Tomohiro Terada

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

PurposeTransport characteristics and tissue distribution of the rat H+/organic cation antiporter MATE1 (multidrug and toxin extrusion 1) were examined.MethodsRat 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.ResultsThe uptake of a prototypical organic cation tetraethylammonium (TEA) by MATE1-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.ConclusionsThese 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.

Efflux properties of basolateral peptide transporter in human intestinal cell line Caco-2

Small peptides and some pharmacologically active compounds are absorbed from the small intestine by the apical H+-coupled peptide transporter 1 (PEPT1) and the basolateral peptide transporter. Here we investigated the efflux properties of the basolateral peptide transporter in Caco-2 cells using two strategies, efflux measurements and a kinetic analysis of transepithelial transport of glycylsarcosine (Gly-Sar). [14C]Gly-Sar efflux through the basolateral membrane was not affected significantly by the external pH. Both approaches revealed that the basolateral peptide transporter was saturable in the efflux direction, and that the affinity was lower than that in the influx direction. For two peptide-like drugs, there was no difference in substrate recognition by the basolateral peptide transporter between the two sides of the membrane. Using the kinetic parameters of PEPT1 and the basolateral peptide transporter, a computational model of Gly-Sar transport in Caco-2 cells was constructed. The simulation fitted the experimental data well. Our findings suggested that substrate affinity of the basolateral peptide transporter is apparently asymmetric, but pH-dependence and substrate specificity are symmetric for the two directions of transport. The behaviour of Gly-Sar in Caco-2 cells could be predicted by a mathematical model describing the peptide transporters.

Pharmacokinetic Significance of Renal OAT3 (SLC22A8) for Anionic Drug Elimination in Patients with Mesangial Proliferative Glomerulonephritis

PurposeOur 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.MethodshOAT 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.ResultsIn 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.ConclusionsThese 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.

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.

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 [14C]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 Vmax. Moreover, PEPT1 mRNA level was positively related to the activity of [14C]Gly-Sar uptake (r=0.55). Based on these findings, the Vmax 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.