Reduced folate carrier transports thiamine monophosphate: an alternative route for thiamine delivery into mammalian cells

Rongbao Zhao, Feng Gao, and I. David Goldman

Departments of Medicine and Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Although the reduced folate carrier RFC1 and the thiamine transporters THTR-1 and THTR-2 share ~40% of their identity in protein sequence, RFC1 does not transport thiamine and THTR-1 and THTR-2 do not transport folates. In the present study, we demonstrate that transport of thiamine monophosphate (TMP), an important thiamine metabolite present in plasma and cerebrospinal fluid, is mediated by RFC1 in L1210 murine leukemia cells. Transport of TMP was augmented by a factor of five in cells (R16) that overexpress RFC1 and was markedly inhibited by methotrexate, an RFC1 substrate, but not by thiamine. At a near-physiological concentration (50 nM), TMP influx mediated by RFC1 in wild-type L1210 cells was ~50% of thiamine influx mediated by thiamine transporter(s). Within 1 min, the majority of TMP transported into R16 cells was hydrolyzed to thiamine with a component metabolized to thiamine pyrophosphate, the active enzyme cofactor. These data suggest that RFC1 may be one of the alternative transport routes available for TMP in some tissues when THTR-1 is mutated in the autosomal recessive disorder thiamine-responsive megaloblastic anemia.

SLC19A transporters; thiamine-responsive megaloblastic anemia; thiamine pyrophosphate; thiamine homeostasis; vitamin B1 uptake


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THIAMINE (VITAMIN B1), in its coenzyme form thiamine pyrophosphate, plays a critical role in oxidative phosphorylation and in the pentose phosphate pathway. Dietary thiamine deficiency causes beriberi and Wernicke encephalopathy; the latter is often encountered in chronic alcoholism (17). Mutations in the thiamine transporter gene (THTR-1, SCL19A2) are the cause of an autosomal recessive disorder, thiamine-responsive megaloblastic anemia (TRMA), associated with diabetes mellitus and sensoneural deafness (5, 9, 14, 19, 25, 30). This transporter is highly expressed in skeletal muscle and to a lesser extent in heart and placenta. Expression is very low in small intestine and kidney despite higher thiamine uptake in these tissues than muscle (27), suggesting that an alternative transport route(s) for thiamine is present in these organs. Recently, a gene (SLC19A3) homologous to SLC19A2 was identified (7), and its function was established as a second thiamine transporter, THTR-2 (23).

Thiamine is present in human tissues and fluids mainly in three forms: thiamine, thiamine monophosphate (TMP), and thiamine pyrophosphate (TPP). Thiamine is converted to TPP by thiamine pyrophosphokinase, and TPP is hydrolyzed by thiamine pyrophosphatase to TMP, which is further hydrolyzed to thiamine by thiamine monophosphatase (26). No specific metabolic role is known for TMP. Whereas TPP exists intracellularly exclusively, thiamine and TMP are present both intracellularly and extracellularly. In plasma, TMP is present at levels ~80% that of thiamine, whereas TMP concentrations exceed that of thiamine by 65% in cerebrospinal fluid (34). Blood thiamine level is normal in patients with TRMA, suggesting that the majority of intestinal thiamine transport is not dependent on THTR-1 (28).

The reduced folate carrier RFC1 is a major transporter for physiological folate in plasma, 5-methyltetrahydrofolate, as well as in antifolates (22). Inactivation of RFC1 in mice results in early embryonic lethality, but in animals brought to term by administration to dams of high levels of folic acid, death occurs rapidly due to failure of hematopoietic tissues (38). RFC1, THTR-1, and THTR-2 are a family of carriers within the major facilitator superfamily (29). RFC1 shares protein sequence identity of ~40% with both THTR-1 and THTR-2 but is functionally distinct from the thiamine transporters. RFC1 does not transport thiamine, and THTR-1 and THTR-2 do not transport folates (6, 37). However, RFC1 transports TPP, the major thiamine intracellular metabolite, and recent studies from this laboratory (37) demonstrated that the level of RFC1 expressed in murine leukemia cells is inversely related to the extent of net TPP accumulation. Hence, low RFC1 expression augments TPP accumulation in these cells, whereas high expression suppresses it.

Because RFC1 is an anion exchanger and has been shown to transport TPP, the current studies were designed to assess whether TMP is also a substrate for RFC1 in murine leukemia cells. The data indicate that RFC1 mediates transport of TMP, which is then rapidly hydrolyzed to thiamine and subsequently phosphorylated to TPP in these cells. These results provide an additional facet of the complex role that RFC1 may play in thiamine homeostasis.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Chemicals. [3',5',7-3H]methotrexate (MTX) (5.7 Ci/mmol) and [3H]thiamine hydrochloride (20 Ci/mmol) were obtained from Amersham (Arlington Heights, IL), and [3H]TMP (generally labeled, 2.1 Ci/mmol) was custom-made by Moravek Biochemicals (Brea, CA). Unlabeled MTX was provided by Lederle (Carolina, Puerto Rico), and thiamine, TMP, and TPP were purchased from Sigma. Tritiated MTX was purified by high-performance liquid chromatography (HPLC) before use (10). Tritiated thiamine and TMP were used immediately after purity was confirmed by HPLC (37).

Cells lines and culture conditions. The MTXrA line was selected from murine leukemia L1210 cells with a loss of RFC1 function due to a point mutation in the carrier (2, 31). R16 cells have high-level expression of RFC1 obtained by transfection of murine RFC1 cDNA into MTXrA (2, 39). Cells were grown in RPMI 1640 medium containing 5% bovine calf serum (HyClone), 2 mM glutamine, 20 µM 2-mercaptoethanol, penicillin (100 U/ml), and streptomycin (100 µg/ml) at 37°C in a humidified atmosphere of 5% CO2. R16 cells were grown with G418 at a concentration of 750 µg/ml to ensure stable, high-level expression of RFC1.

Transport studies. Cells were harvested, washed twice with HEPES-buffered saline (HBS; 20 mM HEPES, 140 mM NaCl, 5 mM KCl, 2 mM MgCl2, and 5 mM glucose, pH 7.4), and resuspended in HBS to 2.0 × 107 cells/ml. Cell suspensions were incubated at 37°C for 25 min, after which uptake was initiated by the addition of [3H]thiamine, [3H]TMP, or [3H]MTX. Samples were taken at the indicated times. Uptake was terminated by injection of 1 ml of the cell suspension into 10 ml of ice-cold HBS. Cells were collected by centrifugation, washed twice with ice-cold HBS, dried, and digested with 1 N NaOH in an 8-ml vial. After the liquid scintillation fluor was added, radioactivity was assessed in a liquid scintillation spectrometer. For influx experiments, the tritiated compound with or without unlabeled chemicals was added to the cell suspension to initiate uptake. Intracellular tritium was expressed in units of nanomoles per gram dry weight of cells. One milligram of dry cells corresponds to 6 × 106 L1210 cells.

Determination of rates of TMP hydrolysis. [3H]TMP was exposed to 250 µl of cell suspension or to the supernatant obtained by centrifugation of the cell suspension. Either the concentration of [3H]TMP, the density of the cell suspension, or the interval of incubation was varied. TMP hydrolysis was stopped by the addition of 15 µl of 100% trichloroacetic acid. After a brief centrifugation, the supernatant was extracted three times with water-saturated ethyl ether. Unlabeled thiamine, TMP, and TPP, each at a final concentration of 1 mM, were added to the mixture, and 20 µl were loaded on a 20 × 20-cm thin-layer chromatography plate with a 250-µm layer of silica gel (Whatman). The plate was developed by a solvent mixture of diethanolamine-methanol-formic acid-67 mM dibasic sodium phosphate (1:15:1.5:5) for 2.5 h (20). Under these conditions, TPP (Rf = 0.26), TMP (Rf = 0.37), and thiamine (Rf = 0.51) were well separated. The TMP and thiamine spots were traced under ultraviolet light, removed from plates, and added to scintillation vials. The silica gel was incubated in NaOH (2 N, 0.5 ml) for 15 min at room temperature, and tritium was measured on a liquid scintillation spectrometer after introduction of the fluor. When TMP hydrolysis was complete, radioactivity recovered as thiamine was equivalent to radioactivity recovered from TMP in the absence of TMP hydrolysis, indicating a 100% conversion of TMP to thiamine.

Measurement of intracellular thiamine metabolites. R16 cells were exposed to 1 µM [3H]TMP for 1 min after a 25-min incubation in HBS. Cells were washed twice with ice-cold HBS. Half of the cell pellet was processed for dry weight and total cell tritium, as described in Transport studies. The other half was resuspended in 250 µl of HBS, and 15 µl of 100% trichloroacetic acid were added. The digest was processed for thin-layer chromatography as described above to assess levels of thiamine, TMP, and TPP. Cell levels were expressed in units of picomoles per gram dry weight, according to the percentages of total tritium recovered.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Influx of TMP in murine leukemia cells. Initial uptake of TMP was assessed in three murine leukemia cell lines with different levels of RFC1 expression: 1) L1210 wild-type cells; 2) MTXrA, a variant of L1210 cells that has lost RFC1 activity due to a point mutation (A130P) in the third transmembrane domain (2); and 3) R16 cells derived by transfection of murine RFC1 into MTXrA cells with carrier expression and activity nine times greater than that of L1210 cells (39). As illustrated in Fig. 1, influx of TMP in MTXrA cells was ~20% that of L1210 cells, whereas TMP influx in R16 was approximately fivefold higher than that of L1210 cells. Thus influx of TMP correlated with the level of RFC1 expression in these cell lines. The high ordinate intercept in R16 cells could be attributed in part to a rapid deviation from initial rates as simulated by the interrupted line.


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 1.   Initial uptake of thiamine monophosphate (TMP) in R16, L1210, and MTXrA cells. After a 25-min incubation in HEPES-buffered saline (HBS) at 37°C, cells at a density of 2 × 107 cells/ml were exposed to 1 µM [3H]TMP. Data are means ± SD of 3 independent experiments. When not apparent, error bars are smaller than symbols. Interrupted line simulates rapid deviation from initial uptake rates in R16 cells.

Inhibitory effect of MTX and thiamine on [3H]TMP influx. TMP influx in L1210, MTXrA, and R16 cells was assessed in the presence of 100 µM MTX, an inhibitor of RFC1 but not the thiamine transporter. At this MTX concentration, RFC1-mediated TMP influx (1 µM) should be inhibited >90% in L1210 cells, based on an MTX influx Km for RFC1 of ~5.8 µM (39) and a TMP Ki of 25 µM (see Affinity of RFC1 for TMP). TMP influx in these cell lines was also determined in the presence of 10 µM thiamine, an inhibitor of THTR-1 but not RFC1. At this thiamine concentration (1 µM), 90% of [3H]thiamine influx activity should be blocked based on the thiamine influx Km of 0.96 ± 0.17 µM that we determined in L1210 cells. As shown in Fig. 2, 100 µM MTX abolished ~90% of TMP influx in R16 cells over a 25-s interval of uptake, whereas 10 µM thiamine had no inhibitory effect at all, indicating that virtually all TMP influx in R16 cells was mediated exclusively by RFC1 under these conditions. Because RFC1 is highly expressed in this cell line, the component of TMP influx mediated by this process is maximized. MTX also inhibited >50% of TMP influx in L1210 cells with lesser RFC1 expression but had no effect at all on TMP influx in MTXrA cells, which lack functional RFC1. In the presence of 10 µM thiamine, however, TMP influx in L1210 and MTXrA cells was reduced by 20 and 37%, respectively. As indicated in Chemical instability of TMP in vitro, the emergence of a thiamine-inhibitable TMP influx component in these cells, with lower or absent RFC1 expression, was subsequently found to be due to the rapid hydrolysis of [3H]TMP to [3H]thiamine.


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 2.   Inhibition of [3H]TMP influx by unlabeled methotrexate (MTX) or thiamine. Cells at a density of 2 × 107 cells/ml were incubated in HBS for 25 min at 37°C. [3H]TMP (1 µM), with or without unlabeled MTX (100 µM) or thiamine (10 µM), was added to the cell suspension. Initial uptakes were measured over 25 or 40 s as shown in Fig. 1. Data are means ± SD of 3 independent experiments.

Comparison of TMP and thiamine influx in L1210 cells at physiological substrate concentrations. Because TMP and thiamine are present in plasma and cerebrospinal fluid at a concentration range of 3-50 nM (34), the relative contribution of TMP and thiamine delivery to cells was assessed by measuring influx of these two substrates at 50 nM over 40 s. TMP influx was ~70% that of thiamine influx (Table 1). More than half of TMP influx was blocked by 100 µM MTX, and half of the remaining activity was further inhibited by 10 µM thiamine. The latter is attributed to inhibition of influx of thiamine derived from the hydrolysis of TMP (see Chemical instability of TMP in vitro). Thiamine influx at 50 nM was decreased by ~75% in the presence of unlabeled thiamine. Thus, in L1210 cells, influx of TMP via RFC1 was approximately half the carrier-mediated influx of thiamine at a physiological substrate concentration.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Comparison of TMP and thiamine influx in L1210 cells at a substrate concentration of 50 nM

Chemical instability of TMP in vitro. Chemical stability of TMP was assessed under the same conditions as in the influx experiments. Cells in HBS at a density of 2 × 107 cells/ml were incubated in the transport buffer for 25 min, after which [3H]TMP was added and the identity of extracellular radiolabel was assessed by thin-layer chromatography. As indicated in Fig. 3A, 80% of [3H]TMP was hydrolyzed to thiamine by 1 min of incubation. By 3 min, no TMP could be detected. When supernatant was separated from the suspension after 25 min of incubation, after which [3H]TMP was added, hydrolysis of TMP was also detected but at a reduced rate. There was no detectable degradation of TMP in HBS alone over a 5-min incubation. These findings suggest that TMP hydrolysis is mediated by phosphatases associated with the L1210 cell membrane and secreted into the suspension buffer.


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 3.   Dependence of TMP hydrolysis on incubation time, cell density, and substrate concentration. A: L1210 cells at a density of 2 × 107 cells/ml were incubated in HBS for 25 min at 37°C. A portion of cell suspension was centrifuged to obtain cell-free supernatant. [3H]TMP (1 µM) was added to either cell suspension or supernatant. TMP hydrolysis was stopped by addition of trichloroacetic acid. Thiamine and its derivatives were analyzed by thin-layer chromatography as described in MATERIALS AND METHODS. B: L1210 cells at a density of from 1.5 × 105 to 2 × 107 cells/ml were incubated in HBS at 37°C for 25 min and exposed to 1 µM [3H]TMP for 2 min. Intracellular tritium was analyzed. C: L1210 cells at a density of 2 × 107 cells/ml were incubated in HBS for 25 min at 37°C and exposed to different concentrations of [3H]TMP (20 nM to 100 µM) for 40 s. Data in all 3 panels are means ± SD of 3 independent experiments.

The rate of TMP hydrolysis was determined both as a function of cell density and substrate concentration. After a 2-min incubation with 105 cells/ml, 90% of TMP was unchanged. However, as the cell density was increased, the rate of hydrolysis was also markedly increased (Fig. 3B). The extent of TMP hydrolysis was also dependent on the concentration of substrate (Fig. 3C). The percentage of TMP hydrolysis was constant at ~70% over a concentration range of from 10 nM to 1 µM but decreased to 10% as the TMP concentration increased from 1 to 100 µM, suggesting saturation of the phosphatase(s).

Affinity of RFC1 for TMP. In measuring the affinity of RFC1 for TMP, R16 cells were used to maximize the component of influx attributable to TMP uptake via RFC1 because this carrier is highly expressed in this cell line. Also, MTX influx can be measured over a very short interval, minimizing hydrolysis of TMP. As shown in Fig. 4, [3H]MTX influx was decreased by 35, 49, and 59% in the presence of 15, 30, and 45 µM TMP, respectively. Assuming that TMP inhibits MTX influx competitively as described for TPP (37), the influx Ki of TMP for RFC1 was calculated to be 25.9 ± 0.9 µM based on an MTX influx Km of 5.8 µM in R16 cells (39).


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 4.   Effect of TMP on MTX influx in R16 cells. R16 cells at a density of 2 × 107 cells/ml were incubated in HBS buffer for 25 min at 37°C before 1 µM [3H]MTX, with or without 15, 30, or 45 µM unlabeled TMP, was added to initiate uptake. Data are means ± SD of 3 independent experiments.

Formation of [3H]TMP metabolites in R16 cells. Most of the thiamine transported into L1210 cells is converted to TPP, the active cofactor for several enzymes (37). As indicated in Fig. 5, about half of TMP transported into R16 cells was converted to thiamine within 1 min; 15% was identified as TPP and 35% remained as TMP. MTX, at a concentration of 100 µM, suppressed intracellular levels of thiamine, TMP, and TPP; the greatest suppression was observed for thiamine. In contrast, 10 µM thiamine had no effect on total cell tritium, with only negligible effects on cell levels of tritiated TMP or TPP. These observations indicate that [3H]thiamine appearing in cells exposed to [3H]TMP was due to TMP hydrolysis to thiamine within cells and not to influx of thiamine from the extracellular compartment. These data also demonstrate that transport of TMP via RFC1 results in the delivery of a precursor of the active thiamine cofactor TPP into cells.


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 5.   Accumulation of tritiated thiamine, TMP, and thiamine pyrophosphate (TPP) in R16 cells upon exposure to [3H]TMP. After a 25-min incubation of R16 cells (2 × 107 cells/ml) in HBS at 37°C, [3H]TMP was added to cell suspension at a concentration of 1 µM, with or without 100 µM MTX or 10 µM thiamine. Incubation continued for 1 min. Total cell tritium was measured as in Transport studies, whereas the percentage of tritiated thiamine, TMP, and TPP was determined by thin-layer chromatography. Data are means ± SD of 3 independent experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Recent studies from this laboratory demonstrated that TPP is a good substrate for RFC1 and that its transport into cells correlates with the level of RFC1 expression (37). Because TPP is present exclusively in the intracellular compartment where it is formed by thiamine pyrophosphokinase, RFC1 action with respect to this substrate is asymmetrical. This asymmetry results in the unidirectional export of TPP, depression of the intracellular TPP level, and the hydrolysis of TPP to thiamine in the extracellular space. On the basis of this consideration, cells that express high levels of RFC1 would be at a disadvantage under conditions of thiamine deficiency.

We now demonstrate that RFC1 also transports TMP, a normal constituent of plasma and cerebrospinal fluid, present at concentrations nearly equivalent to thiamine. Once transported into cells, TMP is converted to thiamine by thiamine monophosphatase, followed by phosphorylation of thiamine to TPP by thiamine pyrophosphokinase (Fig. 6). Hence, the level of RFC1 expression will determine the rate of delivery of this thiamine precursor into cells. Under conditions of thiamine deficiency, when blood levels of thiamine and TMP are low, this added transport via RFC1 might not be important. However, when the thiamine carrier THTR-1 is not functional as occurs in TRMA (33), RFC1 could be an important alternative route for thiamine uptake into cells, along with other potential transport pathways for thiamine such as THTR-2 (23) (Fig. 6). Indeed, the pattern of RFC1 tissue expression may be one of the determinants of the selective organ and metabolic defects associated with TRMA-macrocytic anemia, sensoneural deafness, and diabetes mellitus. It is interesting in this regard that dietary thiamine deficiency leads to cardiac (beriberi) and brain (Wernicke's syndrome) abnormalities, whereas TRMA leads to functional defects in only a limited number of other tissues.


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 6.   A model for transcellular flux and intracellular metabolism of thiamine, TMP, and TPP in nonpolarized cells. Thiamine (T) uptake and efflux are mediated by the facilitative thiamine transporters THTR-1 and THTR-2, whereas influx and efflux of TMP are mediated by RFC1. Because TPP is not present extracellularly, RFC1-mediated TPP transport is unidirectional out of cells. TPP is synthesized from thiamine by thiamine pyrophosphokinase (TPK). TPP is hydrolyzed by thiamine pyrophosphatase (TPPase) to TMP, which is further hydrolyzed to thiamine by thiamine monophosphatase (TMPase). TPP is also hydrolyzed to thiamine by other phosphatases (26).

The high level of expression of RFC1 in the apical brush border of the choroid plexus suggests that this may be an important route of TMP transport, because TMP is present at higher levels than is thiamine in cerebrospinal fluid (35). High expression of RFC1 in the brush border of small intestinal cells could be a route for absorption of TMP and TPP present in the gut (35). However, because of the high phosphatase level in intestinal brush border, these phosphorylated compounds are probably hydrolyzed to thiamine before they are absorbed. Our results suggest that at least part of TMP transport across rat everted jejunal sacs reported previously could be mediated by RFC1 (11). However, RFC1 expressed in intestine has a low pH optimum (4, 18, 24), and the extent to which TMP could be transported by this mechanism under these conditions remains unclear. On the other hand, the transport activity of THTR-1 and THTR-2 is optimal at high pH (6), so that the extent to which thiamine is transported via this carrier is also uncertain at the pH present in the microenvironment of intestinal villi (21).

Unlike THTR-1, RFC1 is an anion exchanger. Folates are bivalent anions with negative charges at the two carboxyl groups of the glutamate moiety. RFC1 is inhibited by a variety of inorganic and organic anions, including the organic phosphates such as the adenine nucleotides (12, 16, 36). It is the asymmetrical distribution of organic phosphates across cell membranes, with their high intracellular electrochemical potential, that is thought to be the energy source for the uphill transport of folates into many mammalian cells (12, 16, 36). Because TPP and TMP have comparable affinities for RFC1 but differ in total charge, the data suggest that the thiamine moiety may play an important role in binding to the carrier. On the other hand, thiamine itself is not transported by RFC1 at all, likely due to thiamine's positive charge, which is reversed by phosphorylation. Further evidence for the role of this moiety in binding to RFC1 comes from the observation that other organic anions, such as ADP and AMP, that share the same charge have a much lower affinity for RFC1 (15). It remains to be determined whether there are critical charged residues in RFC1 vs. THTR-1 and THTR-2 that determine the charge specificity of these carriers for folates and thiamine as has been demonstrated for the rat organic anion transporter rOAT3 (8).

These studies demonstrate the high degree of chemical instability of TMP in cell suspensions. Hence, it is unclear why this substance is present at levels comparable to thiamine in plasma. It is possible that TMP is bound to circulating proteins that protect it from degradation and/or that this component is restored by the slow but constant exit of TMP and/or TPP from cells with subsequent hydrolysis by thiamine pyrophosphatase (Fig. 6). These findings indicate that previous suggestions that thiamine and TMP share the same transport route, based on TMP competition with radiolabeled thiamine for transport, are erroneous (1, 3, 13, 32). It is highly likely that apparent inhibitory effects are due to rapid hydrolysis of TMP to thiamine that, in turn, inhibits radiotracer thiamine uptake into cells. It is clear that any studies focused on the transport of TMP or TPP must be designed to minimize, and correct for, the rapid degradation of these compounds to thiamine.


    ACKNOWLEDGEMENTS

This work was supported by National Cancer Institute Grant CA-82621.


    FOOTNOTES

Address for reprint requests and other correspondence: I. D. Goldman, Albert Einstein College of Medicine Cancer Research Center, Chanin 2, 1300 Morris Park Ave., Bronx, NY 10461 (E-mail: igoldman{at}aecom.yu.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published February 13, 2002;10.1152/ajpcell.00547.2001

Received 15 November 2001; accepted in final form 8 February 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Bettendorff, L, and Wins P. Mechanism of thiamine transport in neuroblastoma cells. Inhibition of a high affinity carrier by sodium channel activators and dependence of thiamine uptake on membrane potential and intracellular ATP. J Biol Chem 269: 14379-14385, 1994[Abstract/Free Full Text].

2.   Brigle, KE, Spinella MJ, Sierra EE, and Goldman ID. Characterization of a mutation in the reduced folate carrier in a transport defective L1210 murine leukemia cell line. J Biol Chem 270: 22974-22979, 1995[Abstract/Free Full Text].

3.   Casirola, D, Ferrari G, Gastaldi G, Patrini C, and Rindi G. Transport of thiamine by brush-border membrane vesicles from rat small intestine. J Physiol 398: 329-339, 1988[Abstract].

4.   Chiao, JH, Roy K, Tolner B, Yang CH, and Sirotnak FM. RFC-1 gene expression regulates folate absorption in mouse small intestine. J Biol Chem 272: 11165-11170, 1997[Abstract/Free Full Text].

5.   Diaz, GA, Banikazemi M, Oishi K, Desnick RJ, and Gelb BD. Mutations in a new gene encoding a thiamine transporter cause thiamine-responsive megaloblastic anaemia syndrome. Nat Genet 22: 309-312, 1999[ISI][Medline].

6.   Dutta, B, Huang W, Molero M, Kekuda R, Leibach FH, Devoe LD, Ganapathy V, and Prasad PD. Cloning of the human thiamine transporter, a member of the folate transporter family. J Biol Chem 274: 31925-31929, 1999[Abstract/Free Full Text].

7.   Eudy, JD, Spiegelstein O, Barber RC, Wlodarczyk BJ, Talbot J, and Finnell RH. Identification and characterization of the human and mouse SLC19A3 gene: a novel member of the reduced folate family of micronutrient transporter genes. Mol Genet Metab 71: 581-590, 2000[ISI][Medline].

8.   Feng, B, Dresser MJ, Shu Y, Johns SJ, and Giacomini KM. Arginine 454 and lysine 370 are essential for the anion specificity of the organic anion transporter, rOAT3. Biochemistry 40: 5511-5520, 2001[ISI][Medline].

9.   Fleming, JC, Tartaglini E, Steinkamp MP, Schorderet DF, Cohen N, and Neufeld EJ. The gene mutated in thiamine-responsive anaemia with diabetes and deafness (TRMA) encodes a functional thiamine transporter. Nat Genet 22: 305-308, 1999[ISI][Medline].

10.   Fry, DW, Yalowich JC, and Goldman ID. Rapid formation of poly-gamma-glutamyl derivatives of methotrexate and their association with dihydrofolate reductase as assessed by high pressure liquid chromatography in the Ehrlich ascited tumor cell in vitro. J Biol Chem 257: 1890-1896, 1982[Free Full Text].

11.   Gastaldi, G, Casirola D, Patrini C, Ricci V, Laforenza U, Ferrari G, and Rindi G. Intestinal transport of thiamin and thiamin monophosphate in rat everted jejunal sacs: a comparative study using some potential inhibitors. Arch Int Physiol Biochim Biophys 96: 223-230, 1988[ISI].

12.   Goldman, ID. The characteristics of the membrane transport of amethopterin and the naturally occurring folates. Ann NY Acad Sci 186: 400-422, 1971[ISI][Medline].

13.   Grassl, SM. Thiamine transport in human placental brush border membrane vesicles. Biochim Biophys Acta 1371: 213-222, 1998[ISI][Medline].

14.   Gritli, S, Omar S, Tartaglini E, Guannouni S, Fleming JC, Steinkamp MP, Berul CI, Hafsia R, Jilani SB, Belhani A, Hamdi M, and Neufeld EJ. A novel mutation in the SLC19A2 gene in a Tunisian family with thiamine-responsive megaloblastic anaemia, diabetes and deafness syndrome. Br J Haematol 113: 508-513, 2001[ISI][Medline].

15.   Henderson, GB, Grzelakowska-Sztabert B, Zevely EM, and Huennekens FM. Binding properties of the 5-methyltetrahydrofolate/methotrexate transport system in L1210 cells. Arch Biochem Biophys 202: 144-149, 1980[ISI][Medline].

16.   Henderson, GB, and Zevely EM. Structural requirements for anion substrates of the methotrexate transport system of L1210 cells. Arch Biochem Biophys 221: 438-446, 1983[ISI][Medline].

17.   Kril, JJ. Neuropathology of thiamine deficiency disorders. Metab Brain Dis 11: 9-17, 1996[ISI][Medline].

18.   Kumar, CK, Nguyen TT, Gonzales FB, and Said HM. Comparison of intestinal folate carrier clone expressed in IEC-6 cells and in Xenopus oocytes. Am J Physiol Cell Physiol 274: C289-C294, 1998[Abstract/Free Full Text].

19.   Labay, V, Raz T, Baron D, Mandel H, Williams H, Barrett T, Szargel R, McDonald L, Shalata A, Nosaka K, Gregory S, and Cohen N. Mutations in SLC19A2 cause thiamine-responsive megaloblastic anaemia associated with diabetes mellitus and deafness. Nat Genet 22: 300-304, 1999[ISI][Medline].

20.   Levorato, C, and Cima L. Thin-layer chromatography and determination of thiamine salts, phosphoric esters, disulphides, and their respective thiochromes. J Chromatogr 32: 771-773, 1968[Medline].

21.   Lucas, ML, Schneider W, Haberich FJ, and Blair JA. Direct measurement by pH-microelectrode of the pH microclimate in rat proximal jejunum. Proc R Soc Lond B Biol Sci 192: 39-48, 1975[ISI][Medline].

22.   Matherly, LH. Molecular and cellular biology of the human reduced folate carrier. Prog Nucleic Acid Res Mol Biol 67: 131-162, 2001[ISI][Medline].

23.   Rajgopal, A, Edmondson A, Goldman ID, and Zhao R. SLC19A3 encodes a second thiamine transporter, ThTr2. Biochim Biophys Acta 1537: 175-178, 2001[ISI][Medline].

24.   Rajgopal, A, Sierra EE, Zhao R, and Goldman ID. Expression of the reduced folate carrier SLC19A1 in IEC-6 cells results in two distinct transport activities. Am J Physiol Cell Physiol 281: C1579-C1586, 2001[Abstract/Free Full Text].

25.   Raz, T, Labay V, Baron D, Szargel R, Anbinder Y, Barrett T, Rabl W, Viana MB, Mandel H, Baruchel A, Cayuela JM, and Cohen N. The spectrum of mutations, including four novel ones, in the thiamine-responsive megaloblastic anemia gene SLC19A2 of eight families. Hum Mutat 16: 37-42, 2000[ISI][Medline].

26.   Rindi, G, and Laforenza U. Thiamine intestinal transport and related issues: recent aspects. Proc Soc Exp Biol Med 224: 246-255, 2000[Abstract/Free Full Text].

27.   Rindi, G, Reggiani C, Patrini C, Gastaldi G, and Laforenza U. Effect of ethanol on the in vivo kinetics of thiamine phosphorylation and dephosphorylation in different organs of rat-II. Acute effects. Alcohol Alcohol 27: 505-522, 1992[Abstract].

28.   Rogers, LE, Porter FS, and Sidbury JBJ Thiamine-responsive megaloblastic anemia. J Pediatr 74: 494-504, 1969[ISI][Medline].

29.  Saier MH Jr,. Beatty JT, Goffeau A, Harley KT, Heijne WH, Huang SC, Jack DL, Jahn PS, Lew K, Liu J, Pao SS, Paulsen IT, Tseng TT, and Virk PS. The major facilitator superfamily. J Mol Microbiol Biotechnol 1: 257-279, 1999.

30.   Scharfe, C, Hauschild M, Klopstock T, Janssen AJ, Heidemann PH, Meitinger T, and Jaksch M. A novel mutation in the thiamine responsive megaloblastic anaemia gene SLC19A2 in a patient with deficiency of respiratory chain complex I. J Med Genet 37: 674-679, 2000[Abstract/Free Full Text].

31.   Schuetz, JD, Matherly LH, Westin EH, and Goldman ID. Evidence for a functional defect in the translocation of the methotrexate transport carrier in a methotrexate-resistant murine L1210 leukemia cell line. J Biol Chem 263: 9840-9847, 1988[Abstract/Free Full Text].

32.   Spector, R. Thiamine transport in the central nervous system. Am J Physiol 230: 1101-1107, 1976[Abstract/Free Full Text].

33.   Stagg, AR, Fleming JC, Baker MA, Sakamoto M, Cohen N, and Neufeld EJ. Defective high-affinity thiamine transporter leads to cell death in thiamine-responsive megaloblastic anemia syndrome fibroblasts. J Clin Invest 103: 723-729, 1999[Abstract/Free Full Text].

34.   Tallaksen, CM, Bohmer T, Karlsen J, and Bell H. Determination of thiamin and its phosphate esters in human blood, plasma, and urine. Methods Enzymol 279: 67-74, 1997[ISI][Medline].

35.   Wang, Y, Zhao R, Russell RG, and Goldman ID. Localization of the murine reduced folate carrier as assessed by immunohistochemical analysis. Biochim Biophys Acta 1513: 49-54, 2001[ISI][Medline].

36.   Yang, CH, Sirotnak FM, and Dembo M. Interaction between anions and the reduced folate/methotrexate transport system in L1210 cell plasma membrane vesicles: directional symmetry and anion specificity for differential mobility of loaded and unloaded carrier. J Membr Biol 79: 285-292, 1984[ISI][Medline].

37.   Zhao, R, Gao F, Wang Y, Diaz GA, Gelb BD, and Goldman ID. Impact of the reduced folate carrier on the accumulation of active thiamin metabolites in murine leukemia cells. J Biol Chem 276: 1114-1118, 2000[Medline].

38.   Zhao, R, Russell RG, Wang Y, Liu L, Gao F, Kneitz B, Edelman W, and Goldman ID. Rescue of embryonic lethality in reduced folate carrier-deficient mice by maternal folic acid supplementation reveals early neonatal failure of hematopoietic organs. J Biol Chem 276: 10224-10228, 2001[Abstract/Free Full Text].

39.   Zhao, R, Seither R, Brigle KE, Sharina IG, Wang PJ, and Goldman ID. Impact of overexpression of the reduced folate carrier (RFC1), an anion exchanger, on concentrative transport in murine L1210 leukemia cells. J Biol Chem 272: 21207-21212, 1997[Abstract/Free Full Text].


Am J Physiol Cell Physiol 282(6):C1512-C1517
0363-6143/02 $5.00 Copyright © 2002 the American Physiological Society