Identification and functional characterization of the first nucleobase transporter in mammals: implication in the species difference in the intestinal absorption mechanism of nucleobases and their analogs between higher primates and other mammals

Abstract

Nucleobases are important compounds that constitute nucleosides and nucleic acids. Although it has long been suggested that specific transporters are involved in their intestinal absorption and uptake in other tissues, none of their molecular entities have been identified in mammals to date. Here we describe identification of rat Slc23a4 as the first sodium-dependent nucleobase transporter (rSNBT1). The mRNA of rSNBT1 was expressed highly and only in the small intestine. When transiently expressed in HEK293 cells, rSNBT1 could transport uracil most efficiently. The transport of uracil mediated by rSNBT1 was sodium-dependent and saturable with a Michaelis constant of 21.2 microM. Thymine, guanine, hypoxanthine, and xanthine were also transported, but adenine was not. It was also suggested by studies of the inhibitory effect on rSNBT1-mediated uracil transport that several nucleobase analogs such as 5-fluorouracil are recognized by rSNBT1, but cytosine and nucleosides are not or only poorly recognized. Furthermore, rSNBT1 fused with green fluorescent protein was mainly localized at the apical membrane, when stably expressed in polarized Madin-Darby canine kidney II cells. These characteristics of rSNBT1 were almost fully in agreement with those of the carrier-mediated transport system involved in intestinal uracil uptake. Therefore, it is likely that rSNBT1 is its molecular entity or at least in part responsible for that. It was also found that the gene orthologous to the rSNBT1 gene is genetically defective in humans. This may have a biological and evolutional meaning in the transport and metabolism of nucleobases. The present study provides novel insights into the specific transport and metabolism of nucleobases and their analogs for therapeutic use.

FIGURE 1.

Sequence analysis of rSNBT1. The deduced amino acid sequence of rSNBT1/Slc23a4 was aligned with those of SVCT1/Slc23a1, SVCT2/Slc23a1, and Slc23a3 of rat, using the program of ClustalW, and processed to visualize using the program BOXSHADE. Identical amino acids and conservative changes are indicated by reversed and shaded characters, respectively. Boxed residues indicate the putative transmembrane domains. Amino acids of the NAT signature motif (Glu³⁹⁷ to Arg⁴⁰⁶) are indicated by circles, and putative N-glycosylation sites (Asn¹⁵² and Asn³⁹⁰) are indicated by asterisks.

FIGURE 2.

Analyses of the expression of the mRNA of rSNBT1 in various rat tissues by Northern blot (A) and RT-PCR (B) and in various segments of the rat small intestine by Northern blot (C). Total RNA (10 μg) from each tissue or segment was probed with ³²P-labeled cDNA of rSNBT1 in Northern blot analysis. The bands of ethidium bromide-stained 28 S rRNA are also shown as references. Total RNA (1 μg) from each tissue was reverse-transcribed and then amplified by PCR using a set of specific primers for rSNBT1 in RT-PCR analysis. The bands of GAPDH are also shown as references.

FIGURE 3.

Uptake of several nucleobases and analogs in HEK293 cells transiently expressing rSNBT1. rSNBT1-specific uptake was evaluated by dividing the uptake in rSNBT1-transfected cells by that in mock cells. Uptakes of [³H]adenine (5 nm), [¹⁴C]guanine (2 μm), [³H]hypoxanthine (5 nm), [³H]xanthine (10 nm), [³H]thymine (5 nm), [³H]uracil (2 nm), [³H]thymidine (5 nm), [³H]uridine (10 nm), and [¹⁴C]ascorbate (5 μm) were evaluated at 37 °C and pH 7.4 for 1 min in both cells. The uptake rates (pmol/min/mg of protein) for unity, which were evaluated in mock cells, were 0.0546 for adenine, 21.5 for guanine, 0.163 for hypoxanthine, 0.0370 for xanthine, 0.0172 for thymine, 0.00740 for uracil, 0.257 for thymidine, 0.170 for uridine, and 19.8 for ascorbate. Data are represented as the means ± S.E. (n = 4).

FIGURE 4.

Functional characteristics of rSNBT1 transiently expressed in HEK293 cells. A, time courses of the uptake of [³H]uracil (2 nm) in rSNBT1-transfected cells (closed circles) and mock cells (open circles). B, effect of extracellular ions on the rSNBT1-specific uptake rate of [³H]uracil (2 nm). NaCl in the control medium was replaced as indicated. C, Na⁺ dependence of the rSNBT1-specific uptake rate of [³H]uracil. The Hill coefficient was estimated to be 1.07 by analysis using the Hill equation. D, concentration dependence of the rSNBT1-specific uptake rates of [³H]uracil (circles) and [³H]xanthine (squares). The K_m and V_max are 21.2 ± 2.8 μm and 737 ± 23 pmol/min/mg of protein, respectively, for uracil as the computer-fitted parameters with S.E. and 83.0 ± 9.0 μm and 1110 ± 46 pmol/min/mg of protein, respectively, for xanthine. In all experiments, uptake measurements were conducted at 37 °C and pH 7.4 and, in panels B, C, and D, for a 1-min period. Data are represented as the means ± S.E. (n = 4). *, p < 0.05 when compared with the value for control.

FIGURE 5.

Effect of various compounds on uracil transport mediated by rSNBT1 transiently expressed in HEK293 cells. A, inhibitory effect of nucleobases on the rSNBT1-specific uptake rate of [³H]uracil (2 nm) evaluated in the presence of varied concentrations of cytosine (open circles), guanine (closed circles), hypoxanthine (open squares), thymine (closed squares), and 5-FU (open triangle). The IC₅₀ values are 80.7 ± 17.1, 17.4 ± 3.1, 12.7 ± 0.6, and 69.0 ± 11.4 μm, respectively, for guanine, hypoxanthine thymine, and 5-FU as the computer-fitted parameters with S.E. B, inhibitory effect of various compounds (0.1 mm) on the rSNBT1-specific uptake rate of [³H]uracil (2 nm). In all experiments, uptake measurements were conducted for a 1-min period at 37 °C and pH 7.4. The control values for normalization were 74.9 and 72.5 fmol/min/mg of protein, respectively, in panels A and B. Data are represented as the means ± S.E. (n = 4). *, p < 0.05 when compared with the value for control. NBMPR, nitrobenzylthioinosine.

FIGURE 6.

Cellular localization of GFP-rSNBT1 stably expressed in polarized MDCKII cells. The confocal laser-scanning microscopic image shows predominant apical membrane localization of GFP-rSNBT1 (green) and nuclei stained with 4′,6-diamino-2-phenylindole (white).

FIGURE 7.

rSNBT1 activity in the everted sacs of the rat small intestine. A, time course of the uptake of [³H]uracil (1. 3 nm) in ileal everted sacs. B, Na⁺ dependence of the uptake clearance of [³H]uracil (1.3 nm) in jejunal and ileal everted sacs. NaCl in the control uptake buffer (closed bars) was replaced with KCl (open bars). C, concentration dependence of the uptake clearance of [³H]uracil in ileal everted sacs. The uptake was evaluated at varied uracil concentrations, which were adjusted by adding unlabeled uracil, and the uptake rate was divided by concentration to estimate the uptake clearance. The values of V_max, K_m, and CL_m,d are 1.26 ± 0.09 nmol/min/100 mg of wtw, 40.3 ± 3.1 μm, and 1.34 ± 0.37 μl/min/100 of mg wtw, respectively, as the computer-fitted parameters with S.E. D, effect of various compounds (0.1 mm) on the uptake clearance of [³H]uracil (1.3 nm) in ileal everted sacs. The control value was 31.5 μl/min/100 mg of wtw. In all experiments, uptake measurements were conducted at 37 °C and pH 7.4 and, in panels B, C, and D, for a 2-min period. Data are represented as the means ± S.E. n = 3 (panels A and B) or 6 (panels C and D). *, p < 0.05 when compared with the value for control; †, p < 0.05 when compared with the value for jejunum. NBMPR, nitrobenzylthioinosine.

FIGURE 8.

Comparison of the SNBT1 gene locus between rat and human. A, relative position and spanning of exons that encode SNBT1 and its mRNA. The scales are set for exons, introns, and mRNA as indicated. B, RT-PCR analysis for the expression of the mRNA of hSNBT1 in the human intestine. cDNAs reverse-transcribed from human small intestine total RNA (1 μg) and human genomic DNA isolated from HEK293 cells were amplified by PCR using a set of specific primers for hSNBT1.