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• W3090716937 abstract "SLC19A2 and SLC19A3, also known as thiamine transporters (THTR) 1 and 2, respectively, transport the positively charged thiamine (vitamin B1) into cells to enable its efficient utilization. SLC19A2 and SLC19A3 are also known to transport structurally unrelated cationic drugs, such as metformin, but whether this charge selectivity extends to other molecules, such as pyridoxine (vitamin B6), is unknown. We tested this possibility using Madin-Darby canine kidney II (MDCKII) cells and human embryonic kidney 293 (HEK293) cells for transfection experiments, and also using Caco-2 cells as human intestinal epithelial model cells. The stable expression of SLC19A2 and SLC19A3 in MDCKII cells (as well as their transient expression in HEK293 cells) led to a significant induction in pyridoxine uptake at pH 5.5 compared with control cells. The induced uptake was pH-dependent, favoring acidic conditions over neutral to basic conditions, and protonophore-sensitive. It was saturable as a function of pyridoxine concentration, with an apparent Km of 37.8 and 18.5 μm, for SLC19A2 and SLC19A3, respectively, and inhibited by the pyridoxine analogs pyridoxal and pyridoxamine as well as thiamine. We also found that silencing the endogenous SLC19A3, but not SLC19A2, of Caco-2 cells with gene-specific siRNAs lead to a significant reduction in carrier-mediated pyridoxine uptake. These results show that SLC19A2 and SLC19A3 are capable of recognizing/transporting pyridoxine, favoring acidic conditions for operation, and suggest a possible role for these transporters in pyridoxine transport mainly in tissues with an acidic environment like the small intestine, which has an acidic surface microclimate. SLC19A2 and SLC19A3, also known as thiamine transporters (THTR) 1 and 2, respectively, transport the positively charged thiamine (vitamin B1) into cells to enable its efficient utilization. SLC19A2 and SLC19A3 are also known to transport structurally unrelated cationic drugs, such as metformin, but whether this charge selectivity extends to other molecules, such as pyridoxine (vitamin B6), is unknown. We tested this possibility using Madin-Darby canine kidney II (MDCKII) cells and human embryonic kidney 293 (HEK293) cells for transfection experiments, and also using Caco-2 cells as human intestinal epithelial model cells. The stable expression of SLC19A2 and SLC19A3 in MDCKII cells (as well as their transient expression in HEK293 cells) led to a significant induction in pyridoxine uptake at pH 5.5 compared with control cells. The induced uptake was pH-dependent, favoring acidic conditions over neutral to basic conditions, and protonophore-sensitive. It was saturable as a function of pyridoxine concentration, with an apparent Km of 37.8 and 18.5 μm, for SLC19A2 and SLC19A3, respectively, and inhibited by the pyridoxine analogs pyridoxal and pyridoxamine as well as thiamine. We also found that silencing the endogenous SLC19A3, but not SLC19A2, of Caco-2 cells with gene-specific siRNAs lead to a significant reduction in carrier-mediated pyridoxine uptake. These results show that SLC19A2 and SLC19A3 are capable of recognizing/transporting pyridoxine, favoring acidic conditions for operation, and suggest a possible role for these transporters in pyridoxine transport mainly in tissues with an acidic environment like the small intestine, which has an acidic surface microclimate. Pyridoxine is one of the vitamin B6 compounds, which include the structurally analogous pyridoxal and pyridoxamine. Because of its hydrophilic nature as a water-soluble vitamin, it cannot easily permeate across the cellular membrane by simple diffusion and, hence, is required for intestinal absorption a specialized carrier-mediated transport system (1Hediger M.A. Clémençon B. Burrier R.E. Bruford E.A. The ABCs of membrane transporters in health and disease (SLC series): introduction.Mol. Aspects Med. 2013; 34 (23506860): 95-10710.1016/j.mam.2012.12.009Crossref PubMed Scopus (376) Google Scholar). Indeed, carrier-mediated transport of pyridoxine has been observed in the Caco-2 cell line, a human intestinal epithelial cell model (2Said H.M. Ortiz A. Ma T.Y. A carrier-mediated mechanism for pyridoxine uptake by human intestinal epithelial Caco-2 cells: regulation by a PKA-mediated pathway.Am. J. Physiol. Cell Physiol. 2003; 285 (12867360): C1219-C122510.1152/ajpcell.00204.2003Crossref PubMed Scopus (64) Google Scholar). Although transporters have been identified for a variety of water-soluble vitamins (1Hediger M.A. Clémençon B. Burrier R.E. Bruford E.A. The ABCs of membrane transporters in health and disease (SLC series): introduction.Mol. Aspects Med. 2013; 34 (23506860): 95-10710.1016/j.mam.2012.12.009Crossref PubMed Scopus (376) Google Scholar), none have been identified as yet for pyridoxine, although transport properties consistent with a carrier-mediated mechanism have been noted in many different cell types (3Said H.M. Ortiz A. Vaziri N.D. Mechanism and regulation of vitamin B6 uptake by renal tubular epithelia: studies with cultured OK cells.Am. J. Physiol. Renal Physiol. 2002; 282 (11832427): F465-F47110.1152/ajprenal.00267.2001Crossref PubMed Scopus (20) Google Scholar, 4Said Z.M. Subramanian V.S. Vaziri N.D. Said H.M. Pyridoxine uptake by colonocytes: a specific and regulated carrier-mediated process.Am. J. Physiol. Cell Physiol. 2008; 294 (18353902): C1192-C119710.1152/ajpcell.00015.2008Crossref PubMed Scopus (39) Google Scholar, 5Srinivasan P. Ramesh V. Wu J. Heskett C. Chu B.D. Said H.M. Pyridoxine and pancreatic acinar cells: transport physiology and effect on gene expression profile.Am. J. Physiol. Cell Physiol. 2019; 317: C1107-C111410.1152/ajpcell.00225.2019Crossref PubMed Scopus (6) Google Scholar). In the current study, we explored whether SLC19A2 and SLC19A3, known thiamine transporter 1 (THTR1) and THTR2 (6Dutta B. Huang W. Molero M. Kekuda R. Leibach F.H. Devoe L.D. Ganapathy V. Prasad P.D. Cloning of the human thiamine transporter, a member of the folate transporter family.J. Biol. Chem. 1999; 274 (10542220): 31925-3192910.1074/jbc.274.45.31925Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 7Rajgopal A. Edmondnson A. Goldman I.D. Zhao R. SLC19A3 encodes a second thiamine transporter ThTr2.Biochim. Biophys. Acta. 2001; 1537: 175-17810.1016/S0925-4439(01)00073-4Crossref PubMed Scopus (127) Google Scholar), respectively, can also transport pyridoxine, based upon the fact that both pyridoxine and thiamine (vitamin B1) have cationic charge and that other structurally unrelated cationic drugs, such as metformin, fedratinib, and trimethoprim, are substrates for these transporters (8Liang X. Chien H.C. Yee S.W. Giacomini M.M. Chen E.C. Piao M. Hao J. Twelves J. Lepist E.I. Ray A.S. Giacomini K.M. Metformin is a substrate and inhibitor of the human thiamine transporter, THTR-2 (SLC19A3).Mol. Pharm. 2015; 12 (26528626): 4301-431010.1021/acs.molpharmaceut.5b00501Crossref PubMed Scopus (62) Google Scholar, 9Giacomini M.M. Hao J. Liang X. Chandrasekhar J. Twelves J. Whitney J.A. Lepist E.I. Ray A.S. Interaction of 2,4-diaminopyrimidine-containing drugs including fedratinib and trimethoprim with thiamine transporters.Drug Metab. Dispos. 2017; 45 (27803021): 76-8510.1124/dmd.116.073338Crossref PubMed Scopus (19) Google Scholar). The data indicate that pyridoxine is indeed also their substrate. We first examined the uptake of pyridoxine at its trace concentration of 5 nm in transient transfectant human embryonic kidney 293 (HEK293) cells expressing each of SLC19A2 and SLC19A3. The trace concentration was used so that the potential transport activities could be detected most effectively, as transporters generally operate more efficiently at lower concentrations. This low concentration is also physiologically relevant, as pyridoxin concentration can be in the nanomolar range at the lowest end in body fluids, typically in plasma (10Lumeng L. Lui A. Li T.K. Plasma content of B6 vitamers and its relationship to hepatic vitamin B6 metabolism.J. Clin. Invest. 1980; 66 (7419716): 688-69510.1172/JCI109906Crossref PubMed Scopus (82) Google Scholar). As shown in Fig. 1, the uptake of pyridoxine was found to be much greater in cells expressing those SLC19As than in mock cells at an acidic pH of 5.5, suggesting that those SLC19As can transport pyridoxine efficiently. On the other hand, pyridoxine uptake was not altered by transient introduction of SLC19A1, suggesting that this transporter, which is another member of SLC19A family and known as reduced folate carrier 1, does not have such a capability for pyridoxine transport. For detailed characterization of pyridoxine transport by SLC19A2 and SLC19A3, we prepared stable transfectant Madin-Darby canine kidney II (MDCKII) cells expressing each of them. As shown in Fig. 2, the uptake of pyridoxine (5 nm) was much greater in cells expressing those SLC19As than in mock cells at pH 5.5, further demonstrating the pyridoxine transport capability of both transporters, which was initially found in the transient transfectant HEK293 cells. As pyridoxine uptake increased in proportion to time up to 3 min in all the transfectant and mock cells, the uptake period was set to be 3 min in subsequent experiments for the evaluation of pyridoxine transport by those SLC19As in the initial uptake phase. The uptake of pyridoxine was almost equilibrated at 30 min in both SLC19A2-transfected and SLC19A3-transfected cells. Using the cellular volumes of 6.5 ± 1.1 and 6.4 ± 1.1 μl/mg of protein (mean ± S.E., n = 3), respectively, estimated from the equilibrium uptake of 3-O-methyl-d-glucose (3-O-MG) in those cells, the apparent intracellular concentrations of pyridoxine were estimated to be 43 and 52 nm, respectively. They were about 10 times higher than the pyridoxine concentration in the extracellular solution, suggesting that those SLC19As could possibly operate in an active transport mode that enables pyridoxine to be concentrated in the cells. In this analysis, the cellular volume was estimated as the uptake/concentration ratio of 3-O-MG (11Fujimoto N. Inoue K. Hayashi Y. Yuasa H. Glycerol uptake in HCT-15 human colon cancer cell line by Na+-dependent carrier-mediated transport.Biol. Pharm. Bull. 2006; 29 (16394529): 150-15410.1248/bpb.29.150Crossref PubMed Scopus (14) Google Scholar), as the uptake of this unmetabolizable analog of d-glucose could be quickly equilibrated in MDCKII cells at the intracellular concentration equal to the concentration in the extracellular solution by nonconcentrative transport, in which facilitative glucose transporters have been suggested to be involved (12Pascoe W.S. Inukai K. Oka Y. Slot J.W. James D.E. Differential targeting of facilitative glucose transporters in polarized epithelial cells.Am. J. Physiol. Cell Physiol. 1996; 271 (8769994): C547-C55410.1152/ajpcell.1996.271.2.C547Crossref PubMed Google Scholar, 13Quan Y. Jin Y. Faria T.N. Tilford C.A. He A. Wall D.A. Smith R.L. Vig B.S. Expression profile of drug and nutrient absorption related genes in Madin-Darby Canine Kidney (MDCK) cells grown under differentiation conditions.Pharmaceutics. 2012; 4 (24300234): 314-33310.3390/pharmaceutics4020314Crossref PubMed Scopus (18) Google Scholar). Consistent with that suggestion, 3-O-MG uptake was confirmed to be Na+-independent and quite rapid, being equilibrated within 10 min, in those transfectant MDCKII cells. Both SLC19A2 and SLC19A3 have been reported to operate in a pH-dependent manner, favoring neutral to basic conditions over acidic conditions, for thiamine transport (6Dutta B. Huang W. Molero M. Kekuda R. Leibach F.H. Devoe L.D. Ganapathy V. Prasad P.D. Cloning of the human thiamine transporter, a member of the folate transporter family.J. Biol. Chem. 1999; 274 (10542220): 31925-3192910.1074/jbc.274.45.31925Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 7Rajgopal A. Edmondnson A. Goldman I.D. Zhao R. SLC19A3 encodes a second thiamine transporter ThTr2.Biochim. Biophys. Acta. 2001; 1537: 175-17810.1016/S0925-4439(01)00073-4Crossref PubMed Scopus (127) Google Scholar). However, for pyridoxine transport, they were both found to operate in an opposite manner of pH dependence, favoring acidic conditions over neutral to basic conditions, as indicated by an increase in the specific uptake of pyridoxine (5 nm) by each transporter with a decrease in pH in stable transfectant MDCKII cells (Fig. 3). Moreover, the specific uptake by SLC19A2 and SLC19A3 were both extensively reduced by protonophores of carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and carbonyl cyanide m-chlorophenylhydrazone (CCCP), suggesting that they may operate for pyridoxine transport in a manner coupled with proton or, at least, facilitated by an inward proton gradient (Fig. 4). Supporting these additionally, the specific uptakes by both SLC19As were, as assessed using uptake solution in which NaCl was replaced with KCl, reduced by intracellular acidification for dissipation of H+ gradient by nigericin (10 μm), a K+/H+-exchanging protonophore, and also by an NH4Cl prepulse method, in which the cells were pretreated with substrate-free uptake solution containing NH4Cl (20 mm) and subsequently with the one free of NH4Cl (14Ohta K. Inoue K. Hayashi Y. Yuasa H. Molecular identification and functional characterization of rat multidrug and toxin extrusion type transporter 1 as an organic cation/H+ antiporter in the kidney.Drug Metab. Dispos. 2006; 34 (16928787): 1868-187410.1124/dmd.106.010876Crossref PubMed Scopus (68) Google Scholar). The values of specific uptake rate were 42.9 ± 2.0% of control (39.5 ± 2.3 fmol/min/mg of protein) and 38.6 ± 3.3% of control (71.6 ± 3.8 fmol/min/mg of protein), respectively, for SLC19A2 and SLC19A3 in the experiments using nigericin, and 62.9 ± 7.7% (47.6 ± 5.2 fmol/min/mg of protein) and 43.7 ± 6.6% of control (65.9 ± 11.2 fmol/min/mg of protein), respectively, in those by NH4Cl prepulse, where the values are presented as the mean ± S.E. (n = 4) and all the differences are significant at p < 0.05 compared with respective control values. It should also be noted in addition that pyridoxine, which is a basic compound with the pKa of 5.1 (15dos Santos T.D A.D. Costa D.O.D. Pita S.S.D.R. Semaan F.S. Potentiometric and conductimetric studies of chemical equilibria for pyridoxine hydrochloride in aqueous solutions: simple experimental determination of pKa values and analytical applications to pharmaceutical analysis.Eclet. Quim. J. 2010; 35: 81-8610.1590/S0100-46702010000400010Crossref Scopus (27) Google Scholar), becomes progressively cationized with a decrease in pH in the near neutral to acidic pH range, coinciding to the pH-dependent increase in the specific uptake of pyridoxine. Hence, the increase in the cationized fraction of pyridoxine may also be involved in that, implicating the preferential transport of its cationized form by those SLC19As as a possibility. Thus, those SLC19As were suggested to operate in a different mode for pyridoxine than for thiamine, discriminating them. Although the mechanism of the substrate-dependent difference in pH dependence is unknown at this time, we conducted all the other experiments in this study at pH 5.5 as an acidic pH where both SLC19As can operate efficiently.Figure 4Effect of protonophores on pyridoxine uptake by SLC19A2 and SLC19A3 stably expressed in MDCKII cells. The specific uptake of [3H]pyridoxine (5 nm) by SLC19A2 (A) and SLC19A3 (B) was evaluated for 3 min at pH 5.5 and 37 °C in the presence of a protonophore (100 μm), or in its absence (control), after pretreatment for 5 min with, or without, the protonophore under the same condition. The control values were 47.5 and 87.8 fmol/min/mg of protein, for SLC19A2 and SLC19A3, respectively. Data are presented as the mean ± S.D. (n = 4). *, p < 0.05 compared with control, as assessed by ANOVA followed by Dunnett's test.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The replacement of NaCl in the medium with various salts (KCl, Na-gluconate, K-gluconate, and mannitol) had insignificant or only modest impacts on the specific uptake of pyridoxine (5 nm) by SLC19A2 (Fig. 5A) and SLC19A3 (Fig. 5B). The specific uptake by SLC19A2 was reduced slightly by replacement of NaCl with KCl and, to a comparable extent, by replacement with mannitol, suggesting that Na+ may be slightly involved in the SLC19A2 operation but Cl− may not. The specific uptake by SLC19A3 was reduced modestly by replacement of NaCl with mannitol, but not by replacement with KCl, suggesting that Cl− may be slightly involved in the SLC19A3 operation but Na+ may not. However, overall, such effects of those ions seemed to be only modest, suggesting at least that both SLC19A2 and SLC19A3 are unlikely to operate in a manner coupled with Na+ or Cl− for pyridoxine transport. To examine a possibility that some other pyridoxine-related compounds may also be recognized by SLC19A2 and SLC19A3, we here assessed the effect of such compounds (200 μm) on the specific uptake of pyridoxine (5 nm) by those SLC19As in stable transfectant MDCKII cells. As shown in Fig. 6A, the specific uptake by SLC19A2 was extensively inhibited by pyridoxamine and, to a lesser extent, by 4-deoxypyridoxine. Pyridoxal, pyridoxal 5-phosphate (PLP), and 4-pyridoxic acid were, however, found not to inhibit the specific uptake. SLC19A3 exhibited almost the same characteristics, although pyridoxal exhibited a slight inhibitory effect as a minor difference (Fig. 6B). These results suggested the potential interaction of pyridoxamine and 4-deoxypyridoxine with both SLC19As and of pyridoxal with SLC19A3 to elicit the inhibitory effects. Among water-soluble vitamins, the SLC19A2/3 substrate of thiamine was an only and potent inhibitor for both SLC19A2 and SLC19A3 (Fig. 7). All the other vitamins were confirmed not to inhibit the specific uptake by either of those SLC19As. As a note, specific transporters have already been identified for almost all of them, with the only exception being nicotinamide. Focusing on thiamine and the vitamin B6 compounds of pyridoxal and pyridoxamine, analyses were conducted for detailed characterization of their concentration-dependent inhibitory effects on the specific uptake of pyridoxine (5 nm) by SLC19A2 and SLC19A3, as shown in Fig. 8 and Table 1. The IC50 values of thiamine, the most potent inhibitor for the both, were comparable, being 0.71 and 0.99 μm, respectively, for SLC19A2 and SLC19A3. Those of pyridoxamine, the second most potent of the two, were quite different, being much higher for SLC19A2 (55.16 μm) than for SLC19A3 (2.97 μm). Those of pyridoxal, the least potent of the two, were relatively close, although being somewhat higher for SLC19A2 (1.49 mm) than for SLC19A3 (0.52 mm). The IC50 of pyridoxal for SLC19A2 was high enough not to elicit detectable inhibition at its concentration of 200 μm, whereas that for SLC19A3 was at a level sufficient to elicit a modest inhibition, consistent with the results in Fig. 6. All the Hill coefficient (n) values were comparable with unity, suggesting the competitive or noncompetitive type of inhibition conforming to the Michaelis-Menten model.Table 1Parameters for the inhibition of SLC19A2- and SLC19A3-mediated pyridoxine transport by selected compounds in stable transfectant MDCKII cellsCompoundTransporternIC50KiThiamineSLC19A20.84 ± 0.070.71 ± 0.08 µm1.11 ± 0.12 µmSLC19A31.20 ± 0.120.99 ± 0.14 µm0.86 ± 0.12 µmPyridoxamineSLC19A20.96 ± 0.0155.16 ± 5.64 µm90.08 ± 7.77 µmSLC19A30.94 ± 0.122.97 ± 0.85 µm1.57 ± 0.32 µmPyridoxalSLC19A20.97 ± 0.031.49 ± 0.22 mm2.63 ± 0.50 mmSLC19A30.94 ± 0.050.52 ± 0.04 mm0.38 ± 0.03 mm Open table in a new tab Both SLC19A2 and SLC19A3 were found to mediate pyridoxine transport in a highly saturable manner, as indicated by the concentration-dependent profiles of specific pyridoxine uptake (Fig. 9). According to kinetic analyses using the Michaelis-Menten model, the Vmax and Km were 332 pmol/min/mg of protein and 37.8 μm, respectively, for SLC19A2, and 264 pmol/min/mg of protein and 18.5 μm, respectively, for SLC19A3 (Table 2). Thus, the Km values for those SLC19As were comparable, suggesting their comparable affinities for pyridoxine. It should also be noted that the pyridoxine concentration of 5 nm, which was used in regular experiments, was confirmed to be in the linear phase of transport kinetics at concentrations much below the Km values for both SLC19As, where their transport activities were highest and could be evaluated most effectively.Table 2Parameters for the SLC19A2- and SLC19A3-mediated transport of pyridoxine and thiamine in stable transfectant MDCKII cellsCompoundTransporterpHVmaxKmKipmol/min/mg of proteinµmµmPyridoxineSLC19A25.5332 ± 2337.8 ± 6.749.6 ± 1.7SLC19A35.5264 ± 2918.5 ± 1.513.8 ± 2.0ThiamineSLC19A27.4106 ± 92.83 ± 0.42-5.5100 ± 103.66 ± 0.68aStatistical differences were assessed by Student's t test. p < 0.1 compared with the value at pH 7.4.1.11 ± 0.12SLC19A37.465.9 ± 9.52.36 ± 0.37-5.526.9 ± 0.8bStatistical differences were assessed by Student's t test. p < 0.05 compared with the value at pH 7.4.2.33 ± 0.270.86 ± 0.12a Statistical differences were assessed by Student's t test. p < 0.1 compared with the value at pH 7.4.b Statistical differences were assessed by Student's t test. p < 0.05 compared with the value at pH 7.4. Open table in a new tab The profiles of the inhibition of the specific uptake of pyridoxine by thiamine, pyridoxamine, and pyridoxal were all found to conform to the competitive inhibition model (Fig. 9), with the values of inhibition constant (Ki) being comparable with the respective IC50 values determined in Fig. 8 (Table 1). These results suggest that the vitamin B6 compounds of pyridoxamine and pyridoxal may be competing substrates recognized at the pyridoxine-binding site in both SLC19As. Cationic characteristic in addition to structural similarity to pyridoxine may be important for pyridoxine-related compounds to be recognized by SLC19A2 and SLC19A3, as PLP and 4-pyridoxic acid, which are less cationic pyridoxine derivatives because of having anionic moieties, did not inhibit the specific uptake of pyridoxine by either of the SLC19As (Fig. 6), suggesting no affinity for them. Thus, those SLC19As may have some multispecific characteristics within that limitation. 4-Deoxypyridoxine, a pyridoxine derivative that does not have an acidic moiety, may also be recognized as a competing substrate by those SLC19As, as this pyridoxine derivative inhibited specific pyridoxine uptake by them (Fig. 6). To further clarify the interrelation between pyridoxine transport and thiamine transport in the operation of each of SLC19A2 and SLC19A3, we examined the effect of pyridoxine on thiamine transport by those SLC19As. The uptake of thiamine was first examined at a trace concentration of 5 nm, the same low concentration as that in regular assessments of pyridoxine transport, and at pH 7.4, as those SLC19As have been reported to operate more efficiently under neutral to basic conditions than acidic conditions (6Dutta B. Huang W. Molero M. Kekuda R. Leibach F.H. Devoe L.D. Ganapathy V. Prasad P.D. Cloning of the human thiamine transporter, a member of the folate transporter family.J. Biol. Chem. 1999; 274 (10542220): 31925-3192910.1074/jbc.274.45.31925Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 7Rajgopal A. Edmondnson A. Goldman I.D. Zhao R. SLC19A3 encodes a second thiamine transporter ThTr2.Biochim. Biophys. Acta. 2001; 1537: 175-17810.1016/S0925-4439(01)00073-4Crossref PubMed Scopus (127) Google Scholar). As shown in Fig. 10, the uptake was much greater in cells expressing those SLC19As than in mock cells, demonstrating their known ability to transport thiamine. As thiamine uptake increased in proportion to time up to 1 min in all the transfectant and mock cells, the uptake period was set to be 1 min in subsequent experiments for the evaluation of thiamine transport by those SLC19As in the initial uptake phase. The specific uptakes of thiamine (5 nm) by SLC19A2 and SLC19A3 were smaller at pH 5.5 than at pH 7.4 by 34 and 64%, respectively (Table 3). These moderately pH-dependent characteristics of thiamine transport were in agreement with those reported earlier (6Dutta B. Huang W. Molero M. Kekuda R. Leibach F.H. Devoe L.D. Ganapathy V. Prasad P.D. Cloning of the human thiamine transporter, a member of the folate transporter family.J. Biol. Chem. 1999; 274 (10542220): 31925-3192910.1074/jbc.274.45.31925Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 7Rajgopal A. Edmondnson A. Goldman I.D. Zhao R. SLC19A3 encodes a second thiamine transporter ThTr2.Biochim. Biophys. Acta. 2001; 1537: 175-17810.1016/S0925-4439(01)00073-4Crossref PubMed Scopus (127) Google Scholar). The inhibitory effects of pyridoxine (200 μm) on the specific uptakes of thiamine by SLC19A2 and SLC19A3 were quite extensive, reducing them to 24.0 and 8.9% of control, respectively, at pH 5.5, whereas its inhibitory effects were insignificant or only minimal at pH 7.4. These pH-dependent characteristics of the effect of pyridoxine on thiamine transport were consistent with those of pyridoxine transport by those SLC19As, which were indicative of the higher interaction of pyridoxine with them for its greater transport at pH 5.5 than at pH 7.4 (Fig. 3).Table 3Effect of pyridoxine on thiamine uptake by SLC19A2 and SLC19A3 stably expressed in MDCKII cellsTransporterpHThiamine uptake rateNone (Control)Pyridoxinefmol/min/mg of protein% controlSLC19A27.4203 ± 16168 ± 1282.9 ± 5.75.5133 ± 4aStatistical differences were assessed by Student's t test. p < 0.05 compared with the value at pH 7.4.32 ± 1bStatistical differences were assessed by Student's t test. p < 0.05 compared with control.24.0 ± 0.6aStatistical differences were assessed by Student's t test. p < 0.05 compared with the value at pH 7.4.SLC19A37.4170 ± 19100 ± 5bStatistical differences were assessed by Student's t test. p < 0.05 compared with control.58.6 ± 3.05.558 ± 3aStatistical differences were assessed by Student's t test. p < 0.05 compared with the value at pH 7.4.5 ± 0.3bStatistical differences were assessed by Student's t test. p < 0.05 compared with control.8.9 ± 0.5aStatistical differences were assessed by Student's t test. p < 0.05 compared with the value at pH 7.4.a Statistical differences were assessed by Student's t test. p < 0.05 compared with the value at pH 7.4.b Statistical differences were assessed by Student's t test. p < 0.05 compared with control. Open table in a new tab The saturable profiles of the specific uptake of thiamine by SLC19A2 and SLC19A3 were kinetically assessed at pH 7.4 (Fig. 11) and also at pH 5.5 (Fig. 12), and the estimated parameters are summarized in Table 2. At pH 7.4, the Vmax values were 106 and 65.9 pmol/min/mg of protein, respectively, for SLC19A2 and SLC19A3, and the Km values were 2.83 and 2.36 μm, respectively. These Km values were comparable with the previously reported values of 2.5 and 3.16 μm, respectively (6Dutta B. Huang W. Molero M. Kekuda R. Leibach F.H. Devoe L.D. Ganapathy V. Prasad P.D. Cloning of the human thiamine transporter, a member of the folate transporter family.J. Biol. Chem. 1999; 274 (10542220): 31925-3192910.1074/jbc.274.45.31925Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 8Liang X. Chien H.C. Yee S.W. Giacomini M.M. Chen E.C. Piao M. Hao J. Twelves J. Lepist E.I. Ray A.S. Giacomini K.M. Metformin is a substrate and inhibitor of the human thiamine transporter, THTR-2 (SLC19A3).Mol. Pharm. 2015; 12 (26528626): 4301-431010.1021/acs.molpharmaceut.5b00501Crossref PubMed Scopus (62) Google Scholar). At pH 5.5, the Vmax values were 100 and 26.9 pmol/min/mg of protein, respectively, for SLC19A2 and SLC19A3, and Km values were 3.66 and 2.33 μm, respectively. These Km values were comparable with the Ki values of 1.11 and 0.86 μm, respectively, for the inhibition of pyridoxine transport by thiamine (Table 2), suggesting that the interaction of thiamine with those SLC19As for its transport is possibly linked to its inhibitory action for pyridoxine transport. It is notable that, for SLC19A2, the Km was indicated to be increased with a decrease in pH, whereas Vmax was unchanged. Although the extent of decrease in Km was not large enough to be statistically significant, the trend was indicated at p < 0.1. To the contrary, for SLC19A3, the Vmax was indicated to be decreased, whereas Km was unchanged. Therefore, different mechanisms may be involved in the pH-dependent alterations in thiamine transport by those SLC19As. It should also be noted that the thiamine concentration of 5 nm, which was used in preceding experiments, was confirmed to be in the linear phase of transport kinetics at concentrations much below the Km values for both SLC19As, where their transport activities were highest and could be evaluated most effectively.Figure 12Effect of pyridoxine on concentration-dependent thiamine uptake by SLC19A2 and SLC19A3 stably expressed in MDCKII cells under the acidic conditions. The specific uptake of [3H]thiamine by SLC19A2 (A) and SLC19A3 (B) was evaluated at varied concentrations for 1 min at pH 5.5 and 37 °C in the presence of pyridoxine or absence for control. The profiles are transformed into the Eadie-Hofstee format in the right panels. The estimated values of Vmax, Km, and the inhibition constant (Ki) are presented in Table 2. Data are presented as the mean ± S.E. (n = 3).View Large Image" @default.
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