title: Stereoselective transport of amethopterin enantiomers by the proton-coupled folate transporter.
abstract: Stereoselective transport of methotrexate (L-amethopterin, L-MTX) and its antipode (D-amethopterin, D-MTX) by the proton-coupled folate transporter (PCFT) was examined using PCFT-expressing HEK293 cells (PCFT-HEK293 cells). Uptake of both L-MTX and D-MTX was pH-dependent and decreased with an increase in the extracellular pH from 5.0 to 7.4. The initial uptake rate of L-MTX into PCFT-HEK293 cells followed Michaelis-Menten kinetics with a K(m) value of approximately 5.0 microM. Dixon plots revealed that L-MTX uptake was inhibited competitively by unlabeled L-MTX, D-MTX, and folic acid (FA), with K(i) values of approximately 3.6, 180, and 2.1 microM, respectively. The initial uptake rate of D-MTX into PCFT-HEK293 cells also followed Michaelis-Menten kinetics with a K(m) value of 211 microM. The V(max) value of D-MTX was similar to that of L-MTX. The present study revealed that the transport of MTX enantiomers by PCFT is highly stereoselective with the uptake clearance of L-MTX being approximately 40-fold greater than that of D-MTX. It was also revealed that this high stereoselectivity results from the difference in K(m) values, and not V(max) values, between the enantiomers. The observed stereoselectivity was consistent with the differences in the intestinal absorption of MTX enantiomers in humans.
authors.affiliation: School of Pharmacy, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo, Japan.
authors.forename: Tomoya
authors.initials: T
authors.lastname: Narawa
authors.forename: Tomoo
authors.initials: T
authors.lastname: Itoh
mesh_terms: D000465:Algorithms; D001692:Biological Transport; D002460:Cell Line; D002853:Chromatography, Liquid; D005492:Folic Acid / Q000378:metabolism; D005493:Folic Acid Antagonists / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007408:Intestinal Absorption; D007668:Kidney / Q000166:cytology / Q000196:embryology; D007700:Kinetics; D013058:Mass Spectrometry; D026901:Membrane Transport Proteins / Q000378:metabolism*; D008727:Methotrexate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*; D058979:Proton-Coupled Folate Transporter; D013237:Stereoisomerism; D014162:Transfection
chemical_list: D005493:Folic Acid Antagonists; D026901:Membrane Transport Proteins; D058979:Proton-Coupled Folate Transporter; C505058:SLC46A1 protein, human; D005492:Folic Acid; D008727:Methotrexate