Abstract
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 Km of 1.67 μM 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.
Footnotes
-
This study was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology, Japan and by a Grant-in-Aid for Research in Nagoya City University.
-
Y.N. and K.I. contributed equally to this work.
-
A portion of this work was previously presented: Nakai Y, Inoue K, Hatakeyama M, Hayashi Y, and Yuasa H (2006) Molecular identification and functional characterization of a pH-sensitive folate transporter in human intestine, in 21st Annual Meeting of the Japanese Society for the Study of Xenobiotics; 2006 Nov 29–Dec 1; Tokyo, Japan. p 277, Japanese Society for the Study of Xenobiotics, Tokyo, Japan.
-
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
-
doi:10.1124/jpet.107.122606.
-
ABBREVIATIONS: MTX, methotrexate; DMARD, disease-modifying antirheumatic drug; RFC, reduced folate carrier; PCFT, proton-coupled folate transporter; HCP, heme carrier protein; hPCFT, human proton-coupled folate transporter; NSAID, nonsteroidal anti-inflammatory drug; HEK, human embryonic kidney; PCR, polymerase chain reaction; GFP, green fluorescent protein; PBS, phosphate-buffered saline; BSA, bovine serum albumin; THF, tetrahydrofolate; 5-MTHF, 5-methyltetrahydrofolate; TPP, thiamine pyrophosphate; SSZ, sulfasalazine; mPCFT/HCP1, mouse ortholog of PCFT/HCP1; OAT, organic anion transporter.
- Received March 12, 2007.
- Accepted May 1, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
JPET articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|