Identification and characterization of rabbit small intestinal villus cell brush border membrane Na-glutamine cotransporter

Abstract

Glutamine, the primary metabolic fuel for the mammalian small intestinal enterocytes, is primarily assimilated by Na-amino acid cotransporters. Although Na-solute cotransport has been shown to exist in the brush border membrane (BBM) of the absorptive villus cells, the identity of Na-glutamine cotransport in rabbit small intestinal villus cells was unknown. Na-dependent glutamine uptake is present in villus BBM vesicles. An intravesicular proton gradient did not stimulate this Na-dependent glutamine uptake, whereas Li⁺ did not significantly suppress this uptake. These observations in concert with amino acid substitution studies suggested that Na-glutamine cotransporter in the villus cell BBM was the newly identified cotransporter B⁰AT1 (SLC6A19). Quantitative real-time PCR identified the message for this cotransporter in villus cells. Thus a full-length cDNA of B⁰AT1 was cloned and expressed in MDA-MB-231 cells. This expressed cotransporter exhibited characteristics similar to those observed in villus cells from the rabbit small intestine. Antibody was generated for B⁰AT1 that demonstrated the presence of this cotransporter protein in the villus cell BBM. Kinetic studies defined the kinetic parameters of this cotransporter. Thus this study describes the identification, cloning, and characterization of the Na-amino acid cotransporter responsible for the assimilation of a critical amino acid by the absorptive villus cells in the mammalian small intestine.

assimilation of amino acids is a major function of the mammalian small intestine. Various amino acids are actively transported by a multitude of transport systems across the brush border membrane (BBM) of small intestinal enterocytes into systemic circulation (6). These systems are L, y⁺, y⁺L, b^0,+, A, ASC, B⁰, B^0,+, and X⁻_AG. They can be differentiated functionally by their substrate specificity and driving forces. Among these, systems L, y⁺, and b^0,+ are Na independent; system y⁺L is Na independent for cationic amino acids and Na dependent for neutral amino acids; systems A, ASC, B⁰, and X⁻_AG are Na dependent; and system B^0,+ is Na and Cl dependent. Furthermore, recent cloning studies have shown that many of these transport system actually consists of two or more subtypes (5, 6, 13). Na-dependent amino acid cotransporters are energized by a transmembrane electrochemical Na⁺ gradient maintained by Na⁺-K⁺-ATPase on the basolateral membrane of the enterocytes.

In the intestine, heterogeneity of expression of amino acid cotransporters may be secondary to entry of different kinds of food that interact for cotransporter expression. In the mammalian small intestine, larger neutral amino acids have been shown to be transported by three different transport systems, namely system B⁰, system B^0,+ (Na-dependent transporter for neutral and cationic amino acids), and system ASC (Na-dependent transporter for midsize neutral amino acids; Refs. 11, 12). In addition, proton-amino acid cotransporter 1 (PAT1) transports small neutral amino acids (1). The activity of the intestinal system B⁰ has also been observed in the renal cortex (9, 14). Recently, successful identification of a mouse B⁰-type transporter was reported by Broer and colleagues (4). This transporter was named B⁰AT1 (SLC6A19) and belongs to a cluster of orphan transporters within the family of Na-dependent neurotransmitters and amino acid transporters (SLC6). When expressed in Xenopus oocytes, the B⁰AT1 cDNA induces a Na-dependent uptake of neutral amino acids.

It is noteworthy, however, that several related gene products, two in human (products of SLC6A18 and 20) and three in mice (SLC6A18 and two genes corresponding to human SLC6A20), are expressed in the kidney brush border and, to some extent, in the small intestine as well. Interestingly, whereas the gene product of SLC6A19 (B⁰AT1) is expressed mainly in the early part of the proximal tubule, the product of Slc6a18 is most abundant in the late proximal segments. In contrast, the gene products derived from the SLC6A20 homologues appear to be expressed all along the proximal tubule (20). It was hypothesized that these gene products perform B⁰-type Na-amino acid cotransport similar to that of B⁰AT1, but with somewhat different selectivity, apparent affinity, and axial distribution. However, it is not known which Na-dependent amino acid cotransporter is responsible for the assimilation of glutamine in the mammalian small intestine.

Given the reported heterogeneity of Na-dependent amino acid cotransporters, the aims of the present study were to identify the predominant Na-dependent glutamine cotransporter in the mammalian intestinal absorptive villus cells, then clone, express, and characterize it.

MATERIALS AND METHODS

Reagents.

l-Glutamine (Glu), l-alanine (Ala), l-serine (Ser), l-methyl-aminoisobutyric acid (MeAIB), l-arginine (Arg), l-tryptophan (Trp), l-histidine (His), l-asparagine (Asn), l-aspartate (Asp), l-glutamate (Glu), and β-hydroxybutyric acid from Sigma Chemical (St. Louis, MO) and insulin from Novo Nordisk (Clayman, NC) were purchased. HEPES buffer, Leibovitz's L-15 medium powder (L-15), bovine fetal serum, and all supplements were supplied from Invitrogen (Grand Island, NY) and [³H]glutamine from Amersham Biosciences (Little Chalfont, UK).

Cell isolation.

Pathogen-free New Zealand White male rabbits weighing 2–2.5 kg were euthanized according to the Guiding Principles for the Care and Use of Laboratory Animals according to a protocol approved by the West Virginia University IACUC. Villus cells were isolated from normal rabbit small intestine by a Ca²⁺ chelation technique as previously described (16). Briefly, a 3-ft section of ileum was filled with buffer containing (in mM) 0.15 EDTA, 112 NaCl, 25 NaHCO₃, 2.4 K₂HPO₄, 0.4 KH₂PO₄, 2.5 L-glutamine, 0.5 β-hydroxybutyrate, 0.5 dithiothreitol, and gassed with 95% O₂-5% CO₂, pH 7.4, at 37°C. The intestine was incubated in this solution for 3 min and gently palpitated for another 3 min to facilitate cell dispersion. The fluid was then drained from the ileal loop, phenylmethylsulfonyl fluoride was added, and the suspension was centrifuged at 100 g for 3 min. Cells used for BBM vesicle (BBMV) preparation were frozen immediately in liquid nitrogen and stored at −80°C until required. Previously established criteria were used to validate good separation of villus cells. These criteria included 1) marker enzymes (e.g., thymidine kinase, alkaline phosphatase), 2) transporter specificity (e.g., Na-glucose cotransport and Na⁺/H⁺ exchange are present on the BBM of villus cells), and 3) morphological differences (e.g., villus cells are larger, with better developed BBM compared with crypt cells).

BBMV preparation.

BBMV from rabbit intestinal villus cells were prepared by CaCl₂ precipitation and differential centrifugation as previously described (17). Briefly, frozen villus cells were thawed and suspended in 2 mM Tris·HCl buffer (pH 7.0) containing 50 mM mannitol. The suspension was homogenized and 10 mM CaCl₂ was added. The suspension was then centrifuged at 8,000 g for 15 min, and the resulting supernatant was centrifuged at 20,000 g for 30 min. The pellet was then resuspended in 10 mM Tris·HCl buffer (pH 7.5) containing 100 mM mannitol and homogenized. Vesicles were formed by adding MgCl₂ to 10 mM. The homogenate was centrifuged at 2,000 g for 15 min to remove debris, and the BBMV were precipitated by centrifugation at 27,000 g for 15 min. BBMV were resuspended in a medium appropriate to each experiment. BBMV purity was assured by marker enzyme (e.g., alkaline phosphatase) enrichment.

Uptake studies in BBMV.

Vesicle uptake studies were performed by the rapid-filtration technique as previously described (17). In brief, 5 μl of BBMV resuspended in 100 mM choline chloride, 0.10 mM MgSO₄, 50 mM HEPES-Tris (pH 7.4), 50 mM mannitol, and 75 mM KCl were incubated in 95 μl of reaction medium that contained 50 mM HEPES-Tris buffer (pH 7.4), 2 nmol glutamine, 10 μCi [³H]glutamine, 0.10 mM MgSO₄, 75 mM KCl, 50 mM mannitol, 100 mM of either NaCl or choline chloride, 10 μM valinomycin, and 100 μM carbonyl cyanide p-(tri-fluoromethoxy) phenyl-hydrazone. At the desired time, uptake was arrested by mixing with ice-cold stop solution (50 mM HEPES-Tris buffer, 0.10 mM MgSO₄, 75 mM KCl, and 100 mM choline chloride, 2 mM glutamine, pH 7.4). The mixture was filtered on a 0.45-μm Millipore (HAWP) filter and washed two times with 5 ml of ice-cold stop solution. Filters with BBMV were dissolved in 5 ml of Ecoscint A and kept in the dark for 12 h, and radioactivity was determined in a Beckman Coulter LS 6500 scintillation counter.

RTQ-PCR studies.

Total RNA was isolated from villus cells by use of TRIzol reagent from Invitrogen Life Technologies. Real-time quantitative PCR (RTQ-PCR) was performed using total RNA isolated by a two-step method. First-strand cDNA synthesis from total RNA was performed by using SuperScript III from Invitrogen Life Technologies using an equal mixture of oligo(dT) primer and random hexamers. The cDNA generated was used as template for real-time PCR using TaqMan Universal PCR master mix from Applied Biosystems (Foster City, CA) according to the manufacturer's protocol.

B⁰AT1 specific primer and probe sequences were as follows: forward primer 5′-GGATCCTGCTGTGCCTCA-3′; reverse primer 5′-CGAGGTAGGGTAGCGTAGAAGT-3′; TaqMan probe 5′-6FAM- CACCATCCGCGGAATCGAGACAAC-TAMRA-3′.

RTQ-PCR for rabbit β-actin was run along with the B⁰AT1 RTQ-PCR as an endogenous control using rabbit β-actin-specific primers and probes. The expression of β-actin was used to normalize the expression levels of B⁰AT1 between the individual samples. The sequences of the rabbit β-actin primers and probes were as follows: forward primer 5′-GCTATTTGGCGCTGGACTT-3′; reverse primer 5′-GCGGCTCGTAGCTCTTCTC-3′; TaqMan probe 5′-FAM- AAGAGATGGCCACGGCCGCGAAC-TAMRA-3′.

Final concentrations of primers and probes for B⁰AT1 and β-actin were 500 and 100 nM, respectively. The parameters for RTQ-PCR were: 95°C for 15 s, and 58°C for 1 min. All experiments were performed in triplicate and repeated at least thrice with RNA obtained from separate animals. Serial dilution experiments of cDNA were performed to establish that the efficiency of PCR was the same between β-actin and B⁰AT1 transporters (data not shown).

Rabbit B⁰AT1 cloning.

Full-length B⁰AT1 was obtained by PCR using Taq polymerase PCR kit from Promega (Madison, WI). For the purpose of transient transfection, full-length B⁰AT1 was cloned into pcDNA3.1/Zeo(+), a mammalian expression vector from Invitrogen Life Technologies (Carlsbad, CA). Rabbit B⁰AT1 sequence corresponding to the NH₂ and COOH termini of the protein were obtained by aligning the mouse B⁰AT1 protein sequence against the rabbit genomic sequence available from the public genomic sequence source and identifying the genomic sequence of maximum homology to the mouse B⁰AT1 protein sequence. Thus the nucleotide sequence was obtained and used to design PCR primers specific to rabbit B⁰AT1 (GenBank accession number EU095941). The upstream and downstream primer sequences are as follows: upstream primer 5′-GCC GCC ACC ATG GTG AGG CTG GTG CTG-3′; downstream primer 5′-TCT AGA TCA GTA CTT GAG GTC CCC ATT-3′. Forward primer contained Kozak consensus sequence upstream of the start codon (GCC GCC ACC) to aid in efficient transcription and the downstream primer contained the stop codon followed by XbaI restriction site to aid in unidirectional cloning into the expression vector pcDNA3.1/Zeo(+). The underlined sequence in the upstream primer is the Kozak consensus sequence followed by the start codon. The resultant PCR product was cloned into pGEM-T Easy vector from Promega (Madison, WI) and the B⁰AT1 insert was released by digesting with EcoRI and was inserted into EcoRI-digested pcDNA3.1/Zeo(+). Both sense and antisense strands of the cDNAs were sequenced by primer walking by use of the custom sequencing service from Agencourt Bioscience (Beverly, MA).

Expression and characterization of B⁰AT1.

The normal, diploid, human breast adenocarcinoma (MDA-MB-231) cell line (HTB-26, American Type Culture Collection, Manassas, VA/ Rockville, MD) was used between passages 5 and 20. Density of cells were ∼0.4 × 10⁶ cells/well, grown on 12-well plastic plate (Corning, NY) in L-15 containing 2 mM l-glutamine, 10% vol/vol fetal bovine serum, and 0.02% insulin, without any antibiotics in a humidified atmosphere at 37°C. To determine the function of the novel sequence of B⁰AT1, MDA-MB-231 cells were transiently transfected with B⁰AT1 cDNA. When the cells attained 70 to 90% confluency, generally at 24 h postseeding, 1.6 μg B⁰AT1 plasmid was transiently transfected into the cells by use of Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. pcDNA 3.1 vector without the B⁰AT1 cDNA was used as control. The transfected cells were used for transporter assay 72 h posttransfection.

Amino acid transport studies were performed in plastic plates using triplicate wells for each time point. Cells were rinsed once with oxygenated Leibovitz's (L-15, 100% oxygen) medium (pH 7.4; pH was adjusted with tetramethyl ammoniun (TMA)-OH) supplemented with 10% fetal calf serum, 20 mM HEPES, 200 U/l insulin (0.02%) and incubated with L-15 at room temperature for 1 h. Cells were washed once with TMA-HEPES buffer (4.7 mM KCl, 1 mM MgSO₄, 1.2 mM KH₂PO_4, 20 mM HEPES, 125 mM CaCl₂, 130 mM TMA-Cl, pH 7.4; pH was adjusted with TMA-OH) and incubated with TMA-HEPES (Na-free) buffer for 10 min in room temperature. Cells were treated with reaction mixture containing 2 nmol of cold glutamine and [³H]glutamine, in TMA-HEPES buffer (Na-free) and with 130 mM NaCl (Na-HEPES) for a specified time. The reaction was stopped and washed twice with ice-cold TMA-HEPES (Na-free) buffer. NaOH (1 M, 800 μl) was added in each well and incubated for 30 min at 70°C to digest the cells. Cell extracts were transferred into a scintillation vial and 5 ml of scintillation fluid (Ecoscint A) was added and counted [³H]glutamine in a scintillation counter.

Western blot studies.

Polyclonal antibody against B⁰AT1 was raised in goat by using the custom antibody services provided by Invitrogen. Antigenic peptide with the sequence-CDRFNKDIEFMIGHKPN-coupled to Keyhole limpet hemocyanin was used as the immunogen. Western blotting for B⁰AT1 was performed according to the standard protocols (2). Briefly, plasma membranes from villus cells were solubilized in RIPA buffer (50 mM Tris·HCl pH 7.4, 1% Igepal, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 mM Na₃VO₄, 1 mM NaF) containing protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). Equal volume of 2× SDS/sample buffer (100 mM Tris, 25% glycerol, 2% SDS, 0.01% bromphenol blue, 10% 2-ME, pH 6.8) was added and the proteins were separated on a 4–20% Ready Gel (Bio-Rad Laboratories, Hercules, CA). The separated proteins were transferred on to polyvinylidene difluoride membrane and probed with the primary antibody against rabbit B⁰AT1. Secondary antibody coupled to horseradish peroxidase was used to monitor the binding of the primary antibody. ECL Western blotting detection reagent (GE Healthcare Bio-Sciences, Piscataway, NJ) was used to detect the B⁰AT1 specific signal. The resultant chemiluminescent signals were quantitated by using a Molecular Dynamics (Sunnyvale, CA) densitometric scanner. All experiments were performed in triplicate.

IHC studies.

Localization of B⁰AT1 protein was determined by immunohistochemistry (IHC). Normal rabbit small intestine was fixed in 10% (vol/vol) neutral-buffered formalin and embedded in paraffin blocks according to the standard protocols. Sections 4 μm thick were made by using a microtome and were mounted on glass slides. The sectioned tissues were deparaffinized in xylene and were hydrated gradually by graded ethanol. The sectioned tissues were incubated in 10 mM sodium citrate buffer, pH 6.0, at 95°C for 5 min to retrieve the antigens. Nonspecific binding sites in tissue sections were blocked by incubation with bovine normal serum (10%, 20 min, room temperature). The tissue sections were incubated overnight (4°C) with 1:200 diluted B⁰AT1 primary antibody. Excess antibody was removed by washing with PBS (5 min, 3 times) and incubated with (1:1,000) bovine anti-goat IgG secondary antibody coupled to tetramethyl rhodamine isothiocyanate (TRITC; Santa Cruz Biotechnology, Santa Cruz, CA) at room temperature for 1 h. Excess secondary antibody was removed by washing with PBS (5 min, 3 times) and the tissue sections were mounted with ProLong Gold Antifade Reagent (Invitrogen Life Technologies). Finally, the signals generated by TRITC were observed under Zeiss LSM510 confocal microscope and photographed.

Protein determination.

Total protein was measured by using the Bio-Rad protein assay kit with BSA as standard.

Statistical analysis.

Data were analyzed by Student's t distribution using mean values and the associated standard errors. A comparative P value of less than 0.05 was considered significant.

RESULTS

Na-dependent glutamine uptake in villus cell BBMV.

To determine the presence of Na-glutamine cotransport in the BBM of villus cells, glutamine uptake was performed in the presence or absence of Na at given time points (Fig. 1). Na-dependent glutamine uptake, defined as ³[H]glutamine uptake in the presence of Na minus uptake in the absence of Na, was present in villus cells. These data indicate that an active Na-dependent glutamine cotransporter is present in villus cell BBM from the rabbit small intestine.

Fig. 1.

Effect of extravesicular Na⁺ on [³H]glutamine uptake in villus cell brush border membrane (BBM) vesicles (BBMV) from rabbit small intestine. BBMV were preloaded with the uptake buffer containing 100 mM choline chloride. Glutamine uptake was performed at room temperature under voltage-clamped conditions with the extravesicular uptake buffer containing 100 mM NaCl (+Na) or ChCl (−Na) and 10 μCi [³H]glutamine (see materials and methods for details). Uptake of [³H]glutamine was significantly stimulated by extravesicular Na⁺. These data indicate that an active, Na-glutamine cotransport system is present in the BBM of rabbit small intestinal villus cell.

Effect of intravesicular pH on glutamine uptake.

To determine the effect of pH on Na-dependent glutamine uptake in villus cell BBMV, experiments were performed with or without an inward directed pH gradient. As shown in Fig. 2, Na-dependent glutamine uptake was 72.6 ± 5.5 pmol·mg protein⁻¹·9 s when intravesicular pH was equal to extravesicular pH (7.4). Furthermore, when intravesicular pH was 6.0 and extravesicular pH was 7.4, Na-dependent glutamine uptake was 65.5 ± 4.2 pmol·mg protein⁻¹·9 s. These data reveal that Na-dependent glutamine cotransporters in villus cell BBMV are not stimulated by intravesicular proton load. Also, these data indicate that Na-dependent glutamine cotransporter in the BBM villus cell is not a proton-amino acid antiporter. Thus these data are consistent with the observation that the Na-dependent glutamine cotransporter in the BBM of villus cell is not system N.

Fig. 2.

Effect of intravesicular pH on Na-dependent [³H]glutamine uptake in villus cell BBMV from rabbit small intestine. BBMV were preloaded with either pH 6.0 or 7.4 buffer containing 100 mM choline chloride. Glutamine uptake was performed at 9 s in room temperature under voltage-clamped conditions with pH 7.4. Extravesicular uptake buffer contained 100 mM NaCl and 10 μCi [³H]glutamine. Lower intravesicular pH (6.0) did not stimulate glutamine uptake compared with intravesicular pH 7.4 (n = 6, P = NS). These data demonstrate that small intestinal villus cell Na-glutamine cotransporter is an Na-dependent amino acid cotransporter and not an amino acid/proton antiporter. Thus the Na-dependent glutamine cotransporter in the BBM of villus cell is not system N.

Effect of Li⁺ on glutamine uptake.

To determine the identity of Na-dependent glutamine cotransporter present in villus cell BBM, uptake studies in villus cell BBMV were performed in presence of Na⁺ or Li⁺. Na-dependent glutamine uptake was 97.4 ± 7.5 pmol·mg protein⁻¹·30 s. In contrast, when Li⁺ was substituted for Na⁺, glutamine uptake was 2.2 ± 0.1 pmol·mg protein⁻¹·30 s (Fig. 3). These data demonstrate that Li⁺, a monovalent cation, significantly inhibited glutamine uptake in villus cell BBMV. Since Na-dependent glutamine cotransport in the BBM of villus cell is Li intolerant, Na-dependent glutamine cotransporter in the BBM of villus cell is unlikely to be system A.

Fig. 3.

The villus cell Na-glutamine cotransporter is not Li⁺ tolerant. BBMV were preloaded with pH 7.4 buffer containing 100 mM choline chloride. Glutamine uptake was performed at 30 s in room temperature under voltage-clamped conditions with extravesicular uptake buffer containing 100 mM NaCl or LiCl and 10 μCi [³H]glutamine. Li⁺ was unable to replace Na⁺ as a monovalent cation (n = 6, *P < 0.01). These data indicated that the villus cell glutamine cotransporter is Li⁺ intolerant, which rules out the presence of system A transporter.

Substrate specificity of Na-dependent glutamine cotransporter in villus cell BBMV.

To determine which Na-dependent glutamine cotransporter is predominantly present in the villus cell BBM, uptake studies in villus cell BBMV were carried out using different amino acid substrates at a concentration of 10 mM. Substrate specificity of Na-dependent glutamine cotransporters in villus cell BBMV is shown in Table 1. Inhibition studies demonstrated that MeAIB inhibited Na-glutamine uptake in BBMV 59%, whereas Gln, Ala, Ser, and Arg inhibited Na-glutamine uptake from 58 to 66%, and His inhibited it 70%. Thus Na-dependent [³H]glutamine uptake was inhibited by all the amino acid substrates examined, except tryptophan. Tryptophan inhibited the uptake the most (88%). These data indicated that the predominant Na-dependent glutamine cotransporter in the BBM of rabbit intestinal villus cells is B⁰AT1.

Table 1.

Substrate specificity of Na-dependent glutamine cotransporters in rabbit small intestinal villus cell BBMV

Competing Amino Acids	Substrate concentration, mM	Na-Dependent [3H]Gln Uptake, pmol/mg protein	% Inhibition
None		97.37±6.44	0
Gln	10	32.96±2.48	66.15
Ala	10	36.19±2.63	62.83
Ser	10	40.94±3.16	57.95
MeAIB	10	40.05±3.42	58.86
Arg	10	37.89±3.06	61.08
Trp	10	11.98±0.51	87.69
His	10	29.42±0.03	69.78

Uptake of 10 μCi[³H]glutamine was performed at conditions with intravesicular choline chloride buffer (pH 7.4) and extravesicular NaCl buffer (pH 7.4) in the absence or presence of 10 mM unlabeled amino acid substrates at 30 s in room temperature. Glutamine (Gln) uptake values without any inhibitor were used as control (100% uptake) and inhibitions obtained by various cold substrates were expressed as percent of control (n = 6). Tryptophan (Trp) inhibited the maximum Gln uptake compared with others, indicating that B⁰AT1 is most likely the major Na-dependent Gln cotransporter in rabbit small intestinal villus cell. BBMV, brush border membrane vesicle.

Kinetics of glutamine cotransport in villus cell BBMV.

To determine the maximal rate of uptake (V_max) and affinity (1/K_m) for Na-dependent glutamine uptake, kinetic studies were performed. Figure 4 demonstrates the kinetics of Na-dependent glutamine uptake in villus cell BBMV. It shows the uptake of glutamine as a function of varying concentrations of extravesicular glutamine. As the extravesicular concentration of glutamine was increased, the uptake of glutamine was stimulated and subsequently became saturated. When these data were analyzed with GraphPad Prism 4 software, the kinetic parameters derived from these data demonstrated that the V_max of glutamine was 4.7 ± 0.3 pmol·mg protein⁻¹·6 s, and the K_m for glutamine was 34.3 ± 2.6 mM.

Fig. 4.

Kinetics of the Na-dependent [³H]glutamine uptake in villus cell BBMV from rabbit small intestine. Data are representative of 3 experiments with uptake done in triplicates. Uptake of Na-dependent [³H]glutamine as a function of varying concentrations of extravesicular glutamine is shown. Uptake for all concentrations was measured at 6 s. Isosmolarity was maintained by adjusting the concentrations of mannitol. As extravesicular glutamine concentrations was increased, uptake of Na-dependent [³H]glutamine was stimulated and subsequently became saturated. Analysis of these data with GraphPad Prism 4 software (Michaelis-Menten) yielded the kinetic parameters. The maximal rate of uptake (V_max) of glutamine was 4.7 ± 0.2 pmol·mg protein⁻¹·6 s⁻¹ and the affinity (K_m) for glutamine was 34.3 ± 2.6 mM.

Molecular identity of rabbit villus cell BBM B⁰AT1.

To determine the molecular identity and characteristics of the rabbit small intestinal villus cell BBM Na-glutamine cotransport, we cloned the rabbit villus cell B⁰AT1 cDNA. First, using the rabbit genomic sequence available from the public database, we identified the nucleotide sequence corresponding to the NH₂ and COOH termini of rabbit B⁰AT1. Then we designed rabbit B⁰AT1 specific primers. The sense primer harbored Kozak consensus sequence upstream of the start codon, and the antisense primer harbored the stop codon. The open reading frame of B⁰AT1 thus obtained was 1,905 base pairs long (GenBank accession number Bankit1009248 EU095941). The cDNA encoded a putative protein of 634 amino acids long. Compared with human B⁰AT1, rabbit B⁰AT1 protein exhibited 84% identity and 93% similarity (Fig. 5A). Rabbit B⁰AT1 contained nine predicted transmembrane domains. Rabbit B⁰AT1 protein did not have NH₂-terminal signal peptide as indicated by pSORT results, and the location of the NH₂ terminus was predicted to be intracellular.

Fig. 5.

A: amino acid comparison of human (hB⁰AT1) and rabbit B⁰AT1 (rB⁰AT1). B: B⁰AT1 mRNA abundance in villus cells compared with crypt cell from rabbit intestine (n = 9, *P < 0.01). These data indicate that Na-dependent glutamine cotransporter, B⁰AT1 is primarily expressed in villus cell in the rabbit intestine.

Real-time PCR for B⁰AT1 transporter.

To determine the distribution of B⁰AT1 message along the villus-crypt axis of the rabbit intestine, RTQ-PCR was performed using B⁰AT1-specific primers and probes. As shown on Fig. 5B, B⁰AT1 message is roughly 11 times more in villus cells (P < 0.01, n = 3) compared with the crypt cells. Thus this Na-glutamine cotransporter is predominantly expressed in the absorptive villus cells in the rabbit small intestine.

Western blotting studies.

Since mRNA levels do not necessarily correlate with immunoreactive B⁰AT1 protein, Western blot studies were performed in crypt and villus cell BBMV using anti-B⁰AT1 polyclonal antibody. A representative Western blot is shown in Fig. 6A where the position of the 75-kDa protein corresponding to B⁰AT1 is marked with an arrow. Figure 6B shows the quantitative data of B⁰AT1 protein expression in villus and crypt cells. These data indicated that B⁰AT1 protein expression is four times higher in the villus cells compared with the crypt cells (P < 0.05, n = 3). Taken together, the functional and molecular data indicate that B⁰AT1 is the major Na-glutamine cotransporter expressed in the villus but not crypt cells in the rabbit intestine.

Fig. 6.

Western blot analysis for the Na-dependent glutamine cotransporter B⁰AT1 in rabbit small intestinal villus cell BBM was compared with crypt cell BBM. A: data are representative of 3 such experiments. B⁰AT1 was probed by the primary goat polyclonal antibody for rabbit B⁰AT1. B: the intensity of the bands was quantitated by use of a densitometric scanner; see materials and methods for details. Data represent means ± SE, n = 3. These results indicate that Na-dependent glutamine cotransporter B⁰AT1 is predominantly present in rabbit small intestinal villus cell BBM.

Immunohistochemical analysis.

To conclusively demonstrate that B⁰AT1 is localized to the villus cells, immunolocalization experiments were performed by using anti-B⁰AT1 polyclonal antibody. Figure 7A shows a representative IHC photograph, and the results of these experiments showed that B⁰AT1 is localized specifically to the villus, not the crypt cells. Specificity of the signals obtained in the villus cells by B⁰AT1 antibody is presented in a parallel experiments by preincubating the primary antibody with 100-fold excess immunogenic peptide prior to the addition to the tissue sections (Fig. 7B). Excess peptide completely abolished the B⁰AT1 antibody binding, demonstrating that the signal obtained by the primary antibody is specific to B⁰AT1.

Fig. 7.

Immunohistochemical analysis of the expression of B⁰AT1 in normal rabbit small intestinal segments. Tissue sections were subjected to immunostaining in the absence and in the presence of the blocking peptides. A: localization of B⁰AT1 to the villus cells. B: no signal was detected in the presence of a 100-fold excess blocking peptide, which indicates that the antibody was specific to B⁰AT1.

Characterization of the cloned rabbit B⁰AT1 cotransporter.

Next, to determine the function of the cloned rabbit B⁰AT1 cotransporter, MDA-MB-231 cells were transiently transfected with rabbit B⁰AT1 cDNA. MDA-MB-231 was chosen because Na-dependent [³H]glutamine uptake was lowest in MDA-MB-231 cells compared with other cells, including IEC-18 and IA-XsSBR cells (CRL-1589 and CRL-1677, American Type Culture Collection). As shown in Figure 8, Na-dependent [³H]glutamine uptake was 141 ± 10.4 nmol·mg protein⁻¹·2 min in pCDNA 3.1-transfected cells. In contrast, Na-dependent [³H]glutamine uptake was 239 ± 18.2 nmol·mg protein⁻¹·2 min in B⁰AT1 cDNA-transfected cells (n = 6, P < 0.05). Thus these data indicated that the villus cell BBM Na-dependent glutamine cotransporter B⁰AT1 is indeed expressed in MDA-MB-231 cells.

Fig. 8.

Rabbit B⁰AT1 expression and Na-dependent [³H]glutamine uptake in MDA-MB-231 cells. Cells were transiently transfected with vector (pcDNA 3.1) or rabbit B⁰AT1 cDNA. Glutamine uptake was performed for 2 min at room temperature with the uptake buffer containing 130 mM Na- or TMA-Cl and 10 μCi [³H]glutamine (n = 6). Na-dependent glutamine uptake was obtained as glutamine uptake with Na-free buffer deducted from the uptake in the presence of Na. To determine the B⁰AT1 expression-dependent values, Na-dependent glutamine uptake in cells transfected with vector was deducted from Na-dependent glutamine uptake in B⁰AT1 cDNA-transfected cells. B⁰AT1 cDNA-transfected cells showed higher Na-dependent [³H]glutamine uptake compared with the vector-transfected cells. These data indicate that rabbit B⁰AT1, the Na-dependent glutamine cotransporter, is highly expressed in MDA-MB-231 cells.

Substrate specificity of the cloned rabbit B⁰AT1.

To determine whether the cloned B⁰AT1 cDNA exhibits the same characteristics of the native one, substrate inhibition studies were performed. Substrate specificity following transient transfection of MDA-MB-231 cells by rabbit B⁰AT1 cDNA is shown in Table 2. [³H]glutamine uptake was performed with the buffer containing 130 mM Na- or TMA-Cl 10 μCi labeled substrate and 2 nmol unlabeled substrate (glutamine) at 2 min (n = 6). Uptake buffer also contained 5 mM of MeAIB and Arg to suppress endogenous amino acid uptake. Glutamine uptake values without any inhibitors was used as control (100% uptake) and inhibitions obtained with various cold substrates were expressed as percent of control. Na-dependent B⁰AT1 cDNA specific glutamine uptake was inhibited more than 70% with 10 mM concentration of Gln, Trp, Ser, and Asn but less than 22% by Glu and Asp. Among the substrates, Trp inhibited maximally whereas Asp minimally inhibited glutamine uptake in B⁰AT1 cDNA expressed cells. These data indicated that B⁰AT1 can be highly expressed in MDA-MB-231 cells following transient transfection and demonstrates functions similar to that in rabbit small intestinal villus cells.

Table 2.

Substrate specificity of rabbit B⁰AT1 cotransporter

Amino Acids	Substrate Concentration, mM	Na-Dependent Gln Uptake, nmol/mg protein	% Control, inhibition
Control	Gln: 0.002; MeAIB: 5; Arg: 5	127.3±9.65	0
Gln	10	33.10±2.59	73.99
Trp	10	24.6±1.94	80.67
Ser	10	28.63±2.13	77.50
Asn	10	25.72±2.11	79.79
Glu	10	104.88±7.36	17.60
Asp	10	99.59±6.16	21.76

MDA-MB-231 cells were transiently transfected with vector [pcDNA 3.1] or rabbit B⁰AT1 cDNA. [³H]glutamine uptake was performed with the buffer containing 100 mM Na- or TMA-Cl, 10 μCi [³H]Gln and 2 nM unlabeled substrate at 2 min in room temperature (n = 6). Uptake buffer also contained 5 mM of l-methyl-aminoisobutyric acid (MeAIB) and Arg to suppress endogenous amino acid uptake in MDA-MB-231 cell. [³H]Gln uptake values obtained with extracellular Na-free buffer were used as background and deducted from the total uptake values obtained in the presence of Na to calculate Na-dependent glutamine uptake. Na-dependent Gln uptake values without any inhibitor was used as control (100% uptake) and inhibitions obtained by various cold substrates were expressed as percent of control (n = 6). Trp inhibited the maximum, and in contrast Glu and Asp inhibited the minimum Gln uptake compared with others, indicating that rabbit B⁰AT1 is highly expressed in MDA-MB-231 cells and is the major Gln cotransporter.

DISCUSSION

In the mammalian small intestine amino acids are primarily absorbed by Na-dependent amino acid cotransporters. These cotransporters are secondarily active depending on Na⁺-K⁺-ATPase to provide the favorable transcellular Na⁺ gradient (7, 8). Furthermore, to date they have been shown to be present on the BBM of absorptive villus cells but not secretory crypt cells in the mammalian small intestine (1). Glutamine is an important amino acid because it is the preferred nutrient for the intestinal enterocytes (7, 21). A number of amino acid transport systems have been reported in the intestine of various animals and human and some of these are capable of transporting glutamine (10, 11, 15). However, specifically which Na amino acid cotransporter is primarily responsible for the assimilation of glutamine in the normal mammalian small intestine was previously unknown.

This study for the first time has identified, cloned, and characterized the Na-dependent glutamine cotransporter in the absorptive villus cells of the rabbit small intestine. Na-stimulated glutamine uptake was present in villus cell BBM (Fig. 1). Both system N and system A have been reported to be present in different organs of various species and are capable of transporting glutamine (3, 4, 9, 10). However, both system N and system A are stimulated by an intravesicular proton load (5). Our studies demonstrated that the Na-glutamine cotransporter in the rabbit intestine villus cell BBM is not stimulated by an intravesicular proton load (Fig. 2). Thus the rabbit intestinal villus cell Na-glutamine cotransporter is not system N or system A. Furthermore, our studies demonstrated that the villus cell BBM Na-glutamine cotransporter is not lithium tolerant (Fig. 3). These data also indicate that this Na-glutamine cotransporter also does not belong to system A. Subsequently the substrate inhibition studies of the Na-dependent glutamine cotransporter in the villus cell BBM demonstrated that the system that is most likely responsible for Na-glutamine cotransport in the villus cell BBM is B⁰AT1 (Table 1). Kinetic studies demonstrated that the Na-glutamine cotransporter in the villus cell BBM follows Michaelis-Menten kinetics. The V_max of this cotransporter was 4.66 pmol·mg protein⁻¹ at 6 s. The K_m for this cotransporter was 34.3 mM (Fig. 4).

In view of the data to this point, which suggested that the rabbit intestinal villus cell BBM Na-glutamine cotransporter is B⁰AT1, primers and probes were designed for quantitative PCR of this cotransport process. RTQ-PCR demonstrated that B⁰AT1 mRNA is abundantly present in villus cells compared with the crypt cells from the normal rabbit small intestine (Fig. 5B). This cotransporter was then cloned. To characterize this cloned cotransporter it was transiently expressed in MDA-MB-231 cells. The expressed cotransporter also had substrate specificity that is consistent with B⁰AT1 properties. Specifically, it is known that Gln, Trp, Ser, and Asn are substrates for B⁰AT1, and indeed all four of these amino acids significantly inhibited the transport of radiolabeled glutamine by the expressed cotransporter (Table 2). However, it is known that Glu and Asp are not substrates for B⁰AT1, and once again these two amino acids did not inhibit the uptake of glutamine by the expressed cotransporter. Given that the data indicated that B⁰AT1 is the predominant Na-amino acid cotransporter responsible for glutamine assimilation in the villus cells of the rabbit intestine, polyclonal antibody for B⁰AT1 was raised in goat. Western blot and immunohistochemical studies using this antibody demonstrated that B⁰AT1 is indeed present in the BBM of the rabbit intestinal villus, but not crypt cells (Figs. 6A and 7).

Thus this study has identified, cloned, and characterized the Na glutamine cotransporter responsible for glutamine absorption in the villus cell of the rabbit small intestine. It is well established that glutamine is the preferred substrate for the mammalian small intestinal enterocytes. Thus it is important for the normal growth and maturation of the intestine. Perhaps it is even more vitally important in pathophysiological states of the small intestine where there is substantial loss of the absorptive surface such as conditions characterized by chronic inflammation [e.g., inflammatory bowel disease (IBD)]. In these conditions, return to health of the mucosa will undoubtedly require the preferred nutrient glutamine. Identification and characterization of the primary transporter responsible for glutamine assimilation in the normal small intestine will allow us to explore the alterations in this transporter during chronic intestinal inflammation, which would potentially allow for more efficacious treatment modalities for this very important intestinal disorder.

It is likely that B⁰AT1 is affected in chronic intestinal inflammation because other amino acid assimilation pathways have previously been demonstrated to be affected. For example, Na-neutral amino acid cotransport, specifically Na-alanine cotransport, mediated by ASCT1 in the rabbit small intestine is inhibited during chronic intestinal inflammation (18). The inhibition in ASCT1 is not secondary to an alteration in the Na⁺ extruding capacity of the villus cell but rather a direct effect on the cotransporter at the BBM level of the villus cells. It was demonstrated that the mechanism of inhibition of ASCT1 was likely secondary to a decrease in the affinity of the cotransporter for alanine without an alteration in the number of cotransporters on the BBM (18). Likewise it was also previously demonstrated that the proton dipeptide cotransporter PEPT1, yet another pathway of assimilation of amino acids in the intestine, was also significantly altered during chronic intestinal inflammation (18). PEPT1 was present in villus but not crypt cells of the rabbit small intestine. PEPT1 was significantly inhibited during chronic intestinal inflammation. Further kinetic studies demonstrated that the mechanism of this inhibition was secondary to a reduction in the affinity of the cotransporter rather than an alteration in the number of cotransporters on the BBM of villus cells (18).

Fortunately as it relates to the treatment of IBD, these alterations in the chronically inflamed intestine lend themselves to modulation. For example, it was previously demonstrated that a very common modality of treatment of IBD, specifically corticosteroids, had significant effects on the altered ASCT1 during chronic intestinal inflammation (19). It was demonstrated that corticosteroids reversed the inhibition of ASCT1 during chronic intestinal inflammation. Furthermore, it was of note that the mechanism of reversal was specifically secondary to a restoration in the affinity of the cotransporter without altering the number of cotransporter in the chronically inflamed rabbit intestine. This suggested that corticosteroids, being a broad-spectrum immune modulator, more than likely inhibited the immune-inflammatory mediator that was responsible for the inhibition of this Na-amino acid cotransporter. Although these studies demonstrated that amino acid and dipeptide transport are significantly affected during chronic intestinal inflammation, how the amino acid that is the primary metabolite for intestinal epithelial cells might be affected in this condition was unknown. The reason for this was that it was previously unknown specifically which Na-amino acid cotransporter was responsible for the assimilation of glutamine in the mammalian small intestine. Having identified this cotransporter as B⁰AT1, this study now provides the means to further delineating its role in chronic intestinal inflammation.

In conclusion, this study has for the first time identified the Na-amino acid cotransporter in the mammalian small intestinal absorptive villus cells responsible for the assimilation of the most important nutrient for the enterocytes as B⁰AT1. B⁰AT1 is functionally present in villus cells; it was cloned and characterized from the rabbit intestinal villus cells. Finally, the functional protein and its message were also shown to be present in the rabbit small intestinal villus, but not crypt cells.

GRANTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Research Grants DK-45062 and DK-58034 to U. Sundaram.