Identification of the amino acid residues involved in the species-dependent differences in the pyridoxine transport function of SLC19A3.

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.

Involvement of proton-coupled SLC49A4-mediated transport in the export of lysosomally trapped pyrilamine.

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.

pH-dependent pyridoxine transport by SLC19A2 and SLC19A3: Implications for absorption in acidic microclimates.

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.

Proinflammatory cytokines inhibit thiamin uptake by human and mouse pancreatic acinar cells: involvement of transcriptional mechanism(s).

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.

Posttranscriptional regulation of thiamin transporter-1 expression by microRNA-200a-3p in pancreatic acinar cells.

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.

Current Understanding of the Intestinal Absorption of Nucleobases and Analogs.

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.

Organic anion transporter 1 (OAT1/SLC22A6) enhances bioluminescence based on d-luciferin-luciferase reaction in living cells by facilitating the intracellular accumulation of d-luciferin.

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 HEK293 cells stably expressing Pyrearinus termitilluminans luciferase (HEK293/eLuc) markedly enhanced BL intensity in the presence of d-luc. When OAT1 was transiently expressed in HEK293 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 µM. 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 µM. 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.

Role of Equilibrative Nucleobase Transporter 1/SLC43A3 as a Ganciclovir Transporter in the Induction of Cytotoxic Effect of Ganciclovir in a Suicide Gene Therapy with Herpes Simplex Virus Thymidine Kinase.

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.

GWAS of clinically defined gout and subtypes identifies multiple susceptibility loci that include urate transporter genes.

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.

Phenobarbital and Insulin Reciprocate Activation of the Nuclear Receptor Constitutive Androstane Receptor through the Insulin Receptor.

Phenobarbital (PB) antagonized insulin to inactivate the insulin receptor and attenuated the insulin receptor downstream protein kinase B (AKT)-forkhead box protein O1 and extracellular signal-regulated kinase 1/2 signals in mouse primary hepatocytes and HepG2 cells. Hepatic AKT began dephosphorylation in an early stage of PB treatment, and blood glucose levels transiently increased in both wild-type and constitutive androstane receptor (CAR) knockout (KO) mice. On the other hand, blood glucose levels increased in wild-type mice, but not KO mice, in later stages of PB treatment. As a result, PB, acting as an insulin receptor antagonist, elicited CAR-independent increases and CAR-dependent decreases of blood glucose levels at these different stages of treatment, respectively. Reciprocally, insulin activation of the insulin receptor repressed CAR activation and induction of its target CYP2B6 gene in HepG2 cells. Thus, PB and insulin cross-talk through the insulin receptor to regulate glucose and drug metabolism reciprocally.

Identification of human-specific amino acid residues governing atenolol transport via organic cation transporter 2.

Organic cation transporter 2 (OCT2/SLC22A2) is predominantly localized on the basolateral membranes of renal tubular epithelial cells and plays a crucial role in the renal secretion of various cationic drugs. Although variations in substrate selectivity among renal organic cation transport systems across species have been reported, the characteristics of OCT2 remain unclear. In this study, we demonstrated that atenolol, a ß1-selective adrenergic antagonist, is transported almost exclusively by human OCT2, contrasting with OCT2s from other selected species. Using chimeric constructs between human OCT2 (hOCT2) and the highly homologous monkey OCT2 (monOCT2), along with site-directed mutagenesis, we identified non-conserved amino acids Val8, Ala31, Ala34, Tyr222, Tyr245, Ala270, Ile394, and Leu503 as pivotal for hOCT2-mediated atenolol transport. Kinetic analysis revealed that atenolol was transported by hOCT2 with a 12-fold lower affinity than MPP+, a typical OCT2 substrate. The inhibitory effect of atenolol on MPP+ transport was 6200-fold lower than that observed for MPP+ on atenolol transport. Additionally, we observed weaker inhibitory effects on MPP+ transport compared to atenolol transport with ten different OCT2 substrates. Altogether, this study suggests that eight hOCT2-specific amino acids constitute the low-affinity recognition site for atenolol transport, indicating differences in OCT2-mediated drug elimination between humans and highly homologous monkeys. Our findings underscore the importance of understanding species-specific differences in drug transport mechanisms, shedding light on potential variations in drug disposition and aiding in drug development.

Disrupted in renal carcinoma 2 (DIRC2/SLC49A4) is an H+-driven lysosomal pyridoxine exporter.

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.

Animal species differences in the pyridoxine transport function of SLC19A3: Absence of Slc19a3-mediated pyridoxine uptake in the rat small intestine.

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.

Functional characterization of human organic anion transporter 10 (OAT10/SLC22A13) as an orotate transporter.

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.

Identification and functional characterization of the first nucleobase transporter in mammals: implication in the species difference in the intestinal absorption mechanism of nucleobases and their analogs between higher primates and other mammals.

Nucleobases are important compounds that constitute nucleosides and nucleic acids. Although it has long been suggested that specific transporters are involved in their intestinal absorption and uptake in other tissues, none of their molecular entities have been identified in mammals to date. Here we describe identification of rat Slc23a4 as the first sodium-dependent nucleobase transporter (rSNBT1). The mRNA of rSNBT1 was expressed highly and only in the small intestine. When transiently expressed in HEK293 cells, rSNBT1 could transport uracil most efficiently. The transport of uracil mediated by rSNBT1 was sodium-dependent and saturable with a Michaelis constant of 21.2 microM. Thymine, guanine, hypoxanthine, and xanthine were also transported, but adenine was not. It was also suggested by studies of the inhibitory effect on rSNBT1-mediated uracil transport that several nucleobase analogs such as 5-fluorouracil are recognized by rSNBT1, but cytosine and nucleosides are not or only poorly recognized. Furthermore, rSNBT1 fused with green fluorescent protein was mainly localized at the apical membrane, when stably expressed in polarized Madin-Darby canine kidney II cells. These characteristics of rSNBT1 were almost fully in agreement with those of the carrier-mediated transport system involved in intestinal uracil uptake. Therefore, it is likely that rSNBT1 is its molecular entity or at least in part responsible for that. It was also found that the gene orthologous to the rSNBT1 gene is genetically defective in humans. This may have a biological and evolutional meaning in the transport and metabolism of nucleobases. The present study provides novel insights into the specific transport and metabolism of nucleobases and their analogs for therapeutic use.

Evaluation of 4',6-diamidino-2-phenylindole as a fluorescent probe substrate for rapid assays of the functionality of human multidrug and toxin extrusion proteins.

Multidrug and toxin extrusion protein 1 (MATE1) and MATE2-K are organic cation/H(+) antiporters that have recently been identified and suggested to be responsible for the brush border secretory transport of many cationic drugs in renal tubules. We here report our finding that 4',6-diamidino-2-phenylindole (DAPI) can be used as a probe substrate for rapid assays of the functionality of the human MATEs, hMATE1, and hMATE2-K, by taking advantage of its fluorescent nature. The specific cellular uptakes of DAPI by cloned hMATE1 and hMATE2-K, which were assessed by fluorescence intensity, were found to be rapid and saturable with the Michaelis constants of 1.13 and 3.16 microM, respectively, indicating that DAPI is a good substrate of both hMATEs. It was found that many organic cations inhibit the specific uptake of DAPI by hMATE1 and hMATE2-K, and the extents of inhibition are in good correlation with those of inhibition of the specific uptake of [(3)H]cimetidine as a typical substrate, indicating comparable performances of both substrates as probes in identifying inhibitors. Thus, DAPI can be an alternative probe substrate that enables fluorometric rapid assays of the functionality of both hMATEs. It was also found that the other major renal organic cation transporters, human organic cation transporter 2 (hOCT2), hOCT3, human novel organic cation transporter 1 (hOCTN1), and hOCTN2, cannot transport DAPI, although hOCT1, which is mainly expressed in the liver, can. Therefore, the DAPI uptake assay can be a method specific to the hMATEs among organic cation transporters in the human kidney.

Functional characteristics of the human ortholog of riboflavin transporter 2 and riboflavin-responsive expression of its rat ortholog in the small intestine indicate its involvement in riboflavin absorption.

Riboflavin transporter (RFT) 2 has recently been identified as a transporter that may be, mainly based on the functional characteristics of its rat ortholog (rRFT2), involved in the intestinal absorption of riboflavin. The present study was conducted to further examine such a possible role of RFT2, focusing on the functional characteristics of its human ortholog (hRFT2) and the response of rRFT2 expression in the small intestine to deprivation of dietary riboflavin. When transiently expressed in human embryonic kidney 293 cells, hRFT2 could transport riboflavin efficiently in a pH-sensitive manner, favoring acidic pH and without requiring Na(+). Riboflavin transport by hRFT2 was saturable with a Michaelis constant of 0.77 µmol/L at pH 6.0, and inhibited by some riboflavin derivatives, such as lumiflavin. It was also inhibited, to a lesser extent, by some cationic compounds, such as ethidium. Thus, hRFT2 was suggested to, together with a finding that its mRNA is highly expressed in the small intestine, have characteristics as an intestinal RFT. Furthermore, feeding rats a riboflavin-deficient diet caused an upregulation of the expression of rRFT2 mRNA in the small intestine, presumably as an adaptive response to enhance riboflavin absorption, which would involve rRFT2, and its apically localized characteristic was suggested by the observation of rRFT2 tagged with green fluorescent protein stably expressed in polarized Madin-Darby canine kidney II cells. All these results combined indicate that RFT2 is a transporter involved in the epithelial uptake of riboflavin in the small intestine for its nutritional utilization.

Functional Analysis of the Role of Equilibrative Nucleobase Transporter 1 (ENBT1/SLC43A3) in Adenine Transport in HepG2 Cells.

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.

Pharmacokinetic functions of human induced pluripotent stem cell-derived small intestinal epithelial cells.

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.

Functional characteristics of two human MATE transporters: kinetics of cimetidine transport and profiles of inhibition by various compounds.

PURPOSE: Human multidrug and toxin extrusion protein 1 (hMATE1) and hMATE2-K are organic cation/H+ antiporters that have recently been identified and suggested to be involved in the renal brush border secretion of various organic cations. Information about functional characteristics of them has been accumulating, but still insufficient to fully understand their functions and respective roles. The present study was conducted to help clarify them. METHODS: The cDNA of hMATE1 was isolated from human brain cDNA by RT-PCR and hMATE2-K cDNA was from human kidney cDNA. HEK293 cells were stably transfected with hMATE1 and hMATE2-K, and the cellular uptakes of [3H]cimetidine and [14C]tetraethylammonium (TEA) were evaluated. RESULTS: It was first found that both hMATE1 and hMATE2-K can transport cimetidine with high affinities, indicated by small Michaelis constants of 8.00 mM and 18.18 mM, respectively. These were much smaller than those for TEA (366 mM and 375 mM, respectively, for hMATE1 and hMATE2-K). Subsequent investigation using cimetidine as a probe substrate into the profiles of inhibition of the two hMATEs by various compounds indicated that they are similar in principle but different to some extent in substrate recognition, reflecting the modest differences in amino acid sequences between them. In fact, cimetidine transport by hMATE1 was correlated to that by hMATE2-K, which is 65% similar to hMATE1, but not as good as to that by rat MATE1, which is 86% similar. CONCLUSIONS: Cimetidine was demonstrated to be a high affinity substrate of both hMATEs. Subsequent evaluation of the inhibition of hMATEs by various compounds indicated no major difference in function or role between hMATE1 and hMATE2-K.