused on peroxisome proliferator-activated receptor alpha (PPARalpha), which plays a pivotal role in the adaptive response to fasting in the liver and other tissues. In 48-h fasted rats, the expression level of PPARalpha mRNA in the small intestine markedly increased, accompanied by the elevation of serum free fatty acids, which are endogenous PPARalpha ligands. Oral administration of the synthetic PPARalpha ligand WY-14643 to fed rats increased the mRNA level of intestinal PEPT1. Furthermore, treatment of the human intestinal model, Caco-2 cells, with WY-14643 resulted in enhanced PEPT1 mRNA expression and uptake activity of glycylsarcosine. In the small intestine of PPARalpha-null mice, augmentation of PEPT1 mRNA during fasting was completely abolished. In the kidney, fasting did not induce PEPT1 expression in either PPARalpha-null or wild-type mice. Together, these results indicate that PPARalpha plays critical roles in fasting-induced intestinal PEPT1 expression. In addition to the well-established roles of PPARalpha, we propose a novel function of PPARalpha in the small intestine, that is, the regulation of nitrogen absorption through PEPT1 during fasting.

Acyl-CoA Oxidase↗

PubMed2006

Th2 cytokines down-regulate TLR expression and function in human intestinal epithelial cells.

TLRs serve important immune and nonimmune functions in human intestinal epithelial cells (IECs). Proinflammatory Th1 cytokines have been shown to promote TLR expression and function in IECs, but the effect of key Th2 cytokines (IL-4, IL-5, IL-13) on TLR signaling in IECs has not been elucidated so far. We stimulated human model IECs with Th2 cytokines and examined TLR mRNA and protein expression by Northern blotting, RT-PCR, real-time RT-PCR, Western blotting, and flow cytometry. TLR function was determined by I-kappaBalpha phosphorylation assays, ELISA for IL-8 secretion after stimulation with TLR ligands and flow cytometry for LPS uptake. IL-4 and IL-13 significantly decreased TLR3 and TLR4 mRNA and protein expression including the requisite TLR4 coreceptor MD-2. TLR4/MD-2-mediated LPS uptake and TLR ligand-induced I-kappaBalpha phosphorylation and IL-8 secretion were significantly diminished in Th2 cytokine-primed IECs. The down-regulatory effect of Th2 cytokines on TLR expression and function in IECs also counteracted enhanced TLR signaling induced by stimulation with the hallmark Th1 cytokine IFN-gamma. In summary, Th2 cytokines appear to dampen TLR expression and function in resting and Th1 cytokine-primed human IECs. Diminished TLR function in IECs under the influence of Th2 cytokines may protect the host from excessive TLR signaling, but likely also impairs the host intestinal innate immune defense and increases IEC susceptibility to chronic inflammation in response to the intestinal microenvironment. Taken together, our data underscore the important role of Th2 cytokines in balancing TLR signaling in human IECs.

Cell Line, Tumor↗

PubMed2006

The transcription factor Cdx2 regulates the intestine-specific expression of human peptide transporter 1 through functional interaction with Sp1.

H(+)/peptide cotransporter 1 (PEPT1, SLC15A1) localized at the brush-border membranes of intestinal epithelial cells plays important roles in the intestinal absorption of small peptides and a variety of peptidemimetic drugs. We previously demonstrated that transcription factor Sp1 functions as a basal transcriptional regulator of human PEPT1. However, the factor responsible for the intestine-specific expression of PEPT1 remains unknown. In the present study, we investigated the effect of the intestinal transcription factors on the transcription of the PEPT1 gene and found that only Cdx2 markedly trans-activated the PEPT1 promoter. However, the promoter region responsible for this effect lacked a typical Cdx2-binding sequence, but instead, possessed some Sp1-binding sites. In vitro experiments using Caco-2 cells showed that (1) mutation of the Sp1-binding site diminished the effect of Cdx2, (2) co-expression of Cdx2 and Sp1 synergistically trans-activated the PEPT1 promoter and (3) Sp1 protein was immunoprecipitated with Cdx2 protein. These results raise the possibility that Cdx2 modulates the PEPT1 promoter by interaction with Sp1. The significance of Cdx2 in vivo for PEPT1 regulation was shown by the determination of mRNA levels of Cdx2 and PEPT1 in human tissue. In gastric samples, some with intestinal metaplasia, the levels of PEPT1 and Cdx2 mRNA were highly correlated. Taken together, the present study suggests that Cdx2 plays a key role in the transcriptional regulation of the intestine-specific expression of PEPT1, possibly through interaction with Sp1.

Binding Sites↗

PubMed2006

Transport characteristics of a novel peptide transporter 1 substrate, antihypotensive drug midodrine, and its amino acid derivatives.

Midodrine is an oral drug for orthostatic hypotension. This drug is almost completely absorbed after oral administration and converted into its active form, 1-(2',5'-dimethoxyphenyl)-2-aminoethanol) (DMAE), by the cleavage of a glycine residue. The intestinal H+-coupled peptide transporter 1 (PEPT1) transports various peptide-like drugs and has been used as a target molecule for improving the intestinal absorption of poorly absorbed drugs through amino acid modifications. Because midodrine meets these requirements, we examined whether midodrine can be a substrate for PEPT1. The uptake of midodrine, but not DMAE, was markedly increased in PEPT1-expressing oocytes compared with water-injected oocytes. Midodrine uptake by Caco-2 cells was saturable and was inhibited by various PEPT1 substrates. Midodrine absorption from the rat intestine was very rapid and was significantly inhibited by the high-affinity PEPT1 substrate cyclacillin, assessed by the alteration of the area under the blood concentration-time curve for 30 min and the maximal concentration. Some amino acid derivatives of DMAE were transported by PEPT1, and their transport was dependent on the amino acids modified. In contrast to neutral substrates, cationic midodrine was taken up extensively at alkaline pH, and this pH profile was reproduced by a 14-state model of PEPT1, which we recently reported. These findings indicate that PEPT1 can transport midodrine and contributes to the high bioavailability of this drug and that Gly modification of DMAE is desirable for a prodrug of DMAE.

Amino Acids↗

PubMed2006

Androgen receptor is responsible for rat organic cation transporter 2 gene regulation but not for rOCT1 and rOCT3.

PURPOSE: Organic cation transporters 1-3 (OCT1-3; Slc22a1-3) mediate the membrane transport of organic cations in the kidney. We previously reported that rat (r)OCT2 expression in the kidney was regulated by testosterone. In this study, we examined the transcriptional mechanisms underlying the testosterone-dependent regulation of rOCT2 expression. METHODS: Approximately 3000-bp fragments of the rOCT1-3 promoter region were isolated, and promoter activities were measured in the renal epithelial cell line LLC-PK1 with the coexpression of rat androgen receptor. RESULTS: Among reporter constructs tested, only rOCT2 promoter activity was stimulated by testosterone. This stimulation was suppressed by nilutamide, an antiandrogen drug. Reporter assays using deletion constructs and mutational constructs of putative androgen response elements (ARE) in the rOCT2 promoter region suggested that two AREs, located at approximately -3000 and -1300, respectively, play an important role in the induction by testosterone. CONCLUSIONS: Testosterone induces the expression of rOCT2, but not of rOCT1 and rOCT3, via the AR-mediated transcriptional pathway. This is the first study to address the transcriptional mechanisms of testosterone-dependent gene regulation of the Slc22 family.

Androgens↗

PubMed2006

Effect of novel NOP receptor ligands on food intake in rats.

Nociceptin/orphanin FQ (N/OFQ), the endogenous ligand for the NOP opioid receptor, stimulates feeding in rats. The present study evaluated the effect of three newly synthesized NOP receptor agonists and two NOP receptor antagonist on food intake. Freely feeding rats were tested with intracerebroventricular (ICV) injections of the NOP receptor agonists OS-500, OS-462 and OS-461. OS-500 and OS-462 evoked a hyperphagic effect more potent and far more pronounced than that of N/OFQ, while OS-461 was ineffective. OS-500 and OS-462 were also tested by intraperitoneal injection, but were unable to evoke hyperphagia following this route of administration. The NOP receptor antagonist NC-797 and UFP-101 did not modify feeding in freely feeding rats while fully antagonized the hyperphagic effect of N/OFQ. Pre-treatment with UFP-101 but not with NC-797 antagonized the hyperphagic effect of OS-462 and OS-500. The present findings indicate that OS-500, OS-462 may act as potent and long-lasting NOP receptor agonists, whereas UFP-101 and NC-797 show antagonistic properties. The higher efficacy of UFP-101 in blocking the hyperphagic effect of OS-462 and OS-500 may be linked to the better pharmacokinetic profile of this antagonist compared to NC-797. Overall, the results indicate that these compounds may represent valuable pharmacological tools to investigate the role of the brain N/OFQ system.

Animals↗

PubMed2006

Unequivocal synthesis of (Z)-alkene and (E)-fluoroalkene dipeptide isosteres to probe structural requirements of the peptide transporter PEPT1.

[reaction: see text] Described is a novel synthetic route for dipeptide isosteres containing (Z)-alkene and (E)-fluoroalkene units as cis-amide bond equivalents via organocopper-mediated reduction of gamma-acetoxy- or gamma,gamma-difluoro-alpha,beta-unsaturated-delta-lactams. The synthesized isosteres were evaluated in terms of their affinities for the peptide transporter PEPT1. trans-Amide isosteres tended to possess higher affinities for PEPT1 as compared to the corresponding cis-amide bond equivalents.

Alkenes↗

PubMed2006

Molecular cloning, functional characterization and tissue distribution of rat H+/organic cation antiporter MATE1.

PURPOSE: Transport characteristics and tissue distribution of the rat H+/organic cation antiporter MATE1 (multidrug and toxin extrusion 1) were examined. METHODS: Rat MATE1 cDNA was isolated by polymerase chain reaction (PCR) cloning. Transport characteristics of rat MATE1 were assessed by HEK293 cells transiently expressing rat MATE1. The mRNA expression of rat MATE1 was examined by Northern blot and real-time PCR analyses. RESULTS: The uptake of a prototypical organic cation tetraethylammonium (TEA) by MATEI-expressing cells was concentration-dependent, and showed the greatest value at pH 8.4 and the lowest at pH 6.0-6.5. Intracellular acidification induced by ammonium chloride resulted in a marked stimulation of TEA uptake. MATE1 transported not only organic cations such as cimetidine and metformin but also the zwitterionic compound cephalexin. MATE1 mRNA was expressed abundantly in the kidney and placenta, slightly in the spleen, but not expressed in the liver. Real-time PCR analysis of microdissected nephron segments showed that MATE1 was primarily expressed in the proximal convoluted and straight tubules. CONCLUSIONS: These findings indicate that MATE1 is expressed in the renal proximal tubules and can mediate the transport of various organic cations and cephalexin using an oppositely directed H+ gradient.

Animals↗

PubMed2005

Regulation of human peptide transporter 1 (PEPT1) in gastric cancer cells by anticancer drugs.

Human peptide transporter 1 (PEPT1) mediates the cellular uptake of di- and tripeptides and peptide-like drugs in the small intestine. In the present study, we examined the regulation of PEPT1 by anticancer drugs in the gastric cancer cell line MKN45. PEPT1 was expressed and functioned in MKN45 cells. The transport activity and mRNA expression of the facilitative glucose transporter 1 (GLUT1) were significantly decreased by 5-fluorouracil treatment, but those of PEPT1 were slightly increased. Cisplatin treatment affected neither PEPT1 nor GLUT1 activity. In conclusion, PEPT1 expressed in MKN45 cells are resistant against the cellular injury induced by 5-fluorouracil and cisplatin.

Antimetabolites, Antineoplastic↗

PubMed2005

Prediction of glycylsarcosine transport in Caco-2 cell lines expressing PEPT1 at different levels.

H(+)-coupled peptide transporter 1 (PEPT1) and the basolateral peptide transporter mediate the absorption of small peptides and peptide-like drugs in the small intestine. Recently, we constructed a mathematical model to simulate glycylsarcosine (Gly-Sar) transport in Caco-2 cells. In this study, we attempted to adjust our model to a change in the expression level of PEPT1. To obtain cell lines expressing PEPT1 at different levels, recloning of Caco-2 cells was performed, and nine clones were isolated. Compared with parental cells, clones 1 and 9 exhibited the lowest and the highest levels of [(14)C]Gly-Sar uptake from the apical side, respectively, whereas activities of the basolateral peptide transporter were comparable. Kinetic analysis demonstrated that the difference in the activity of PEPT1 was accounted by variations in V (max). Moreover, PEPT1 mRNA level was positively related to the activity of [(14)C]Gly-Sar uptake (r=0.55). Based on these findings, the V (max) value of PEPT1 was defined as a variable using the amount of PEPT1 mRNA as an index of the expression level. With this improved model, Gly-Sar transport in clones 1 and 9 was well-predicted, suggesting that our model can simulate Gly-Sar transport in cells expressing PEPT1 at different levels.

Biological Transport↗

PubMed2005

Expression profiles of various transporters for oligopeptides, amino acids and organic ions along the human digestive tract.

Various transporters such as H+/peptide cotransporter PEPT1 are expressed in the intestine, and play important physiological and pharmacological roles in the body. Present study was performed to examine the expression profile of 20 kinds of transporters (PEPT1 and 2, P-glycoprotein, amino acid transporters and organic ion transporters) along the human digestive tract, especially focusing on PEPT1. Using normal mucosal specimens, real-time polymerase chain reactions were carried out. Immunoblot analyses were also performed for PEPT1 expression. PEPT1 mRNA was highly expressed in the small intestine (duodenum>jejunum>ileum) compared to other tissues, and some patients showed a significant level of expression in the stomach. The expressional pattern of PEPT1 in the stomach and histological diagnosis indicated that gastric PEPT1 originated from the intestinal metaplasia. The amino acid transporters showed unique mRNA expression levels and distributions in the digestive tract. For example, the expression levels of B(0)AT1, a Na+-dependent and chloride-independent neutral amino acid transporter, were increased from the duodenum to ileum, which pattern is completely inverted to that for PEPT1. There is little expression of organic ion transporters except for organic cation/carnitine transporter OCTN2. In conclusion, PEPT1 was abundantly expressed in the small intestine, and the reciprocal expression of PEPT1 and B(0)AT1 may serve for the efficient absorption of protein digestive products.

Aged↗

PubMed2005

Pharmacokinetic significance of renal OAT3 (SLC22A8) for anionic drug elimination in patients with mesangial proliferative glomerulonephritis.

PURPOSE: Our previous studies showed that the mRNA level of human organic anion transporter (hOAT) 3 in the kidney was correlated with the rate of elimination of an anionic antibiotic cefazolin. However, the correlation coefficient was not so high. In the present study, therefore, we enrolled more patients to examine whether additional factors were responsible for the correlation. METHODS: hOAT mRNA levels in renal biopsy specimens were quantified using the real-time polymerase chain reaction method. The elimination rates for the free fraction of cefazolin were determined in patients with various renal diseases. RESULTS: In the present study, the coefficient of correlation between the hOAT3 mRNA level and the elimination rates for the free fraction of cefazolin was not so high in the patients overall as in our previous study (r = 0.536). However, following the classification of renal diseases, a better correlation was obtained in patients with mesangial proliferative glomerulonephritis (r = 0.723). In contrast, multiple regression analyses including gender, age, and liver function did not result in any improvements in the correlation coefficients. CONCLUSIONS: These results suggest that the hOAT3 mRNA level is a significant marker of pharmacokinetics with which to predict the rate of elimination of cefazolin in patients with mesangial proliferative glomerulonephritis.

Adolescent↗

PubMed2005

Characterization of the human peptide transporter PEPT1 promoter: Sp1 functions as a basal transcriptional regulator of human PEPT1.

H+-coupled peptide transporter 1 (PEPT1, SLC15A1) localized at the brush-border membranes of intestinal epithelial cells plays an important role in the intestinal absorption of small peptides and a variety of peptidemimetic drugs. PEPT1 is regulated by various factors, including hormones, dietary conditions, some pharmaceutics, and diurnal rhythm. But there is little information about the transcriptional regulation of PEPT1. In the present study, therefore, we cloned the human (h)PEPT1 promoter region and examined its promoter activity using a human intestinal cell line, Caco-2. Deletion analysis of the hPEPT1 promoter suggested that the region spanning -172 to -35 bp was essential for basal transcriptional activity. This region lacked a TATA-box but contained some GC-rich sites that supposedly bind with the transcription factor Sp1. Mutational analysis revealed that three of these putative Sp1 sites contributed to the transcriptional activity. EMSA showed that Sp1 bound to two GC-rich sites. Furthermore, inhibition of Sp1 binding by mithramycin A treatment significantly reduced the transcriptional activity. Finally, overexpression of Sp1 increased the transcriptional activity in a dose-dependent manner. This study reports the first characterization of the hPEPT1 promoter and shows the significant role of Sp1 in the basal transcriptional regulation of hPEPT1.

Caco-2 Cells↗

PubMed2005

Computational modelling of H+-coupled peptide transport via human PEPT1.

H+-coupled peptide transporter 1 (PEPT1) mediates the transport of small peptides and peptide-like drugs in a pH- and voltage-dependent manner. Here, we investigated the transport mechanisms of PEPT1 for neutral and charged substrates by experimental studies and computational simulation. Uptake studies revealed that the Michaelis-Menten constant (Km) of glycylsarcosine (Gly-Sar), a neutral substrate, decreased with a fall in pH from 7.4 to 5.5, but at pH 5.0, the Km increased again. In contrast, the Km value of an anionic substrate, ceftibuten, declined steadily with decreasing pH. Based on these findings and information from the literature, we hypothesized the transport mechanisms in which (1) H+ binds to not only the H+-binding site, but also the substrate-binding site; and (2) H+ at the substrate-binding site inhibits the interaction of neutral and cationic substrates, but is necessary for that of anionic substrates. To validate these hypotheses, a computational model was constructed and various properties of substrate transport by PEPT1 were simulated. Our model reproduced the voltage dependence, hyperbolic saturation and bell-shaped pH-profile of Gly-Sar transport. Moreover, the various transport properties of negatively and positively charged substrates were also reconstructed. These findings indicated that the inferred mechanisms are able to sufficiently interpret the transport of both neutral and charged substrates by PEPT1.

Anions↗