Tomoji Maeda

INVOLVEMENT OF URIC ACID TRANSPORTER IN INCREASED RENAL CLEARANCE OF THE XANTHINE OXIDASE INHIBITOR OXYPURINOL INDUCED BY A URICOSURIC AGENT, BENZBROMARONE

Benzbromarone has been reported to increase the renal clearance of oxypurinol, an active metabolite of allopurinol. We examined the renal transport of oxypurinol to determine whether such a change in renal clearance could be explained by altered transporter-mediated reabsorption. Since the first step of reabsorption takes place at the renal epithelial apical membrane, we focused on membrane transporters. Benzbromarone is an inhibitor of reabsorption of uric acid mediated by the uric acid transporter (URAT) URAT1 (SLC22A12), which is expressed at the apical membrane of proximal tubular cells in humans. Uptake of oxypurinol by Xenopus oocytes injected with complementary RNA of URAT1 was significantly higher than that by water-injected oocytes, and the uptake was saturable, with a Km of about 800 μM. Moreover, benzbromarone inhibited the oxypurinol uptake by URAT1 at concentrations as low as 0.01 μM. The uptake of oxypurinol by another organic anion transporter (OAT), OAT4 (SLC22A11), which is also expressed at the apical membrane of proximal tubular epithelial cells, was negligible, whereas the uptake of [3H]estrone-3-sulfate by OAT4 was significantly inhibited by oxypurinol. Furthermore, neither the transport activity of organic cation/carnitine transporter (OCTN) 1 nor OCTN2 was affected by oxypurinol or benzbromarone. These results indicate that URAT1 is involved in renal reabsorption of oxypurinol, and the increment of renal clearance of oxypurinol upon concomitant administration of benzbromarone could be due to drug interaction at URAT1.

Concentration-dependent mode of interaction of angiotensin II receptor blockers with uric acid transporter.

Serum uric acid (SUA) is currently recognized as a risk factor for cardiovascular disease. It has been reported that an angiotensin II receptor blocker (ARB), losartan, decreases SUA level, whereas other ARBs, such as candesartan, have no lowering effect. Because the renal uric acid transporter (URAT1) is an important factor controlling the SUA level, we examined the involvement of URAT1 in those differential effects of various ARBs on SUA level at clinically relevant concentrations. This study was done by using URAT1-expressing Xenopus oocytes. Losartan, pratosartan, and telmisartan exhibited cis-inhibitory effects on the uptake of uric acid by URAT1, whereas at higher concentrations, only telmisartan did, and these ARBs reduced the uptake in competitive inhibition kinetics. On the other hand, candesartan, EXP3174 [2-n-butyl-4-chloro-1-[(2′-(1H-tetrazol-5-yl)biphenyl-4-yI)methyl]imidazole-5-carboxylic acid] (a major metabolite of losartan), olmesartan, and valsartan were not inhibitory. Preloading of those ARBs in the oocytes enhanced the URAT1-mediated uric acid uptake, showing a trans-stimulatory effect. The present study is a first demonstration of the differential effects of ARBs on URAT1 that some ARBs are both cis-inhibitory and trans-stimulatory, depending on concentration, whereas others exhibit either a trans-stimulatory or cis-inhibitory effect alone, which could explain the clinically observed differential effects of ARBs on SUA level. Furthermore, it was found that such differential effects of ARBs on URAT1 could be predicted from the partial chemical structures of ARBs, which will be useful information for the appropriate use and development of ARBs without an increase of SUA.

Expression pattern, subcellular localization and structure--function relationship of rat Tpx-1, a spermatogenic cell adhesion molecule responsible for association with Sertoli cells.

The gene for a testicular cell adhesion protein called Tpx‐1, which mediates the binding of spermatogenic cells to Sertoli cells of the rat in primary culture, was previously cloned. Here the characterization of Tpx‐1 is reported. Tpx‐1 messenger ribonucleic acid (mRNA) became detectable in pachytene spermatocytes and continued to be present throughout development into elongated spermatids, while the amount of Tpx‐1 protein seemed to increase some time after the increment of mRNA. Tpx‐1 protein was also present, although less abundantly, in spermatozoa prepared from the epididymis. Tpx‐1 contains a cluster of hydrophobic amino acid residues near the amino terminus and a cysteine‐rich region in the carboxyl‐terminal half. Tpx‐1 fused with green fluorescence protein was secreted into the medium when expressed in a cultured cell line, depending on the presence of the amino‐terminal hydrophobic region. Moreover, Tpx‐1 was present in the medium of testicular cell primary culture. Structure–function analysis revealed that the amino‐terminal 101 amino acid residues were sufficient for cell adhesion activity, whereas the carboxyl‐terminal cysteine‐rich region was dispensable. In conclusion, Tpx‐1 is produced and secreted from spermatogenic cells at various differentiation stages, and mediates the interaction of those cells with Sertoli cells.

Involvement of Rat and Human Organic Anion Transporter 3 in the Renal Tubular Secretion of Topotecan [(S)-9-Dimethylaminomethyl-10-hydroxy-camptothecin hydrochloride]

Topotecan [(S)-9-dimethylaminomethyl-10-hydroxy-camptothecin hydrochloride] is primarily excreted into urine in humans, with approximately 49% of the dose recovered as total topotecan (topotecan lactone plus topotecan hydroxyl acid form). The renal elimination of topotecan involves tubular secretion in addition to glomerular filtration, but little is known about the molecular mechanism of the renal tubular secretion. In the present study, we investigated the transport characteristics of topotecan hydroxyl acid across the renal basolateral membrane using rat kidney slices and rat or human transporter-expressing Xenopus laevis oocytes. Pravastatin and probenecid significantly inhibited the uptake of topotecan hydroxyl acid by rat kidney slices with Ki values of 10.6 and 8.1 μM, respectively, and p-aminohippurate was weakly inhibitory at high concentrations, whereas excess tetraethylammonium had no effect. The uptake of topotecan hydroxyl acid by oocytes injected with complementary RNA of either rat or human organic anion transporter 3 (rOAT3 or hOAT3) was greater than that of water-injected oocytes. Kinetic analysis showed that the Km values for rOAT3 and hOAT3 were 21.9 and 56.5 μM, respectively. Neither rOAT1 nor hOAT1 stimulated topotecan hydroxyl acid transport. These results suggest that the urinary excretion of topotecan hydroxyl acid is accounted for by transport via OAT3, as well as glomerular filtration, in both rats and humans; therefore, drug-drug interactions involving OAT3 may cause a change in clearance of topotecan.

Nucleoside Transport at the Blood-Testis Barrier Studied with Primary-Cultured Sertoli Cells

Nucleosides are essential for nucleotide synthesis in testicular spermatogenesis. In the present study, the mechanism of the supply of nucleosides to the testicular system across the blood-testis barrier was studied using primary-cultured Sertoli cells from rats and TM4 cells from mice. Uptake of uridine by these cells was time- and concentration-dependent. Uridine uptake was decreased under Na+-free conditions, and the system was presumed to be high affinity, indicating an Na+-dependent concentrative nucleoside transporter (CNT) is involved. On the other hand, nitrobenzylthioinosine, a potent inhibitor of Na+-independent equilibrative nucleoside transporters (ENTs), inhibited uridine uptake by the Sertoli cells in a concentration-dependent manner. Expression of nucleoside transporters ENT1, ENT2, ENT3, CNT1, CNT2, and CNT3 was detected in Sertoli cells by reverse transcriptase-polymerase chain reaction analysis. Inhibition studies of the uptake of uridine by various nucleosides both in the presence and absence of Na+ indicated that the most of those expressed nucleoside transporters, ENTs and CNTs, are involved functionally. These results demonstrated that Sertoli cells are equipped with multiple nucleoside transport systems, including ENT1, ENT2, and CNTs, to provide nucleosides for spermatogenesis.

Uptake transporter organic anion transporting polypeptide 1B3 contributes to the growth of estrogen-dependent breast cancer.

Estrone-3-sulfate is one of the most abundant estrogen precursors in postmenopausal women. We previously showed that estrone-3-sulfate transporters are present in human breast cancer-derived MCF-7 cells (J. Pharmacol. Exp. Ther. 311 (2004) 1032-1037) and that inhibition of estrone-3-sulfate uptake resulted in the suppression of cell growth (Pharm. Res. 22 (2005) 1634-1641); therefore, estrone-3-sulfate transporter should be a novel target for therapy of hormone-dependent breast cancers. The purpose of the present study is to identify the transporter(s) responsible for the uptake of estrone-3-sulfate in breast cancer cells. We obtained two subclones of MCF-7 cells with different estrone-3-sulfate uptake activities and searched for differentially expressed transporter genes by means of DNA microarray analysis. Among several candidate transporters identified, OATP1B3 was further evaluated, since the uptake characteristics of estrone-3-sulfate by MCF-7 cells seemed consistent with the transport properties of OATP1B3. The contribution of OATP1B3 to estrone-3-sulfate uptake by MCF-7 cells was examined by the relative activity factor (RAF) method, and was calculated to amount to 6%. This result suggests that OATP1B3 is one of the transporters contributing to the supply of the estrogen precursor estrone-3-sulfate to estrogen-dependent breast cancer cells.

Mechanism of the Regulation of Organic Cation/Carnitine Transporter 1 (SLC22A4) by Rheumatoid Arthritis-Associated Transcriptional Factor RUNX1 and Inflammatory Cytokines

Recently, it was reported that the organic cation/carnitine transporter 1 (OCTN1, SLC22A4) is associated with chronic inflammatory diseases, such as rheumatoid arthritis (RA) and Crohns disease. OCTN1 in humans is expressed in synovial tissues of individuals with rheumatoid arthritis. Furthermore octn1 in mice is expressed in inflamed joints with collagen-induced arthritis, a model of human arthritis, but not in the joints of normal mice. OCTN1 should be involved in the inflammatory disease and in the present study, the regulatory mechanism of OCTN1 expression was characterized using the human fibroblast-like synoviocyte cell line MH7A, derived from RA patients. A luciferase-reporter gene assay and gel shift assay demonstrated that RUNX1, which is an essential hematopoietic transcription factor associated with acute myeloid leukemia and is related to RA and Sp1, is involved in the regulation of OCTN1 promoter activity. Inflammatory cytokines such as interleukin-1β and tumor necrosis factor-α increased the expression of OCTN1 mRNA. Furthermore, overexpression of nuclear factor-κB (NF-κB) activated promoter activity of OCTN1. These results clearly demonstrate that expression of OCTN1 is regulated by various factors, including RUNX1, inflammatory cytokines, and NF-κB, all of which are also related to the pathogenesis of RA. Further studies on the physiological substrate(s) of OCTN1 should be done to clarify the roles of OCTN1 in these diseases.

Peptide Derivation of Poorly Absorbable Drug Allows Intestinal Absorption Via Peptide Transporter

The purpose of the present study was to examine whether the intestinal absorption of low-permeability drugs could be improved by utilization of the intestinal influx transporter PEPT1. We investigated whether peptide derivatives of poorly absorbable nonamino acid-like drugs might be substrates of PEPT1, using rebamipide (Reb) as a model drug. We synthesized several peptide derivatives of rebamipide and examined their inhibitory effect on the uptake of [(3)H]Gly-Sar by PEPT1-expressing HeLa cells. Some of the peptide derivatives inhibited PEPT1-mediated uptake of [(3)H]Gly-Sar. Next, uptake of the inhibitory peptide derivatives was evaluated in PEPT1-expressing Xenopus oocytes and HeLa cells. Ser(Reb)-Gly exhibited significantly increased uptake by PEPT1-expressing cells in comparison with that by mock cells. The permeability of Ser(Reb)-Gly across a Caco-2 cell monolayer was significantly higher than that of rebamipide itself, and the transport was decreased in the presence of PEPT1 substrates. Further, a rat intestinal perfusion study revealed increased absorption of Ser(Reb)-Gly compared with rebamipide. These results demonstrate that the addition of a dipeptide moiety to a poorly absorbable nonpeptide/nonamino acid-like drug can result in absorption via the intestinal transporter PEPT1, though there is some selectivity as regards the structure of the added peptide moiety.

Decreased Proliferation and Erythroid Differentiation of K562 Cells by siRNA-induced Depression of OCTN1 (SLC22A4) Transporter Gene

PurposeRecently, it was reported that OCTN1 transporter (SLC22A4) is associated with rheumatoid arthritis (RA) and Crohn’s disease. Additionally, we reported that OCTN1 is expressed in hematopoietic cells, preferentially in erythroid cells. Accordingly, we assessed the physiological role of OCTN1 by examining the effect of knockdown of OCTN1 in blood cells using siRNA method.Materials and MethodsVector-based short hairpin RNA (shRNA) was used to establish K562 cell line which shows stably decreased expression of OCTN1. The characteristic of knockdown of OCTN1 in K562 cells was investigated by cell proliferation, cell differentiation, and uptake of ergothioneine that is a good substrate of OCTN1.Results Several clones of K562 cells exhibited significantly reduced expression of OCTN1 mRNA and protein. They also showed a decreased growth rate and butyrate-dependent differentiation to erythrocytes compared with control-vector transfected cells. In addition, uptake of [3H]ergothioneine by K562 cells suggested that Na+-dependent and high-affinity transporter which is similar to the characteristics of OCTN1 is functional. Moreover, uptake of ergothioneine by K562 cells which exhibit decreased-expression of OCTN1 was decreased in comparison with wild type K562 cells.ConclusionsIt was suggested that OCTN1 is involved in the transport of physiological compounds that are important for cell proliferation and erythroid differentiation.

Effect of Pregnane X Receptor Ligand on Pharmacokinetics of Substrates of Organic Cation Transporter Oct1 in Rats

Because rat organic cation transporter 1 (Oct1, SLC22a1) is expressed mainly in the liver and mediates drug transport, its activity may determine the hepatic handling of cationic drugs. Here, we studied the regulation mechanism of the expression of Oct1, focusing on the nuclear receptors. In vitro studies using cultured hepatocytes indicated that expression of Oct1 was up-regulated by treatment with pregnenolone-16α-carbonitrile (PCN) and by overexpression of rat pregnane X receptor (PXR). In addition, isolated rat hepatocytes exhibited an increase of 1-methyl-4-phenylpyridinium (MPP+) uptake on treatment with PCN. When rats were subcutaneously administered PCN, an increase of biliary excretion clearance and distribution volume was observed for drugs such as MPP+, metformin, and tetraethylammonium, although the effects on pharmacokinetic parameters were variable among the tested drugs. In addition, the expression of Oct2 in kidney was increased by treatment with PCN. Thus, PXR ligands appear to regulate the expression of organic cation transporters in rats and thereby to influence the pharmacokinetic properties of cationic drugs. Because PXR ligands include various clinically used drugs, alterations of hepatic drug handling may arise from interactions between cationic drugs that are substrates of Oct1 and ligands of PXR.