Functional Characterization of Human Proton-Coupled Folate Transporter/Heme Carrier Protein 1 Heterologously Expressed in Mammalian Cells as a Folate Transporter
The functional characteristics of human proton coupled folate transporter (hPCFT)/heme carrier protein (HCP) 1 were investigated. hPCFT/HCP1 expressed transiently in human embryonic kidney 293 cells mediated the transport of folate at an acidic extracellular pH of 5.5 in a manner independent of Na(+) and insensitive to membrane potential, but its transport activity was absent at near-neutral pH. Folate transport mediated by hPCFT/hHCP1 at pH 5.5 was saturable with a K(m) of 1.67 microM and extensively inhibited by reduced folates, such as folinate, 5-methyltetrahydrofolate, and methotrexate (MTX). Sulfobro-mophthalein and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid were also found to be potent inhibitors of hPCFT/hHCP1, but hemin was found to exhibit only minimal inhibitory effect. When expressed stably as a protein fused with green fluorescent protein (GFP-hPCFT/HCP1) in MDCKII cells, GFP-hPCFT/HCP1 was mainly localized at the apical membrane, and the cellular accumulation of MTX was higher from the apical side than from the basal side. These functional features of hPCFT/HCP1 are consistent with those of the well characterized carrier-mediated folate transport system in the small intestine, suggesting that hPCFT/HCP1 is responsible for the intestinal absorption of folate and also MTX. We also found that sulfasalazine is a potent inhibitor of hPCFT/HCP1, which would interfere with the intestinal absorption of MTX when coadministered in therapy for rheumatoid arthritis as well as folate.
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- "For proton co-transporters, like those expressed in small intestinal epithelium, optimal transporting activity is usually observed at acidic pH condition which is close to physiological conditions in the intestine. The proton-coupled folate transporter (PCFT), encoded by SLC46A1, incorporates folate by coupling a proton co-transport down the proton gradient at pH 5.5, however, this transport activity is absent at neutral pH (Nakai et al., 2007). Peptide transporters 1 and 2 (PEPT1 and PEPT2, encoded by SLC15A1 and SLC15A2) are similar examples, which mediate absorption small peptides and peptide-like drugs from the intestine and kidney respectively with symport of a proton (Liu et al., 1995; Liang et al., 1995). "
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ABSTRACT: Transporters comprise the largest family of membrane proteins in human organism, including members of solute carrier transporter and ATP-binding cassette transporter families. They play pivotal roles in the absorption, distribution and excretion of xenobiotic and endogenous molecules. Transporters are widely expressed in various human tissues and are routinely evaluated during the process of drug development and approval. Over the past decade, increasing evidence shows that drug transporters are important in both normal physiology and disease. Currently, transporters are utilized as therapeutic targets to treat numerous diseases such as diabetes, major depression, hypertension and constipation. Despite the steady growth of the field of transporter biology, more than half of the members in transporter superfamily have little information available about their endogenous substrate(s) or physiological functions. This review outlines current research methods in transporter studies, and summarizes the drug-transporter interactions including drug-drug and drug-endogenous substrate interactions. In the end, we also discuss the therapeutic perspective of transporters based on their physiological and pathophysiological roles.
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- "Similarly, MDCKII cells stably expressing human PCFT tagged with green fluorescent protein (GFP), which had been prepared as described previously , were grown on 24-well plates (1.5 Â 10 5 cells/mL and 1 mL/well) for 2 days to confluence and, after pretreatment with or without myricetin (100 mM), uptake assays were conducted using [ 3 H]folate as a substrate. Mock cells, which were transfected with empty vector, were used to estimate nonspecific uptake. "
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ABSTRACT: Myricetin is a flavonoid that has recently been suggested to interfere with the intestinal folate transport system. To examine that possibility, focusing on its sustained inhibitory effect on proton-coupled folate transporter (PCFT), the uptake of folate was examined in Caco-2 cells, in which PCFT is known to be in operation, in the absence of myricetin in the medium during uptake period after preincubation of the cells with the flavonoid (100 μM) for 1 h. This pretreatment induced an extensive and sustained reduction in the carrier-mediated component of folate uptake, which was attributable to a reduction in the maximum transport rate (Vmax). Although the affinity of the transporter for folate was increased at the same time as indicated by a reduction in the Michaelis constant (Km), the change in Km was overwhelmed in extent by that in Vmax. Consistent with the finding, folate transport by human PCFT stably expressed in Madin-Darby canine kidney II cells was reduced in a similar manner with simultaneous reductions in Vmax and Km by myricetin pretreatment. Attention may need to be given for a possibility that such a sustained inhibition of PCFT could potentially be a cause of the malabsorption of folate and also antifolate drugs.
Copyright © 2014 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.
Available from: Nobuhiro Mori
- "In brush-border membrane vesicles and everted sacs of rat intestine, MTX was transported in saturable and pH-dependent manner [84,101,102], in which the transport of MTX was greatest at pH 4.5 and comparably high in a range from pH 4.0 to 5.5. Above pH 5.5, the transport decreased with an increase in pH and reached a negligibly small level at a neutral pH and above, indicating that PCFT-mediated transport is highly sensitive to extracellular pH . "
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ABSTRACT: Mizoribine is administered orally and excreted into urine without being metabolized. Many research groups have reported a linear relationship between the dose and peak serum concentration, between the dose and AUC, and between AUC and cumulative urinary excretion of mizoribine. In contrast, a significant interindividual variability, with a small intraindividual variability, in oral bioavailability of mizoribine is also reported. The interindividual variability is mostly considered to be due to the polymophisms of transporter genes. Methotrexate (MTX) is administered orally and/or by parenteral routes, depending on the dose. Metabolic enzymes and multiple transporters are involved in the pharmacokinetics of MTX. The oral bioavailability of MTX exhibits a marked interindividual variability and saturation with increase in the dose of MTX, with a small intraindividual variability, where the contribution of gene polymophisms of transporters and enzymes is suggested. Therapeutic drug monitoring of both mizoribine and MTX is expected to improve their clinical efficacy in the treatment of rheumatoid arthritis.
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