Characterization of a mouse colonic system B0+ amino acid transporter related to amino acid absorption in colon

Shinya Ugawa, Yoko Sunouchi, Takashi Ueda, Eri Takahashi, Yoshitsugu Saishin, and Shoichi Shimada

Department of Anatomy II, Nagoya City University Medical School, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Previous experiments have shown that an amino acid transport system B0+ transporter in cultured colonic epithelial cells mediates amino acid absorption. Here we describe the cloning and functional characterization of a system B0+ transporter selectively expressed in the colon. Using the combination of an expressed sequence tag database search and RT-PCR approaches, we cloned a mouse colonic amino acid transporter, designated mCATB0+. Northern blot analysis revealed that mCATB0+ was selectively expressed in the large intestine. In situ hybridization showed the mCATB0+ mRNA to be localized in absorptive epithelial cells. When expressed in Xenopus oocytes, mCATB0+ exhibited a Na+-dependent stereoselective uptake and a broad specificity for neutral and cationic amino acids, which is characteristic of amino acid transport system B0+. In vivo [3H]glycine uptake assay demonstrated that a system B0+-like transporter protein was expressed on the apical surface of the colonic absorptive cells. Our data suggest that a mouse colonic amino acid transporter mCATB0+ may absorb amino acids from the intestinal contents in the colon.

cDNA cloning; absorptive epithelial cell


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE COLON IS THE FINAL SITE not only for fluid and electrolyte conservation but also for absorption of nutrients in the gastrointestinal system. For example, in patients with short bowel syndrome, functional adaptations such as increased colonic absorption of nutrients and minerals can be observed (10). Recent research has elucidated various cellular and molecular mechanisms for water and electrolyte transport in colon. Amiloride-sensitive sodium channels located in the apical cell membrane, through which sodium ions are taken into the epithelial cells, are a case in point (2, 12). By contrast, little is known about the molecular mechanisms that regulate the colonic absorption of nutrients, including essential amino acids.

Amino acid transport across the plasma membrane is mediated by transporters, which are distinguished primarily by substrate selectivity and ionic dependence (11). In the rabbit small intestine, transport of glycine, lysine, and beta -alanine across the brush-border membrane is mediated by a system B0+ transporter (8), which is characterized by sodium-dependent uptake and broad specificity, with high affinity for neutral and cationic amino acids (11). In cultured colonic epithelial cells (Caco-2), neutral amino acids are absorbed via the system B0+ transporter and system ASC transporter (a sodium-dependent alanine/serine/cysteine transporter), and these amino acids are then released from the cells in an intracellular concentration-dependent manner (1). System B0+ is widely distributed on epithelial cells and mediates amino acid transport on the apical surface (11).

In this study, we describe the molecular cloning and functional expression of a system B0+ transporter selectively expressed in the colon. In addition, we also provide evidence that a system B0+-like amino acid transporter is expressed at the protein level in the apical membrane of the colonic absorptive cells, absorbing amino acids from the intestinal contents in the colon. Our data will be helpful in understanding the molecular mechanisms that underlie amino acid absorption in the colon, which may be crucial in patients with short bowel and severe malabsorption in the remaining small intestine.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Molecular cloning and DNA sequencing. Comparison of the glycine transporter protein sequence (15) with the database of expressed sequence tags (EST) identified one cDNA sequence from total mouse embryos (GenBank accession no. AI006618). This clone showed a considerable similarity to the putative transmembrane domain I-III of sodium-dependent transporters (9). The insert cDNA of the EST clone was subcloned into pBluescript II SK(-) vector (Stratagene), and sequencing was carried out on both strands by the dideoxy chain termination method by using successive synthetic oligonucleotides.

Since the EST clone we obtained was derived from the Sugano mouse embryo library, which was constructed by using a PCR-based method (16), the clone might contain some mutations. To verify the nucleotide sequence, the RT-PCR method was performed. Male mice of the ddy strain were purchased from JAPAN SLC (Shizuoka, Japan) at 10 wk of age (Center for Experimental Animal Sciences at Nagoya City University gave us permission for the experiments). Five micrograms of total RNA extracted from colon were used as a template for the cDNA synthesis. On the basis of the analyzed sequence data, a pair of oligonucleotide primers [5'-GGCGAGGCACACCAAGGGATCCA-3' (forward) and 5'-TAGATATTTTCAAAGGTGACAT-3' (reverse)] was prepared. The PCR fragments obtained from five independent experiments were sequenced.

Northern blot analysis. Twenty micrograms of total RNA isolated from various adult mouse organs were separated by electrophoresis on 1% agarose-formaldehyde gels and blotted onto Hybond-N+ membranes (Amersham Pharmacia). The procedures were basically the same as those described previously (18). The blots were hybridized with a random-primed 32P-labeled fragment of the mouse colonic amino acid transporter B0+ (mCATB0+) cDNA corresponding to nucleotides 767-1782. The hybridization signals were analyzed with a bioimaging analyzer BAS 2500 (FUJIX).

In situ hybridization. Localization of the mCATB0+ mRNA in embryos at embryonic day 18 and in adult mouse colon was determined by in situ hybridization. Mouse embryos were also purchased from JAPAN SLC. The procedures were basically the same as those described previously (18). Fresh-frozen sections (6-µm thick) were cut on a cryostat. To prepare riboprobes, a 1,016-bp mCATB0+ cDNA insert (bases 767-1782) subcloned in the pBluescript II SK(-) vector was used. The specificity of hybridization signals was confirmed by a control study with the sense cRNA probe.

Functional characterization by Xenopus oocyte expression. The coding sequence of mCATB0+ was subcloned by using modified pBluescript (pBsMXT; Stratagene). The multiple cloning site of pBsMXT was flanked by Xenopus beta -globin 5'- and 3'-untranslated regions to promote stable mRNA expression in oocytes. The procedures for two-electrode voltage-clamp recording were basically the same as those described previously (4). Current (I) as a function of substrate concentration ([S]) was fitted by least squares to I = Imax · [S]/(Km + [S]), where Imax is the maximal current and Km is the transport constant.

The transporter function was verified by measuring uptake of [3H]glycine in the Xenopus laevis oocyte system. The procedures were basically the same as those described previously (4, 17). For uptake measurements, the oocytes expressing mCATB0+ were washed in ND96 and incubated in ND96 solution containing 20 µM [3H]glycine for 10 min at room temperature or at 4°C with or without 20 mM unlabeled glycine.

Glycine uptake by mouse colonic epithelial cells in vivo. Male ddy mice (at 10 wk of age) were fasted overnight before the experiments. Anesthesia was induced by an intraperitoneal injection of chloral hydrate (42 mg/kg). After the anesthesia was achieved, a modified Hanks' balanced salt solution (HBSS) containing D-glucose (19 mM) and [3H]glycine (20 µM), with or without 20 mM unlabeled various L-amino acids, was administered onto the luminal surface of the colon by intestinal infusion. Subsequently, 60 min after the administration, the colons (~7 cm in length) were dissected away, cut open longitudinally, and washed in the ice-cold HBSS three times. After the wash, they were digested with proteinase K, and the incorporated radioactivity was counted in a Beckman scintillation counter. Data were analyzed by using the unpaired Student's t-test. The level of significance was set at P < 0.01.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cloning of the full-length mCATB0+ cDNA. Virtual screening of the dbEST database of the National Center for Biotechnology Information (NCBI) with probes corresponding to the glycine transporter sequence led to the identification of a system B0+ transporter. Since this EST clone was synthesized by using a PCR-based method (13), the insert cDNA might contain some mutations. To address this problem, we recloned the same transporter from adult mouse colon, and subsequent sequence analysis revealed one point mutation at position 1449 of the EST clone (A replaced by G). We confirmed that the cDNA was 1,923 bp long with an open reading frame of 1,914 bp encoding a protein of 638 amino acids (Fig. 1), and the corresponding gene was designated mCATB0+. Hydropathy analysis (5) of the primary amino acid sequence of the predicted protein showed the presence of 12 putative transmembrane domains. The predicted mCATB0+ protein lacks an identifiable signal sequence, which is consistent with a cytoplasmic location of the NH2 terminus. Seven potential N-linked glycosylation sites are located at positions 155, 163, 174, 185, 193, and 198 in the putative second extracellular loop and at position 298 in the putative third extracellular loop. There are three potential sites for protein kinase C-dependent phosphorylation. Recently, Sloan and Mager (14) reported the cloning of the human amino acid transporter system B0+ (hATB0+), which shows 88% amino acid identity with mCATB0+. They also submitted the sequence for mouse ATB0+ (mATB0+) to GenBank. Only four amino acids are different between mCATB0+ and mATB0+. As described in DISCUSSION, however, mCATB0+ and hATB0+ showed quite different tissue distribution patterns, raising the sufficient possibility that mCATB0+ is not a mouse counterpart to hATB0+.


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Fig. 1.   Nucleotide and deduced amino acid sequences of mouse colonic amino acid transporter B0+ (mCATB0+). Twelve putative transmembrane domains are underlined. The nonsense codons are marked by asterisks. Potential N-linked glycosylation sites are indicated by squares, and potential protein kinase C phosphorylation sites are indicated by circles.

Localization of mCATB0+ by Northern blot analysis and in situ hybridization. Tissue-specific expression of mCATB0+ mRNA was examined by Northern blot analysis (Fig. 2). In the adult mouse, mCATB0+ mRNA (~4.0 kb long) was abundantly and selectively expressed in the large intestine. Barely detectable signals were observed in the lung. No signals were found in other tissues such as brain, heart, liver, small intestine, kidney, spleen, testis, and skeletal muscle. The level of mCATB0+ mRNA in the lung was far lower than that in the large intestine, indicating that mCATB0+ is almost specifically expressed in the large intestine.


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Fig. 2.   Northern blot analysis of mCATB0+ expression in adult mouse tissues. At bottom, 28S ribosomal RNA bands visualized by ethidium bromide staining are shown.

To determine the detailed localization of mCATB0+ gene expression, we performed in situ hybridization by using an 35S-labeled cRNA probe. Since the corresponding EST clone was derived from a mouse embryo library, we investigated the distribution of mCATB0+ in whole embryos. In embryonic day 18 parasagittal sections, mCATB0+ mRNA was expressed very strongly in the large intestine, particularly in the left side colon, including the rectum (Fig. 3, A and B). The hybridization signals in the lung were barely detectable (Fig. 3A). No detectable hybridization signals were observed in other tissues.


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Fig. 3.   Expression of mCATB0+ mRNA by in situ hybridization. A: autoradiogram of embryonic day 18 parasagittal section. B: embryonic colon hybridized with an antisense probe, shown in a darkfield photomicrograph. C: adult mouse colon hybridized with an antisense probe, shown in a darkfield photomicrograph. Strong expression of mCATB0+ mRNA was observed in the superficial cells of the colonic mucosa and the upper portions of the crypts. D: adult mouse colon hybridized with an antisense probe, shown at high magnification in a brightfield photomicrograph. Dense hybridization signals were accumulated in absorptive epithelial cells in the middle and upper portions of the crypts.

To reveal the cellular distribution of mCATB0+ gene expression, adult mouse colon was also tested. Strong expression of mCATB0+ mRNA was observed in the adult mouse colon, especially in the superficial cells of the colonic mucosa and the upper portions of the crypts (Fig. 3C). At high magnification, dense hybridization signals were mainly accumulated in absorptive epithelial cells (Fig. 3D), which are the principal cell type at the surface of the mucosa in the middle and upper portions of the crypts.

Oocyte expression of mCATB0+. To assess the transport activity, two-electrode voltage-clamp recording was performed. Voltage-clamp measurements showed concentration-dependent inward currents in response to glycine (Km =142.1 ± 19.9 µM) (Fig. 4, A-C). The inward current elicited by glycine superfusion was not seen when lithium was substituted for sodium, indicating that the transport activity was sodium dependent. No detectable currents were observed in water-injected oocytes under the same extracellular conditions.


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Fig. 4.   Inward currents evoked by amino acids in voltage-clamped mCATB0+-expressing Xenopus oocytes. A: sodium-dependent currents induced by various concentrations of glycine in mCATB0+-expressing oocytes. B: saturation analysis of glycine transport. C: Eadie-Hofstee plot of the data from B. D: substrate selectivity and stereoselectivity of mCATB0+. Various amino acids (100 µM) generated inward currents in mCATB0+-expressing oocytes. Data are plotted relative to the current elicited by 100 µM glycine. Values represent means ± SE from 3 different oocytes.

To determine the substrate selectivity, a series of L-amino acids was tested for their ability to elicit the inward currents. Superfusion of mCATB0+ cRNA-injected oocytes voltage clamped at -60 mV with various L-amino acids (100 µM) produced inward currents that were absent in water-injected controls (Fig. 4, A and D). Among the amino acids, nonpolar amino acids such as L-alanine, L-leucine, and L-methionine generated larger inward currents than glycine. The negatively charged amino acids, L-aspartate and L-glutamate, evoked no current. Km values and Imax/ImaxGly (Imax values normalized by the maximum current induced by glycine in the same oocyte) for L-alanine, L-leucine, and L-methionine were 69.0 ± 17.7, 21.6 ± 3.1, and 31.4 ± 5.5 µM and 0.95 ± 0.02, 0.84 ± 0.01, and 0.89 ± 0.03, respectively (means ± SE of 3 separate experiments). mCATB0+-mediated transport was stereoselective because D-amino acids that were able to elicit sodium-dependent currents evoked reduced inward currents compared with those induced by L-amino acids (Fig. 4D). Km values and Imax/ImaxGly values for D-alanine were 208.0 ± 22.7 µM and 0.93 ± 0.02, respectively (means ± SE of 3 separate experiments).

To verify the transport function, the uptake of [3H]glycine was monitored by using a Xenopus oocyte uptake assay. The oocytes injected with the sense cRNA consistently showed more than fourfold higher glycine transport activity than water-injected oocytes (Fig. 5). This accumulation was sodium- and temperature-dependent. These findings were entirely consistent with the results obtained from our electrophysiological experiments.


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Fig. 5.   Sodium- and temperature-dependent uptake of [3H]glycine into mCATB0+-expressing Xenopus oocytes. mCATB0+-expressing oocytes consistently showed >4-fold higher glycine transport activity than water-injected oocytes. The transport of 20 µM [3H]glycine was significantly attenuated in the presence of 20 mM unlabeled glycine.

Glycine uptake in the native mouse colon. The results from our in situ hybridization and electrophysiological experiments suggest that the native mouse colon is able to absorb amino acids from the intestinal contents via the amino acid transporter mCATB0+. To examine whether mCATB0+ protein is expressed on the apical surface of the absorptive cells, we directly administered [3H]glycine, with or without competitive inhibitors, into the colonic lumen by intestinal infusion (Fig. 6). Administration of 20 µM [3H]glycine to the luminal surface of the colon resulted in glycine influx into the absorptive epithelium [total uptake: 136.2 ± 9.6 pmol · animal-1 · h-1 (mean ± SE of 3 separate experiments)]. By contrast, the [3H]glycine influx was reduced to 84.1 ± 3.8 pmol · animal-1 · h-1 (n = 3) in the presence of 20 mM unlabeled glycine. Although the 1,000-fold amounts of unlabeled glycine failed to inhibit the total [3H]glycine uptake completely, the difference was statistically significant (P < 0.01), indicating that there are specific glycine uptake systems in the colonic absorptive cells. The component of specific glycine uptake was completely inhibited in the presence of 20 mM unlabeled L-histidine (a cationic amino acid), whereas coadministration of unlabeled L-aspartate (a negatively charged amino acid) did not display any inhibitory effects. {The [3H]glycine uptake in the presence of 20 mM unlabeled L-histidine and L-aspartate were 41.9 ± 4.4 and 121.0 ± 18.2 pmol · animal-1 · h-1 (n = 3 each), respectively.} These results suggest that the transporter that displays high affinity for neutral and cationic amino acids is expressed in the apical membrane of the colonic absorptive cells.


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Fig. 6.   Glycine uptake by mouse colonic epithelial cells in vivo. The transport of 20 µM [3H]glycine was significantly attenuated in the presence of 20 mM unlabeled glycine or L-histidine but was not attenuated in the presence of 20 mM unlabeled L-aspartate, a substrate specificity similar to that described for amino acid transport system B0+.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We have described here the isolation and characterization of a sodium-dependent amino acid transporter abundantly and selectively expressed in colonic absorptive epithelial cells. On the basis of amino acid sequence homology, mCATB0+ is a member of the family of sodium/chloride-dependent transporters with a characteristic membrane topology of 12 transmembrane helices and potential N-linked glycosylation sites in the putative second extracellular loop (9, 11). The sequence of mCATB0+ shows 39 and 31% identities with human glycine transporter GlyT2 (7) and human L-proline transporter (13), respectively, and shows 88% identity with hATB0+ (14). A common structural feature of mCATB0+ and hATB0+ is a potential N-linked glycosylation site in the putative third extracellular loop that has not been found in other family members, including orphan transporters (9).

When expressed in Xenopus oocytes, mCATB0+ demonstrated a sodium-dependent stereoselective uptake and a broad specificity for neutral and cationic amino acids, which is characteristic of amino acid transport system B0+. mCATB0+ prefers hydrophobic L-amino acids with a large R group and has very low affinity for L-proline, which differs from other nonpolar amino acids in that it has a cyclic structure. Despite these findings, however, mCATB0+ shows an apparent affinity for several D-amino acids. Together with other structural features, a potential N-linked glycosylation site in the putative third extracellular loop may play a key role in accepting such a wide range of amino acid substrates.

Although the transport functions of mCATB0+ and hATB0+ are similar, the tissue distribution patterns of the two transporters are quite different. hATB0+ was expressed abundantly in lung, trachea, and salivary gland and weakly in colon (14), whereas mCATB0+ was expressed intensely in colon and faintly in lung. In human bronchial epithelial cells, hATB0+ on the apical surface may play a significant role in removal of amino acids to maintain a low amino acid concentration in the airway surface liquid (3). Similarly, mCATB0+ in the colon may be involved in the absorption of amino acids from the intestinal contents. Chen et al. (1) reported a system B0+ amino acid transporter on the apical surface in human colonic epithelial cells (Caco-2) involved in amino acid absorption. In Caco-2 cells, the apical uptake of amino acids is dependent on a combination of transport system B0+ and ASC, and the basolateral uptake is more dependent on the system L, sodium-independent system mainly for bulky side-chain amino acids (11). To examine the involvement of mCATB0+ in amino acid absorption in the colon, we investigated the localization of mCATB0+ mRNA in absorptive epithelial cells. Our in situ hybridization experiments showed dense hybridization signals for mCATB0+ localized in absorptive epithelial cells. These findings suggest that mCATB0+ actively absorbs neutral and cationic amino acids into the absorptive cells by using a electrochemical potential gradient, which is generated by high sodium concentrations in the intestinal contents.

The remarkable differences in the tissue distribution patterns of mCATB0+ and hATB0+ raise the sufficient possibility that mCATB0+ is not a mouse homologue of hATB0+. There may be a human homologue of mCATB0+ specifically expressed in the colon. The ability of the normal human small intestine to absorb amino acids is highly efficient, with only small amounts of amino acids reaching the colon. Therefore, it is also possible that hATB0+ in the colon is usually expressed at low levels and that the expression of hATB0+ mRNA can be markedly upregulated in a variety of pathological circumstances as the need for colonic absorption of amino acids arises. Further investigations are needed to examine these hypotheses.

In this study, we have also investigated whether a system B0+ transporter is expressed at the protein level and is functional on the apical surface of the colonic absorptive cells. Figure 6 clearly shows that, despite large numbers of microorganisms that can take up amino acids and metabolize them rapidly (6), the native tissue (mouse colon) can absorb the neutral amino acid glycine and the cationic amino acid L-histidine with high affinity but cannot absorb the negatively charged amino acid L-aspartate via the same pathway, a substrate specificity similar to that described for transport system B0+. The results also showed that the 1,000-fold amounts of unlabeled glycine failed to inhibit the total [3H]glycine uptake completely in vivo (the uptake was found to be ~60% of the original), whereas heterologous expression of the mCATB0+ protein in Xenopus oocytes displayed almost complete inhibition of total [3H]glycine uptake in the presence of cold glycine under the similar treatment conditions of solutions (Fig. 5). The high background radioactivity value of the glycine uptake assay in vivo was probably due to high viscosity of mucins and complex tertiary structures of the colonic epithelium. Alternatively, the colon may have another high Km pathway for glycine uptake, which would not be expected to be as sensitive to competition by unlabeled glycine. Together with the findings of the system B0+ transporter in human enterocyte-like Caco-2 cells and the localization of mCATB0+ mRNA, our in vivo glycine uptake assay suggests that mCATB0+ protein is expressed on the apical surface of the colonic absorptive cells, absorbing amino acids from the intestinal contents in the colon.


    ACKNOWLEDGEMENTS

We thank K. Tanaka and K. Kajita for their skillful technical assistance.


    FOOTNOTES

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EBI Data Bank with accession no. AB033285.

Address for reprint requests and other correspondence: S. Ugawa, Dept. of Anatomy II, Nagoya City Univ. Medical School, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan (E-mail: ugawa{at}med.nagoya-cu.ac.jp).

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.

Received 5 December 2000; accepted in final form 23 March 2001.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Am J Physiol Gastrointest Liver Physiol 281(2):G365-G370
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