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Am J Physiol Gastrointest Liver Physiol 294: G1301–G1310, 2008. First published April 3, 2008; doi:10.1152/ajpgi.00578.2007.

TRANSLATIONAL PHYSIOLOGY

Functional characterization, localization, and molecular identification of rabbit intestinal N-amino acid transporter Jamilur R. Talukder,1 Ramesh Kekuda,1 Prosenjit Saha,1 Puttur D. Prasad,2 Vadivel Ganapathy,2 and Uma Sundaram1 1

Section of Digestive Diseases, Department of Medicine, West Virginia University School of Medicine, Morgantown, West Virginia; and 2Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, Georgia Submitted 14 December 2007; accepted in final form 3 April 2008

Talukder JR, Kekuda R, Saha P, Prasad PD, Ganapathy V, Sundaram U. Functional characterization, localization, and molecular identification of rabbit intestinal N-amino acid transporter. Am J Physiol Gastrointest Liver Physiol 294: G1301–G1310, 2008. First published April 3, 2008; doi:10.1152/ajpgi.00578.2007.—We have characterized the Na-glutamine cotransporter in the rabbit intestinal crypt cell brush border membrane vesicles (BBMV). Substrate specificity experiments showed that crypt cell glutamine uptake is mediated by system N. Real-time PCR experiments showed that SN2 (SLC38A5) mRNA is more abundant in crypt cells compared with SN1 (SLC38A3), indicating that SN2 is the major glutamine transporter present in the apical membrane of the crypt cells. SN2 cDNA was obtained by screening a rabbit intestinal cDNA library with human SN1 used as probe. Rabbit SN2 cDNA encompassed a 473amino-acid-long open reading frame. SN2 protein displayed 87% identity and 91% similarity to human SN2. Functional characterization studies of rabbit SN2 were performed by using vaccinia virusmediated transient expression system. Substrate specificity of the cloned transporter was identical to that of SN2 described in the literature and matched well with substrate specificity experiments performed using crypt cell BBMV. Cloned rabbit SN2, analogous to its human counterpart, is Li⫹ tolerant. Hill coefficient for Li⫹ activation of rabbit SN2-mediated uptake was 1. Taken together, functional data from the crypt cell BBMV and the cloned SN2 cDNA indicate that the crypt cell glutamine transport is most likely mediated by SN2.

a variety of nutrients and solutes are absorbed via Na-solute cotransport processes. Under normal physiological conditions, villus cells are responsible for most of the nutrient absorption, whereas crypt cells act as secretory cells (16, 22). This vectorial distribution of function is reflected in the nutrient transporter distribution in the villus and crypt cells. For example, it has been demonstrated that Na-glucose, Na-amino acid (alanine), and Na-bile acid cotransporters are found on the brush border membrane (BBM) of the villus cells but not in the crypt cells (29 –31). One of the most common pathophysiological states of the small intestine is inflammatory bowel disease (IBD). The Na gradient is the driving force for many Na-nutrient cotransport processes like amino acid, and sugar absorption (10). Using a

rabbit model of chronic intestinal inflammation, our laboratory has shown that the function of several solute transporters, both Na dependent and Na independent, are affected during chronic intestinal inflammation (27). For example, during chronic intestinal inflammation, we observed a decrease in the transporter activity of short-chain fatty acid-bicarbonate exchanger (19), H-dipeptide cotransporter (28), Na-amino acid transporter (29), Na-glucose cotransporter 1 (SGLT-1; 30), and Na-bile acid transporter (31). It was demonstrated that the mechanism of inhibition was secondary to a decrease in the de novo synthesis of SGLT-1 in villus cells (30). In contrast, Na-neutral amino acid cotransport, which is also known to be present only on the BBM of the absorptive villus cells in the normal mammalian small intestine, was inhibited during chronic intestinal inflammation by a decrease in the affinity of the cotransporter for its substrate without a change in the number of cotransporters (29). The most important amino acid in the bloodstream is glutamine. It accounts for 20% of the total amino acid content in the human bloodstream (35). Glutamine is the preferred substrate for the intestinal enterocytes. Glutamine is not only important for the functioning of the small intestinal enterocytes but also critical for mucosal cell integrity and gut barrier (17, 33). It is a precursor for the rapidly growing mucosal cell’s nucleic acid and protein synthesis (7). Furthermore, as important as glutamine is for the normal small intestine, it may be even more important in pathophysiological states of the small intestine to help restore it back to health (12, 13, 18, 23, 32). During chronic intestinal inflammation, villus cells undergo atrophy and crypt cells hypertrophy. Because of decreased villus cell numbers and BBM surface area, absorption of glutamine by the villus cells is likely diminished. Since rapidly dividing crypt cells are in need of glutamine for their metabolic processes, we speculated that under these conditions crypt cells perform at least part of the absorptive functions, especially glutamine. Del Castillo et al. (9), using substrate specificity experiments, reported that the villus and crypt cells express Na⫹-dependent glutamine transporter in the guinea pig small intestine, and they suggested that system ASC (alanine/serine/cysteine transporter) is responsible for the glutamine uptake. However, to date, the identity of

Address for reprint requests and other correspondence: U. Sundaram, Section of Digestive Diseases, Dept. of Medicine, West Virginia Univ., Morgantown, WV 26506 (e-mail: [email protected]).

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.

Na glutamine cotransporter; small intestine; brush border membrane; system N-amino acid transporter IN THE MAMMALIAN SMALL INTESTINE,

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glutamine transporters at the molecular level has not been shown in crypt cells. Given this background, the objectives of this study were to determine the presence of apical Na-glutamine cotransporters in rabbit intestinal crypt cells and to decipher the molecular identity of these transporters. METHODS

Crypt cell isolation. Pathogen-free male New Zealand White rabbits, weighing 2–2.5 kg, were euthanized according to the protocol approved by the West Virginia University animal care and use committee. Crypt cells were isolated from the rabbit small intestine by a calcium chelation technique as previously described (27, 30). A 3-ft section of ileum was removed and washed free of fecal material with a buffer containing (in mM) 112 NaCl, 25 NaHCO3, 2.4 K2HPO4, 0.4 KH2PO4, 2.5 L-glutamine, 0.5 ␤-hydroxybutyrate, and 0.5 dithiothreitol, gassed with 95% O2-5% CO2, pH 7.4 (buffer A). The ileal loop was filled with buffer A and incubated for 10 min at 37°C. This solution was discarded as it contained primarily mucus, bacteria, and other luminal contents. The ileal loop was then filled with buffer A supplemented with 0.15 mM EDTA (buffer B) and was incubated at 37°C for 3 min and gently palpitated for another 3 min to facilitate cell dispersion. The fluid was then drained from the small intestinal loop and the cell suspension was retained. Similarly, five more cell fractions were collected. These six fractions represented the sequential collection of cells from the villus tip to the crypt base. Enzyme markers, morphology, and transporter specificity were used to assure good separation of crypt and villus cells. Phenylmethylsulfonyl fluoride was added to all the fractions to a final concentration of 1 mM, and the suspensions were centrifuged at 500 g for 3 min. The pellets from fractions 5 and 6, which contained crypt cells, were stored at ⫺80°C until used. BBMV preparation. BBM vesicles (BBMV) from rabbit intestinal crypt cells were prepared by CaCl2 precipitation and differential centrifugation as previously described (27, 30). Briefly, frozen crypt cells were thawed and suspended in 2 mM Tris 䡠 HCl buffer (pH 7.0) containing 50 mM mannitol. The suspension was homogenized and CaCl2 was added to 10 mM final concentration. 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 suspended in 10 mM Tris 䡠 HCl buffer, pH 7.5, containing 100 mM mannitol, and homogenized. MgSO4 was added to the suspension to a final concentration of 10 mM. The suspension was centrifuged at 2,000 g for 15 min to remove debris, and the BBMV were collected by centrifugation at 27,000 g for 30 min. BBMV were resuspended in a medium appropriate to each experiment. BBMV purity was assured with marker enzyme enrichment (e.g., alkaline phosphatase). Uptake studies in crypt cell BBMV. BBMV uptake studies were performed by the rapid-filtration technique as previously described (26, 29). In brief, 5 ␮l of BBMV, resuspended in the buffer containing 100 mM choline chloride, 0.1 mM MgSO4, 100 mM HEPES-Tris (pH 7.4), 50 mM mannitol, and 50 mM KCl, were incubated in 95 ␮l of the reaction medium containing 100 mM HEPES-Tris buffer (pH 7.4), 0.2 mM glutamine, 10 ␮Ci [3H]glutamine, 0.1 mM MgSO4, 50 mM KCl, 50 mM mannitol, and 100 mM of either NaCl or choline chloride. Whenever pH 6.0 uptake buffers was needed, 100 mM HEPES-Tris was replaced with 100 mM MES-Tris. The vesicles were voltage clamped with 5.6 ␮M valinomycin and 15 ␮M carbonyl cyanide p-trifluoromethoxyphenylhydrazone. At desired times, uptake was arrested by mixing with ice-cold stop solution (50 mM HEPESTris buffer, pH 7.4, 0.1 mM MgSO4, 50 mM KCl, and 100 mM choline chloride). The mixture was filtered on 0.45-␮m Millipore (HAWP) filters and washed twice with 5 ml of ice-cold stop solution. Filters with BBMV were dissolved in Ecoscint scintillation fluid, and radioactivity retained on the filters was counted in a Beckman Coulter LS 6500 liquid scintillation counter.

RTQ-PCR studies. Total RNA was isolated from crypt cells using TRIzol reagent from Invitrogen Life Technologies (Carlsbad, CA). 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 using SuperScript III from Invitrogen Life Technologies by 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. Rabbit SN2-specific primer and probe sequences were as follows: forward primer, 5⬘-CTGGGACAGAGGGCATTC-3⬘; reverse primer, 5⬘-CGGATTTGATGATGAACAGGT-3⬘; TaqMan probe, 5⬘-FAM-CCACCGTCATCTGTCTGCACAATGTTG-TAMRA-3⬘. The primers and probes for rabbit SN1 were generated by using the rabbit SN1 cDNA sequence (V. Ganapathy, unpublished results). The sequence of SN1-specific primers and probes were as follows: forward primer, 5⬘-GGCAGGGGTTTCCTACAGA-3⬘; reverse primer, 5⬘-CGATGTCTTCCCCTCGAA-3⬘; TaqMan probe, 5⬘-FAM-AGCCCCAGCAAGGAGCCGCACTT-TAMRA-3⬘. RTQ-PCR for rabbit ␤-actin was run along with the SN1 and SN2 RTQ-PCR as an internal control by using rabbit ␤-actin-specific primers and probes. The expression of ␤-actin was used to normalize the expression levels of SN1 and SN2 between the individual samples. The sequences of the rabbit ␤-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 SN1, SN2, and ␤-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 ␤-actin, SN1, and SN2 transporters (data not shown). Western blot studies. Polyclonal antibody against SN2 was raised in chicken by use of the custom antibody services provided by Invitrogen. Antigenic peptide with the sequence CRIVPSDTEPLFSWPK coupled to keyhole limpet hemocyanin was used as the immunogen. Western blotting for SN2 was performed essentially according to the standard protocols (1, 8). Briefly, BBMV from villus and crypt cells were solubilized in RIPA buffer (50 mM Tris 䡠 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-mercaptoethanol, 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 onto a polyvinylidene difluoride membrane and probed with the primary antibody against rabbit SN2. 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 SN2-specific signal. The resultant chemiluminescent signal was detected by autoradiography and was quantitated with a Molecular Dynamics (Sunnyvale, CA) densitometric scanner. All experiments were performed at least thrice. IHC experiments. Immunolocalization of SN2 protein was determined by immunohistochemistry (IHC) technique. Normal rabbit intestinal tissue was fixed in 10% (vol/vol) neutral-buffered formalin (Richard-Allan Scientific, Kalamazoo, MI) and embedded in paraffin. Sections 4 ␮m thick were taken by use of a microtome and the sections were mounted on glass slides. Paraffin was removed from the sections by incubating the slides with xylene, and sections were hydrated gradually by incubating with graded ethanol. Antigen retrieval was performed by incubating the sections with 10 mM sodium citrate buffer, pH 6, at 95°C for 5 min. Nonspecific binding sites in the tissue sections were blocked by incubation with goat normal serum.

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The tissue sections were then incubated overnight with 1:500 diluted SN2 primary antibody. Excess antibody was removed by washing with PBS and incubated with goat anti-chicken IgG secondary antibody coupled to FITC (Santa Cruz Biotechnology, Santa Cruz, CA). Excess secondary antibody was removed by washing with PBS and the tissue sections were mounted by use of ProLong Gold Antifade Reagent (Invitrogen Life Technologies). Finally, the signal generated by FITC was observed under Zeiss LSM510 confocal microscope and photographed. Probe preparation and cDNA library screening. The human SN1 cDNA was used as a probe to screen rabbit intestinal cDNA library. The probe was prepared as described previously (20). The ⬃0.6 kbp cDNA fragment was labeled with [32P]dCTP via the Ready-to-go oligolabeling kit (GE Healthcare Biosciences, Piscataway, NJ). The rabbit intestinal cDNA library (24) was screened with this probe under low-stringency conditions. DNA sequencing. Both sense and antisense strands of the cDNAs were sequenced by primer walking. Sequencing by the dideoxynucleotide chain termination method was performed by Taq DyeDeoxy terminator cycle sequencing with an automated Perkin-Elmer Applied Biosystems 377 Prism DNA sequencer. The sequence was analyzed using the GCG sequence analysis software package GCG, version 10 (Genetics Computer Group, Madison, WI). Functional expression in mammalian cells. The cloned rabbit SN2 cDNA was functionally expressed in human retinal pigment epithelial (HRPE) cells by the vaccinia virus expression technique (21). Uptake measurements were made at 37°C for 15 min with radiolabeled amino acids. The composition of the uptake buffer was 25 mM Tris-HEPES (pH 8.0), 140 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.8 mM MgSO4, and 5 mM glucose. In some experiments, NaCl was substituted with LiCl isoosmotically. Uptake buffers of different pH were made by appropriately adjusting the concentrations of Tris, HEPES and MES. Data presentation. When data were averaged, means ⫾ SE are shown, except when error bars are inclusive within the symbol. All BBMV uptakes and RTQ-PCR were done in triplicate unless otherwise specified. The number (n) for any set of experiments refers to total RNA, vesicle, protein extracts from vesicles, or isolated cell preparations from different animals. Student’s t-test was used for statistical analysis. RESULTS

Glutamine uptake in crypt cell BBMV. To determine whether crypt cells transported glutamine, uptake of glutamine was determined in the BBMV prepared from the crypt cells for a period of 90 s. Crypt cell BBMV showed a Na-dependent uptake of glutamine in the presence of Na⫹ (Fig. 1). The basal level of uptake obtained with the extravesicular buffer without Na⫹ (62.8 ⫾ 4.1 pmol/mg protein per 90 s) was deducted from the uptake value obtained with the Na⫹-containing buffer (100.7 ⫾ 5.1 pmol/mg protein per 90 s) to obtain Na⫹dependent glutamine uptake (37.8 pmol/mg protein per 90 s). These data indicated the presence of a Na⫹-dependent glutamine cotransporter on the BBM of crypt cells. Effect of pH on glutamine uptake in crypt cells. To determine the effect of pH on glutamine uptake in crypt cell BBMV, glutamine uptake was performed with or without an outward directed pH gradient. As shown in Fig. 2, when the intravesicular pH of BBMV was low (pH 6.0), the uptake was significantly higher (30.3 ⫾ 2.4 pmol/mg protein per 9 s) compared with high intravesicular pH of 7.4 (15.0 ⫾ 1.5 pmol/mg protein per 9 s). These data demonstrate that glutamine transporter in crypt cell BBMV is a proton-amino acid

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Fig. 1. Uptake of Na-dependent glutamine uptake in crypt cell brush border membrane vesicles (BBMV) from normal rabbit small intestine at 90 s. Crypt cell BBMV was preloaded with the uptake buffer containing 100 mM choline chloride. Glutamine uptake was performed under voltage-clamped conditions with the extravesicular uptake buffer containing either 100 mM choline chloride (Na⫹-free buffer; ⫺Na) or 100 mM NaCl (⫹Na) and 10 ␮Ci [3H]glutamine (0.2 mM). Na-dependent glutamine uptake was calculated by deducting Na-independent glutamine uptake from total glutamine uptake. These data indicate that an active, Na-glutamine cotransporter is present in crypt cell BBMV.

antiporter. Furthermore, proton dependence indicated that this is a system A or N transport function. Kinetic studies. To determine maximal rate of uptake (Vmax) and affinity (1/Km, where Km is the Michaelis constant) of the Na-glutamine cotransporter, kinetic studies were performed using the BBMV from the crypt cells. As the concentration of extravesicular glutamine was increased, the Na-dependent glutamine uptake increased, subsequently becoming saturated (Fig. 3). Kinetic parameters were calculated by use of GraphPad Prism 4. The Km of the transporter was determined to be 43.7 ⫾ 2.7 mM and Vmax was determined to be 3.87 ⫾ 0.1 pmol/mg protein per 6 s. These data provide further evidence for the carrier-mediated uptake of glutamine. Functional identification of crypt cell BBMV glutamine transporter. To determine the identity of the glutamine transporter at the functional level, substrate inhibition experiments were performed. Figure 2 demonstrated that crypt cell BBMV glutamine transport is a proton-dependent phenomenon. Of all the Na-amino acid cotransporters characterized today, protondependent transport of glutamine occurs via system A or system N. Of these two, system A can be identified by its ability to transport MeAIB whereas system N has a distinct property of its ability to partially substitute Li⫹ in place of Na⫹ as the driving force for substrate transport (15). When we performed glutamine uptake experiments by substituting Na⫹ by Li⫹, BBMV glutamine transporter was functional, showing 93% of glutamine transport activity in Li⫹ (25.8 ⫾ 2.3 pmol/mg protein per 30 s) compared with Na⫹ containing buffer (27.6 ⫾ 2.1 pmol/mg protein per 30 s; Fig. 4). Furthermore, histidine, a very good substrate for system N compared with system A, inhibited glutamine uptake almost completely at 10 mM concentration (Table 1). Taken together, these data show that the major glutamine transporter in crypt cell BBMV is system N. However, it should be pointed out that when glutamine uptake in crypt cell BBMV was performed in the presence of excess cold MeAIB (10 mM), glutamine uptake was inhibited by 26% (Table 1), indicating the possible presence of system A as well. Real-time PCR for system N transporters. To date, two members of system N family of transporters have been iden-

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Fig. 2. Effect of intravesicular pH on [3H]glutamine uptake in crypt cell BBMV. Crypt cell BBMV was preloaded with either pH 6.0 or pH 7.4 buffer containing 100 mM choline chloride. Glutamine uptake was performed under voltage-clamped conditions with pH 7.4 extravesicular uptake buffer containing 100 mM NaCl and 10 ␮Ci [3H]glutamine (0.2 mM). Na-dependent glutamine uptake was calculated by deducting Na-independent glutamine uptake from total glutamine uptake at both pH measurements. Lower intravesicular pH (6.0) stimulated glutamine uptake by ⬃50% compared with intravesicular pH of 7.4. These data indicate that crypt cell Na-glutamine cotransporter is an amino acid-proton antiporter belonging to system A or N.

tified, SN1 and SN2. To identify the subtype of system N transporter present in the crypt cells, we performed RTQ-PCR using specific primers and probes for rabbit SN1 and SN2 on cDNA obtained from the crypt cell total RNA. The RTQ-PCR results showed that in the crypt cells SN1 mRNA expression is only 0.02% as that of SN2 (Fig. 5A). This ruled out the possibility of SN1 being the system N transporter present in the crypt cells. Furthermore, to prove that SN2 message is more in the crypt cells compared with the villus cells, RTQ-PCR was performed using SN2-specific primers and probes. As can be seen from Fig. 5B, SN2 mRNA is roughly 11 times more in crypt cells (P ⬍ 0.05, n ⫽ 3) compared with the villus cells. Taken together, the functional data and molecular data prove

Fig. 4. Crypt cell Na-glutamine cotransporter is Li⫹ tolerant. Crypt cell BBMV was preloaded with pH 7.4 buffer containing 100 mM choline chloride. Uptake of [3H]glutamine was measured under voltage-clamped conditions with pH 7.4 extravesicular uptake buffer containing 100 mM NaCl or LiCl and 10 ␮Ci [3H]glutamine (0.2 mM). Crypt cell Na-glutamine and Li-glutamine cotransport activities were obtained by deducting Na⫹/Li⫹-independent glutamine uptake from total glutamine uptake. As seen in the figure, Li⫹ partially fulfilled the role of Na⫹ as the monovalent cation (n ⫽ 6).

that SN2 is the major pH-dependent glutamine transporter expressed in the crypt cells. Western blotting and IHC studies. To prove that not only SN2 mRNA but also immunoreactive SN2 protein is present in the crypt cells, Western blot studies were performed using crypt and villus cell BBMV and anti-SN2 polyclonal antibody (Fig. 6A, lanes 1 and 2). A representative picture of a Western blot is shown in Fig. 6A, where the position of the 100-kDa protein corresponding to SN2 is marked with an arrow. Specificity of the anti-SN2 polyclonal antibody was shown in a separate experiment by preincubating the primary antibody with 10 ␮g of SN2 antigenic peptides. As can be seen from Fig. 6A, lanes 3 and 4, a 100-kDa band disappeared almost completely, showing that this band is SN2 specific. Since the predicted molecular weight of SN2 based on the amino acid sequence is 52 kDa, crypt cell SN2 seems to be extensively glycosylated. This hypothesis can be supported by the observation that there are five predicted N-glycosylation sites in SN2 Table 1. Substrate specificity of rabbit crypt cell BBMV Na-glutamine cotransporter

Fig. 3. Kinetic parameters of glutamine uptake by BBMV from crypt cells. Uptake of 10 ␮Ci [3H]glutamine uptake was performed as a function of varying glutamine concentrations (n ⫽ 3) for 6 s. Intravesicular buffer was 100 mM choline chloride buffer (pH 7.4), and uptake buffer was 100 mM NaCl buffer (pH 7.4). Na-dependent glutamine uptake was calculated by deducting Na-independent glutamine uptake from total glutamine uptake at all glutamine concentrations. Crypt cell BBMV showed an increase in glutamine uptake with increasing concentrations of the substrate, attaining saturation at higher substrate concentrations. The kinetic parameters were obtained by analyzing the data obtained by Lineweaver-Burk plot.

Competing Amino Acid

Uptake, pmol/ mg protein per 30 s

% Control

None MeAIB 10 mM 50 mM Histidine 10 mM 50 mM

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5.58⫾0.37 1.02⫾0.06

20 4

Uptake of 10 ␮Ci 关3H兴glutamine was performed at conditions with intravesicular choline chloride buffer (pH 7.4) and extravesicular NaCl buffer (7.4) in the absence or presence of 10 mM and 50 mM unlabeled amino acid substrates. Na-dependent glutamine uptake was calculated by deducting Na-independent glutamine uptake from total glutamine uptake. Glutamine 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). Inhibition obtained was consistent with system N transporters, and these data in concert with real-time quantitative PCR (RTQ-PCR) data (Fig. 3) further supported the hypothesis that SN2 is the major Na-glutamine cotransporter in the crypt cells. BBMV, brush border membrane vesicles; MeAIB, ␣-(methylamine)isobutyric acid.

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Fig. 5. Expression of SN1 and SN2 transcript in villus and crypt cells. RTQ-PCR for rabbit SN1 and SN2 was performed using cDNA obtained from the villus and crypt cell total RNA. Rabbit SN1- and SN2-specific primers and TaqMan probes were used for RTQ-PCR, and expression of SN1 and SN2 was normalized against ␤-actin. A: expression of SN1 and SN2 mRNA in normal crypt cells. SN2 expression in crypt cells was given an arbitrary value of 1, and relative expression of SN1 was compared with SN2. These data indicated that SN2 is the major system N transporter subtype present in crypt cells. B: relative expression of SN2 transcript in the villus and crypt cells. SN2 expression in villus cells was given an arbitrary value of 1, and SN2 expression in crypt cells is compared with the expression in villus cells. These data indicated that SN2 is 11 times more in crypt cells compared with the villus cells and is the major system N transporter present in crypt cells.

protein at positions 48, 57, 227, 245, and 296. Figure 6B shows the quantitative data (P ⬍ 0.05, n ⫽ 3) of SN2 protein expression in villus and crypt cells. These data indicated that SN2 protein expression is five times higher in the crypt cells compared with the villus cells. To further prove that SN2 is localized to the crypt cells, immunolocalization experiments were performed using anti-SN2 polyclonal antibody. Figure 6C shows a representative IHC photograph, and the results of these experiments showed that SN2 is localized specifically to the crypt cells but not the villus cells. Specificity of the signal obtained in the crypt cells by SN2 antibody was shown in a parallel experiment by preincubating the primary antibody with 100-fold excess immunogenic peptide prior to the addition to the tissue sections (Fig. 6D). Excess peptide completely abolished the SN2 antibody binding, demonstrating that the signal obtained by the primary antibody is specific to SN2.

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Fig. 6. Expression of immunoreactive SN2 protein in villus and crypt cells. BBMV from crypt and villus cells were solubilized and separated on 4 –20% polyacrylamide gel. The separated proteins were transferred onto polyvinylidene difluoride (PVDF) membrane and probed with SN2-specific polyclonal antibody raised in chicken. A: representative Western blot. B: quantitative data of SN2 expression in villus and crypt cells. To show the specificity of the Western blotting, 10 ␮g of SN2 antigenic peptides were preincubated with the SN2-specific polyclonal antibody for 1 h at 37°C before incubation of the PVDF with the primary antibody (A, lanes 3 and 4). Lanes 1 and 2 represent SN2 in the villus and crypt cells where the SN2 primary antibody did not contain the blocking peptides. The signal obtained by performing Western blotting was scanned with a densitometer, and the relative expression of SN2 in the crypt cells was compared with its expression in the villus cells. The expression of SN2 in the villus cells was given an arbitrary value of 1, and relative expression of SN2 in the crypt cells was compared with this value. These data showed that SN2 protein expressed 5 times more in the crypt cells compared with the villus cells. C and D: results of a representative immunohistochemistry experiment in the absence (C) and in the presence (D) of the blocking peptides. Tissue sections from the normal rabbit small intestine were subjected to immunostaining using anti-SN2 polyclonal antibody; C shows the localization of SN2 to the crypt cells (marked with white arrows). No signal was detected in the presence of a 100-fold excess blocking peptide indicating that the signal observed in C was specific to SN2 (D).

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Molecular features of rabbit SN2. To obtain the crypt cell SN2 cDNA for functional studies, we screened a rabbit intestinal library using human SN1 cDNA as the probe. The rabbit SN2 thus obtained was 2,108 bp long (GenBank accession number EF405800). The cDNA encoded an open reading frame of 473 amino acids. Compared with human SN2, rabbit SN2 protein exhibited 87% identity and 91% similarity (Fig. 7A). Hydrophobicity analysis using the algorithm of Kyte and Doolittle (16a) with a 17-amino acid window size indicated that rabbit SN2 contained 11 predicted transmembrane domains like its human counterpart (Fig. 7B). Rabbit SN2 protein does not have NH2-terminal signal peptide as indicated by pSORT results, and the location of the NH2 terminus is predicted to be extracellular. Functional characterization of rabbit SN2 cDNA clone. By use of vaccinia virus-mediated transient transfection system, the function of the cloned rabbit SN2 was evaluated by using a human cell line HRPE. Previous data of the cloned human SN2 showed that SN2 transported glutamine, asparagine, and

histidine in a Na⫹- or Li⫹-dependent manner (21). To prove that rabbit SN2 transporter also had similar transport characteristics, we first compared the uptake of radiolabeled Asn, Gln, Ser, and His in NaCl-containing buffer (Fig. 8). Since our earlier experiments with human SN2 showed that uptake mediated by SN2 cDNA is maximal at pH 8.0, a characteristic of proton antiporters, uptake buffer of pH 8.0 was used in all our transport experiments. As can be seen from Fig. 8, there was an increase of 1.5-, 1.8-, 1.9-, and 2.9-fold in the uptake of glutamine, serine, asparagine, and histidine, respectively, in SN2-transfected cells compared with vector-transfected cells. SN2 is capable of transporting its substrate in the presence of Li⫹ instead of Na⫹. This also helped to reduce endogenous Na⫹-dependent serine uptake in HRPE cells, making the cDNA-dependent serine uptake much higher compared with uptake in vector-transfected cells. We first investigated Li⫹activation kinetics of rabbit SN2-specific serine uptake. As can be seen from Fig. 9, the cDNA-specific serine uptake increased with increasing Li⫹ concentrations. When the data were fit to

Fig. 7. Amino acid analysis of rabbit SN2. A: amino acid comparison of human (hSN2) and rabbit SN2 (rbSN2). Putative transmembrane domains are underlined and numbered sequentially. B: hydropathy analysis of the amino acid sequence of rabbit SN2 performed using ProtScale, a web-based freeware available from the ExPASy (Expert Protein Analysis System) website, http://www.expasy.ch/tools/protscale.html.

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Table 2. Substrate specificity of rabbit SN2 transiently expressed in HRPE cells Uptake, pmol/106 cells per 15 min Amino Acid

pSPORT

cDNA

Fold

Alanine Histidine Glycine Asparagine Glutamine Serine MeAIB

127⫾7 27⫾1 46⫾1 62⫾2 82⫾2 53⫾2 43⫾1

238⫾8 56⫾1 145⫾6 279⫾13 274⫾10 391⫾7 37⫾1

2 2 3 4 3 7 0.9

HRPE cells, human retinal pigment epithelial cells. Fig. 8. Substrate specificity of rabbit SN2. Human retinal pigment epithelial (HRPE) cells were transiently transfected with vector or rabbit SN2 cDNA. Uptake of radiolabeled amino acids were performed in NaCl containing buffer at 37°C using 0.5 ␮Ci labeled substrate and 10 ␮M unlabeled substrate. Uptake buffer also contained 5 mM leucine to suppress the endogenous amino acid uptake in HRPE cells. SN2 cDNA transfected cells showed higher amino acid uptake compared with the vector-transfected cells with all the amino acids tested.

the Hill equation, we obtained a Hill coefficient of 1.1, indicating that one Li⫹ ion is involved in the activation of rabbit SN2-mediated serine uptake, giving a Li⫹-serine stoichiometry of 1:1 for the activation process. It is noteworthy that when similar experiments were performed with rat brain SN2 cDNA, a Hill coefficient of 1.4 was obtained (20), indicating a similar transporter mechanism in rabbit and human SN2. All further substrate specificity experiments were performed in the presence of 140 mM LiCl. Various radiolabeled amino acid substrates were used in uptake experiments using HRPE cells transfected with vector or SN2 cDNA. Cells transfected with vector alone transported different amino acids to different extents, indicating the presence of various amino acid transporters in these cells (Table 2). However, transfection with SN2 cDNA further enhanced the uptake of these amino acid substrates, ranging from two- to sevenfold. MeAIB, a system A-specific substrate, was not transported. Serine was the best

substrate, with a sevenfold enhancement in the uptake in cDNA transfected cells. Further substrate functional characterization experiments were performed using labeled serine as the substrate. The substrate specificity of the amino acid transport system induced by SN2 cDNA was evaluated further by using various amino acids as inhibitors of serine uptake (Table 3). The cDNAspecific transport was deduced by subtracting the uptake of vector-transfected HRPE cells from the uptake of SN2 cDNAtransfected cells. Serine uptake is inhibited by SN2-specific substrates like glutamine, asparagine, and histidine as well as nonpolar amino acids like glycine and alanine. MeAIB, a system A-specific substrate, did not inhibit serine uptake. DISCUSSION

The presence of Na-glutamine cotransporters in the intestinal villus cells is well documented and many of them have been identified and characterized at both the functional and the molecular levels. On the other hand, Na-glutamine cotransporters have not been described at the molecular level in the intestinal crypt cells, even though the presence of both the Na-dependent and Na-independent processes has been documented in the crypt cell-like IEC-6 cell line (14). The lack of functional information on crypt cell Na-glutamine cotransporters could partly be attributed to the inherent complexity in the Table 3. Substrate specificity of rabbit SN2; inhibition by unlabeled amino acids Uptake of Serine, pmol/ 106 cells per 15 min

Fig. 9. Kinetic parameters of activation by Li⫹. Uptake of radiolabeled serine was performed at increasing concentrations of Li⫹. The uptake was performed at pH 8.5 in the presence of 2 mM leucine. Uptake was performed using 0.5 ␮Ci of radiolabeled and 5 ␮M unlabeled serine for 15 min at 37°C. cDNAspecific uptake of serine was calculated by subtracting SN2 cDNA-specific uptake by vector-specific uptake. Hill plot (inset) was plotted using the data obtained to calculate the stoichiometry of Li⫹ to serine uptake.

Amino Acid

pSPORT

cDNA

cDNA Specific

% control

None Glycine Alanine Asparagine Glutamine Histidine MeAIB

51⫾2 24⫾0.8 20⫾0.4 22⫾0.4 20⫾0.3 26⫾0.2 25⫾0.1

360⫾9 119⫾3 109⫾2 110⫾6 94⫾4 52⫾0.2 316⫾12

309⫾9 95⫾3 89⫾2 87⫾6 74⫾4 26⫾0.2 291⫾12

100 31 29 28 24 8 94

Values are means ⫾ SE for 6 uptake measurements from 2 independent transfections. Unlabeled amino acid substrates (10 mM) were used to inhibit labeled serine uptake, and 0.5 ␮Ci serine with 5 ␮M cold serine was used for uptake in LiCl buffer containing 2 mM leucine. Uptake was done for 15 min at 37°C. SN2 cDNA-specific uptake was calculated by subtracting vector (pSPORT)-specific serine uptake from transiently transfected SN2 cDNAspecific uptake. Uptake values obtained with no inhibitor present was used as a control (100% uptake), and inhibitions obtained by various cold substrates were expressed as percent of control.

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separation of intestinal villus and crypt cells. Our laboratory has earlier described an efficient method for isolating the rabbit intestinal villus and crypt cells (27). In this report, we have demonstrated the presence of a Na-dependent glutamine transporter in the rabbit crypt cell BBMV using the method described earlier to isolate the crypt cells (Fig. 1). Furthermore, we performed saturation kinetic experiments using glutamine as substrate. We calculated the kinetic parameters using these data; the Km was determined to be 43.7 ⫾ 2.7 mM and the Vmax was determined to be 3.87 ⫾ 0.1 pmol/mg protein per 6 s (Fig. 3). These data provided further evidence for a Na-dependent carrier mediated active uptake of glutamine. Amino acids are transported into the cells by several amino acid transporter families and, although overlapping, different families are defined by distinct substrate specificities and ionic requirements. Glutamine is transported by both pH-dependent and pH-independent transporters. To decipher whether glutamine in crypt cell BBMV is transported via pH-dependent or pH-independent transporters, we performed effect of pH gradient on glutamine uptake. When BBMV were preloaded with 100 mM choline chloride buffer, pH 6.0, the glutamine uptake was significantly higher than the uptake seen in pH 7.4 buffer (Fig. 2). This experiment established that the glutamine transport phenomenon in crypt cell BBMV is mediated by a protonamino acid antiporter (Fig. 2). Two transporters belonging to SLC38 family, system A and system N, are known to transport glutamine in a pH-dependent manner. Using basolateral plasma membrane vesicles from rabbit jejunal mucosa, Wilde and Kilberg (34) showed that system A is located on the basolateral membranes and is responsible for glutamine absorption from the circulation into the intestinal epithelial cells, providing the necessary nutrients for the enterocytes. However, the authors have not separated the villus and crypt cells for their experiments and therefore whether system A is present in the basolateral membranes of villus or crypt cells or both is not known. Currently three members of system A (SLC38A1, A2, and A4 also known as ATA1, ATA2, and ATA3, respectively) have been identified. Recent studies have shown that ATA2 is localized specifically to the basolateral membrane of intestinal epithelial cells in the rat (11). System N transporters transport glutamine, histidine, serine, and asparagine along with Na⫹ in exchange for H⫹ (15). The molecular identities of two types of system N are described so far, SN1 (4) and SN2 (20, 24). SN1 and SN2 cannot be differentiated at the functional level since they share identical substrate specificities. However, they differ in their tissue distribution (20). Human SN2 is expressed relatively ubiquitously, with maximum expression in stomach and also in brain, liver, lung, and small intestine. To the contrary, human SN1 is expressed mostly in kidney and liver, and to some extent in the brain. Because of its known expression in the intestine, it is conceivable that SN2 is the major transporter expressed in the rabbit intestinal crypt cells. To lend support to this hypothesis, we performed RTQ-PCR experiments using rabbit SN1- and SN2-specific primers and cDNA obtained from total RNA from villus and crypt cells as templates. As can be seen from Fig. 5A, in crypt cells, SN1 mRNA expression was insignificant compared with SN2 (SN1 expression was 0.02% that of SN2), ruling out the possibility that SN1 is the transporter responsible for the crypt cell glutamine uptake. Furthermore, as

seen from Fig. 5B, SN2 mRNA expression was 11 times more in crypt cells compared with villus cells, further lending credence to the hypothesis that SN2 is the major glutamine transporter in the crypt cells compared with SN1. To prove that not only SN2 transcript but also immunoreactive SN2 protein expression is higher in crypt cells compared with the villus cells, we performed Western blotting studies using BBMV from villus and crypt cells. As can be seen from Fig. 6, A and B, SN2 protein is fivefold higher in the crypt cells, proving unequivocally that SN2, not SN1, is the major Na-dependent and pH-responsive glutamine transporter in the crypt cells. Further proof for the localization of SN2 comes IHC experiments. As seen from Fig. 6, C and D, only the crypt cells but not the villus cells express SN2. There is a constant turnover of intestinal epithelium and new villus cells are generated by the differentiation of crypt cells, which in turn are generated from stem cells located at the bottom of the crypt. Because of cell proliferation, crypt cells are metabolically very active and transcription of many of the villus transporters has its origin in crypt cells. Hence increased SN2 transcription or for that matter translation is not indicative of its function in the crypt cells. For example, when hamster apical, Na-dependent, small intestinal bile acid transporter (IBAT) RNA and protein were mapped by in situ hybridization, IHC, and Western blotting, IBAT mRNA was found to be more abundant near the crypt-villus junction, whereas the protein was expressed evenly along the villus axis (25). To prove that the crypt cell glutamine transporter is indeed mediated by SN2, we embarked on the functional characterization of crypt cell glutamine transporter. Even though both system N and system A transporters are amino acid-proton antiporters, system N transporters can be distinguished from system A by their ability to transport their substrates in the presence of Li⫹. Also, MeAIB is a system A-specific substrate and is not transported by system N (5, 6). First, to rule out the possibility of system A being the crypt cell BBMV glutamine transporter, we performed Li⫹ substitution experiments (Fig. 4). When Na⫹ in the uptake buffer was replaced with Li⫹, we obtained roughly 93% glutamine uptake in crypt cell BBMV compared with the uptake in Na⫹ containing buffer. This indicated that the majority of the glutamine uptake in crypt cell BBMV is carried out by SN2. To further prove that SN2 is the glutamine carrier in the crypt cell BBMV, additional substrate inhibition experiments were carried out. When glutamine uptake in crypt cell BBMV was performed in the presence of 10 and 50 mM MeAIB, a system A transporter-specific substrate, we obtained only a partial inhibition (26 and 42% inhibition at 10 and 50 mM MeAIB concentration, respectively; Table 1). On the other hand, histidine, a preferred system N transporter substrate, inhibited glutamine uptake almost completely at 10 mM concentration (80% inhibition). These substrate specificity experiments, although they did not rule out the presence of system A completely, supported the conjecture that SN2 is the major glutamine transporter in the crypt cells. The partial inhibition of glutamine uptake by MeAIB could be due to two reasons. First, it is possible that a part of the pH-dependent uptake of glutamine in the crypt cells is partially performed by system A since we found system A mRNA in crypt cells, particularly ATA2 (data not shown). Second, since system A has been shown to be the transporter responsible for glutamine uptake in the basolateral membranes of the rabbit intestinal cells (34), it

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is also possible that we have some contamination of BBMV preparation with the basolateral membrane. To further clarify this issue, cellular localization of system A transporter in the crypt cells needs to be investigated by immunohistochemical techniques. One of the many ways with which one could prove unequivocally that SN2 is the major glutamine transporter present in the crypt cells is to obtain SN2 cDNA from rabbit intestinal epithelial cells, express it in a mammalian system, and compare the functional characteristics of the cloned cDNA to that of the crypt cell BBMV glutamine transporter. To date, molecular identification and the functional characterization of rabbit SN2 cDNA have not been done. Toward this goal, we embarked on cloning and functional expression of rabbit intestinal SN2. As expected of an orthologous gene, rabbit SN2 predicted protein displayed 87% identity and 91% similarity to the cloned human SN2 (Fig. 7). In functional studies using human SN2 cDNA transfected HRPE cells, it was shown earlier that SN2 transport function is maximal at pH 8.0 (20). Hence functional characterization studies of rabbit SN2 cDNA transiently transfected into HRPE cells were performed at pH 8, or 8.5 in certain experiments. Even though SN2 cDNA transfected HRPE cells displayed higher uptake of all the radiolabeled amino acids used for the experiment compared with the vector-transfected HRPE cells, there was a significant background amino acid transport when the uptake experiments were performed in the presence of Na⫹, indicating the presence of endogenous Na-amino acid cotransporters in HRPE cells (Fig. 8). Since SN2 is Li⫹ tolerant (Fig. 9) and there are not too many Li⫹-tolerant amino acid transporters, we devised a strategy to maximize the SN2 cDNA-specific transport. First, we used LiCl in the uptake buffer instead of NaCl. In addition, since leucine is not a substrate for SN2, we added 5 mM leucine (or 2 mM in some instance) to the uptake buffer to reduce the basal amino acid uptake activity even further (21). These modifications in the uptake buffer suppressed the constitutively expressed basal amino acid uptake activity in these cells, which made the conditions ideal to measure the activity of the heterologously expressed SN2 cDNA. When Li⫹-activation kinetic data were fit to Hill equation, we obtained a Hill coefficient of 1.1 (Fig. 9 and Fig. 9 inset). This indicated that rabbit SN2, like its rat counterpart, transports its substrate with Li⫹-amino acid stoichiometry of 1:1, suggesting a single Li⫹ binding site in the SN2 protein to each amino acid substrate bound. To compare the functional characteristics of rabbit crypt cell glutamine transporter to that of the cloned rabbit SN2, we performed substrate specificity experiments. This was achieved by performing two separate but complementary experiments. First we looked at the ability of the cloned rabbit SN2 to transport various radiolabeled amino acids. As can be seen from Table 2, when various known radiolabeled amino acids were used, HRPE cells transfected with rabbit SN2 showed higher uptake compared with vector-transfected HRPE cells. Rabbit SN2transfected HRPE cells transported neutral (serine, asparagine, glutamine), nonpolar (glycine and alanine), and charged (histidine) amino acids. Fold activation of amino acid uptake, compared with the vector-transfected HRPE cells, ranged from twofold (alanine and histidine) to sevenfold (serine), indicating that serine is the best substrate for rabbit SN2 transporter under the conditions in which experiments were performed. As ex-

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pected, we did not find any uptake when MeAIB was used as a substrate. Next, we performed inhibition experiments to see whether excess unlabeled SN2 substrates would inhibit radiolabeled serine uptake (Table 3). The results from this experiment confirmed the results we obtained when different labeled amino acids were used as substrates. These experiments showed that functional characteristics of the cloned cDNA was consistent with SN2 in that it is pH activated, is Li⫹ tolerant, and transported well-known system N transporter substrates glutamine, histidine, serine, and asparagine. It should be pointed out that, unlike in crypt cell BBMV where MeAIB inhibited radiolabeled glutamine uptake partially, radiolabeled MeAIB was not transported (Table 2) and serine uptake was not inhibited by excess unlabeled MeAIB (Table 3) in HRPE cells transfected with rabbit SN2 cDNA. This could be because glutamine uptake in HRPE cells transfected with rabbit SN2 cDNA was performed with LiCl-containing buffer in which system A transporter is not active, whereas glutamine uptake in the crypt cell BBMV was done with NaCl-containing buffer in which system A is active. The physiological importance for the presence of glutamine transporter in crypt cells is not clearly understood at present, since crypt cells have access to much less luminal nutrients compared with villus cells. System A, which is present in the basolateral membrane, serves the purpose of obtaining nutrients from the blood stream. Thus we hypothesize that SN2 is the luminal transporter under normal conditions. Furthermore, it may have a backup function under conditions in which villus cells are compromised, for example chronic inflammation such as IBD, in which villus cells are atrophied and crypt cells are hypertrophied and thus have more access to the intestinal lumen. It is possible that under these pathophysiological conditions crypt cells take up the function of amino acid absorption for the intestine from villus cells, at least in part. This may be all the more important, since under these conditions circulating glutamine may be decreased because of the inability of villus cells to absorb nutrition. Thus absorption of nutrients by crypt cells may become necessary to offset the compromised villus function. In an earlier study of bovine cryptosporidiosis (2) in which villus cells are totally lost because of cryptosporidium infection, it was shown that luminally administered glutamine was capable of fully restoring Na⫹ transport. This suggested the presence of a Na-amino acid cotransporter in the bovine crypt cells under the conditions in which villus cells are functionally compromised. The authors suggested that the presence of crypt cell Na-amino acid transporter not only could promote Na⫹ absorption but also could increase the uptake of glutamine, which is required to promote crypt cell proliferation and necessary to restore villus architecture. Also, by using several human hepatoma cell lines, it was earlier shown that SN1 and SN2 were expressed only in well-differentiated cell lines, suggesting a stage-specific absorptive role for these transporters (3). These data suggest a regulatory role for system N family of amino acid transporters during cell proliferation. This observation can be explained by the fact the increased metabolic rate results in the production of acids, increasing the intracellular proton load, which is conducive to system N transporter function. Probably because of this very reason SN2 is expressed highly in the metabolically active crypt cells but not the villus cells, and it is possible that high rate of protein

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degradation leads to the rapid disappearance of SN2 from the villus cells. It is only logical to extend these observations and hypothesize that crypt cell SN2 plays a compensatory role in the crypt-villus growth and differentiation and could play a role in the repair process of the intestine under pathophysiological conditions, for example during chronic intestinal inflammatory disorders. GRANTS This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-45062 and DK-58034 to U. Sundaram. REFERENCES 1. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K. Analysis of proteins. In: Current Protocols in Molecular Biology, edited by Janssen K. New York: John Wiley & Sons, 1995, vol.2, chapt. 10, p. 10.2.1-10.2.16. 2. Blikslager A, Hunt E, Guerrant R, Rhoads M, Argenzio R. Glutamine transporter in crypts compensates for loss of villus absorption in bovine cryptosporidiosis. Am J Physiol Gastrointest Liver Physiol 281: G645– G653, 2001. 3. Bode BP, Fuchs BC, Hurley BP, Conroy JL, Suetterlin JE, Tanabe KK, Rhoads DB, Abcouwer SF, Souba WW. Molecular and functional analysis of glutamine uptake in human hepatoma and liver-derived cells. Am J Physiol Gastrointest Liver Physiol 283: G1062–G1073, 2002. 4. Chaudhry FA, Reimer RJ, Krizaj D, Barber D, Storm-Mathisen J, Copenhagen DR, Edwards RH. Molecular analysis of system N suggests novel physiological roles in nitrogen metabolism and synaptic transmission. Cell 99: 769 –780, 1999. 5. Christensen HN. Role of amino acid transport and countertransport in nutrition and metabolism. Physiol Rev 70: 43–77, 1990. 6. Christensen HN. On the strategy of kinetic discrimination of amino acid transport systems. J Membr Biol 84: 97–103, 1985. 7. Coeffier M, Claeyssens S, Hecketsweiler B, Lavoinne A, Ducrotte P, Dechelotte P. Enteral glutamine stimulates protein synthesis and decreases ubiquitin mRNA level in human gut mucosa. Am J Physiol Gastrointest Liver Physiol 285: G266 –G273, 2003. 8. Coon S, Kim J, Shao G, Sundaram U. Na-glucose and Na-neutral amino acid cotransport are uniquely regulated by constitutive nitric oxide in rabbit small intestinal villus cells. Am J Physiol Gastrointest Liver Physiol 289: G1030 –G1035, 2005. 9. Del Castillo JR, Sulbaran-Carrasco MC, Burguillos L. Glutamine transport in isolated epithelial intestinal cells. Identification of a Na⫹dependent transport mechanism, highly specific for glutamine. Pflu¨gers Arch 445: 413– 422, 2002. 10. Field M. Intestinal ion transport and the pathophysiology of diarrhea. J Clin Invest 111: 931–943, 2003. 11. Ganapathy V, Gupta N, Martindale RG. Digestion and absorbtion. In: Physiology of the Gastrointestinal Tract, edited by Johnson LR. Burlington, MA: Elsevier, 2006, vol.2, chapt. 5, p. 1667–1692. 12. Horvath K, Jami M, Hill ID, Papadimitriou JC, Magder LS, Chanasongcram S. Isocaloric glutamine-free diet and the morphology and function of rat small intestine. J Parenter Enteral Nutr 20: 128 –134, 1996. 13. Inoue Y, Grant JP, Snyder PJ. Effect of glutamine-supplemented total parenteral nutrition on recovery of the small intestine after starvation atrophy. J Parenter Enteral Nutr 17: 165–170, 1993. 14. Inui K, Quaroni A, Tillotson LG, Isselbacher KJ. Amino acid and hexose transport by cultured crypt cells from rat small intestine. Am J Physiol Cell Physiol 239: C190 –C196, 1980. 15. Kilberg MS, Handlogten ME, Christensen HN. Characteristics of an amino acid transport system in rat liver for glutamine, asparagine, histidine, and closely related analogs. J Biol Chem 255: 4011– 4019, 1980.

16. Kockerling A, Fromm M. Origin of cAMP-dependent Cl⫺ secretion from both crypts and surface epithelia of rat intestine. Am J Physiol Cell Physiol 264: C1294 –C1301, 1993. 16a.Kyte J, Doolittle RF. A simple method for displaying the hydropathic character of a protien. J Mol Biol 157: 105–132, 1982. 17. Levy J, Turkish A. Protective nutrients. Curr Opin Gastroenterol 18: 717–722, 2002. 18. Li J, Langkamp-Henken B, Suzuki K, Stahlgren LH. Glutamine prevents parenteral nutrition-induced increases in intestinal permeability. J Parenter Enteral Nutr 18: 303–307, 1994. 19. Manokas T, Fromkes JJ, Sundaram U. Effect of chronic inflammation on ileal short-chain fatty acid/bicarbonate exchange. Am J Physiol Gastrointest Liver Physiol 278: G585–G590, 2000. 20. Nakanishi T, Kekuda R, Fei YJ, Hatanaka T, Sugawara M, Martindale RG, Leibach FH, Prasad PD, Ganapathy V. Cloning and functional characterization of a new subtype of the amino acid transport system N. Am J Physiol Cell Physiol 281: C1757–C1768, 2001. 21. Nakanishi T, Sugawara M, Huang W, Martindale RG, Leibach FH, Ganapathy ME, Prasad PD, Ganapathy V. Structure, function, and tissue expression pattern of human SN2, a subtype of the amino acid transport system N. Biochem Biophys Res Commun 281: 1343–1348, 2001. 22. O’Loughlin EV, Hunt DM, Bostrom TE, Hunter D, Gaskin KJ, Gyory A, Cockayne DJ. X-ray microanalysis of cell elements in normal and cystic fibrosis jejunum: evidence for chloride secretion in villi. Gastroenterology 110: 411– 418, 1996. 23. Platell C, McCauley R, McCulloch R, Hall J. The influence of parenteral glutamine and branched-chain amino acids on total parenteral nutrition-induced atrophy of the gut. J Parenter Enteral Nutr 17: 348 –354, 1993. 24. Rajan DP, Kekuda R, Huang W, Devoe LD, Leibach FH, Prasad PD, Ganapathy V. Cloning and functional characterization of a Na⫹-independent, broad-specific neutral amino acid transporter from mammalian intestine. Biochim Biophys Acta 1463: 6 –14, 2000. 25. Stelzner M, Hoagland V, Somasundaram S. Distribution of bile acid absorption and bile acid transporter gene message in the hamster ileum. Pflu¨gers Arch 440: 157–162, 2000. 26. Sundaram U, Knickelbein RG, Dobbins JW. pH regulation in ileum: Na⫹-H⫹ and Cl⫺-HCO⫺ 3 exchange in isolated crypt and villus cells. Am J Physiol Gastrointest Liver Physiol 260: G440 –G449, 1991. 27. Sundaram U, West AB. Effect of chronic inflammation on electrolyte transport in rabbit ileal villus and crypt cells. Am J Physiol Gastrointest Liver Physiol 272: G732–G741, 1997. 28. Sundaram U, Wisel S, Coon S. Mechanism of inhibition of proton: dipeptide cotransport during chronic enteritis in the mammalian small intestine. Biochim Biophys Acta 1714: 134 –140, 2005. 29. Sundaram U, Wisel S, Fromkes JJ. Unique mechanism of inhibition of Na⫹-amino acid cotransport during chronic ileal inflammation. Am J Physiol Gastrointest Liver Physiol 275: G483–G489, 1998. 30. Sundaram U, Wisel S, Rajendren VM, West AB. Mechanism of inhibition of Na⫹-glucose cotransport in the chronically inflamed rabbit ileum. Am J Physiol Gastrointest Liver Physiol 273: G913–G919, 1997. 31. Sundaram U, Wisel S, Stengelin S, Kramer W, Rajendran V. Mechanism of inhibition of Na⫹-bile acid cotransport during chronic ileal inflammation in rabbits. Am J Physiol Gastrointest Liver Physiol 275: G1259 –G1265, 1998. 32. Tremel H, Kienle B, Weilemann LS, Stehle P, Furst P. Glutamine dipeptide-supplemented parenteral nutrition maintains intestinal function in the critically ill. Gastroenterology 107: 1595–1601, 1994. 33. Van der Hulst RR, van Kreel BK, von Meyenfeldt MF, Brummer RJ, Arends JW, Deutz NE, Soeters PB. Glutamine and the preservation of gut integrity. Lancet 341: 1363–1365, 1993. 34. Wilde SW, Kilberg MS. Glutamine transport by basolateral plasmamembrane vesicles prepared from rabbit intestine. Biochem J 277: 687– 691, 1991. 35. Windmueller HG. Glutamine utilization by the small intestine. Adv Enzymol Relat Areas Mol Biol 53: 201–237, 1982.

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