The reduced folate carrier (RFC1) is a major route for the transport of folates in mammalian cells. The localization of RFC1 in murine tissues was evaluated by immunohistochemical analysis using a polyclonal antibody to the C-terminus of the carrier. There was expression of RFC1 in the brush-border membrane of the jejunum, ileum, duodenum and colon. RFC1 was localized to the basolateral membrane of the renal tubular epithelium. Carrier was detected on the plasma membrane of hepatocytes but not in bile duct epithelial cells. In the choroid plexus RFC1 was highly expressed at the apical surface. It was also expressed in axons and dendrites and on the apical membrane of cells lining the spinal canal. In spleen, RFC1 was detected only in the cells of the red pulp. These data provide insights into the role that RFC1 plays in folate delivery in a variety of tissues. In particular, the localization of carrier may elucidate the role of RFC1 in the vectorial transport of folates across epithelia. The data also indicate that in kidney tubules and choroid plexus the sites of RFC1 expression are different from what has been reported previously for the folate receptor; and while RFC1 is expressed in small intestine, folate receptor is not.

Long-term use of imidacloprid in a wide range of rice-growing areas might be associated with high levels of resistance in N. lugens. Therefore, insecticide resistance management strategies must be developed to prevent further increase in resistance.

Studies were undertaken to characterize a low pH transport activity in a reduced folate carrier (RFC)-null HeLa-derived cell line (R5). This transport activity has a 20-fold higher affinity for pemetrexed (PMX; K t , ϳ45 nmol/L) than methotrexate (MTX; K t , ϳ1 mol/L) with comparable V max values. The K i values for folic acid, ZD9331, and ZD1694 were ϳ 400 -600 nmol/L, and the K i values for PT523, PT632, and trimetrexate were >50 mol/L. The transporter is stereospecific and has a 7-fold higher affinity for the 6S isomer than the 6R isomer of 5-formyltetrahydrofolate but a 4-fold higher affinity for the 6R isomer than the 6S isomer of dideazatetrahydrofolic acid. Properties of RFC-independent transport were compared with transport mediated by RFC at low pH using HepG2 cells, with minimal constitutive low pH transport activity, transfected to high levels of RFC. MTX influx K t was comparable at pH 7.4 and pH 5.5 (1.7 versus 3.8 mol/L), but V max was decreased 4.5-fold. There was no difference in the K t for PMX (ϳ1.2 mol/L) or the K i for folic acid (ϳ130 mol/L) or PT523 (ϳ 0.2 mol/L) at pH 7.4 and pH 5.5. MTX influx in R5 and HepG2 transfectants at pH 5.5 was trans-stimulated in cells loaded with 5-formyltetrahydrofolate, inhibited by Cl ؊ (HepG2-B > R5), Na ؉ independent, and uninhibited by energy depletion. Hence, RFC-independent low pH transport activity in HeLa R5 cells is consistent with a carrier-mediated process with high affinity for PMX. Potential alterations in protonation of RFC or the folate molecule as a function of pH do not result in changes in affinity constants for antifolates. Whereas both activities at low pH have similarities, they can be distinguished by folic acid and PT523, agents for which they have very different structural specificities.

The thiamin transporter encoded by SLC19A2 and the reduced folate carrier (RFC1) share 40% homology at the protein level, but the thiamin transporter does not mediate transport of folates. By using murine leukemia cell lines that express no, normal, or high levels of RFC1, we demonstrate that RFC1 does not mediate thiamin influx. However, high level RFC1 expression substantially reduced accumulation of the active thiamin coenzyme, thiamin pyrophosphate (TPP). This decreased level of TPP, synthesized intracellularly from imported thiamin, resulted from RFC1-mediated efflux of TPP. The reduced folate carrier (RFC1), 1 first cloned in 1994, mediates transport of reduced folates critical to one carbonrequiring biosynthetic reactions in mammalian cells and is a member of the major facilitator superfamily of transporters (1-3). RFC1 also delivers MTX and new generation antifolates into a variety of tumors, particularly those of hematopoietic origin (4). RFC1 exchanges folates with a broad spectrum of inorganic and organic anions, and high extracellular concentrations of a variety of organic phosphates competitively inhibit RFC1-mediated folate influx (5-7). This interaction between RFC1 and organic phosphates results in the uphill transport of folates into cells linked to the organic phosphate gradient across cell membranes (5).Structurally unrelated to the folates, thiamin plays an essential role in glycolysis and oxidative decarboxylation reactions after conversion to the coenzyme thiamin pyrophosphate by thiamin pyrophosphokinase in cells. Thiamin is also transported across cell membranes by a carrier-mediated process (8). Thiamin deficiency, reflected in a decrease in plasma thiamin concentration and TPP levels in erythrocytes, results in a variety of clinical abnormalities including cardiovascular and neurological disorders (9). Thiamin deficiency due to impaired transport results in the thiamin-responsive megaloblastic anemia syndrome, a disorder also associated with deafness and diabetes mellitus (10, 11). Positional cloning with families inheriting this autosomal recessive disease led to the recent identification of the thiamin transporter gene SLC19A2 (12-14).The thiamin transporter encoded by SLC19A2 is highly homologous to RFC1, sharing an amino acid identity of 40% and similarity of 55%, and both are predicted to have 12 transmembrane domains. Despite the similarity between these two proteins, the thiamin transporter, when expressed in HeLa cells, was not found to transport folates (15). In the current report, the impact of RFC1 function on thiamin transport and accumulation of its active coenzyme metabolites was studied in murine leukemia cells. Although RFC1 was not found to transport thiamin, it does transport phosphorylated thiamin derivatives, thereby modulating the intracellular accumulation of active thiamin metabolites. -[3Ј,5Ј,7-3 H]MTX (5.7 Ci/mmol) and [ 3 H]thiamin hydrochloride (20 Ci/mmol) were obtained from Amersham Pharmacia Biotech, and [ 3 H]TPP (generally labeled, 4.2 Ci/mmol) was custo...