HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: 295/1/G1    most recent 00498.2007v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Talukder, J. R. Articles by Sundaram, U. Search for Related Content PubMed PubMed Citation Articles by Talukder, J. R. Articles by Sundaram, U.

TRANSLATIONAL PHYSIOLOGY

Mechanism of leukotriene D₄ inhibition of Na-alanine cotransport in intestinal epithelial cells

Section of Digestive Diseases, Department of Medicine, West Virginia University School of Medicine, Morgantown, West Virginia

Submitted 29 October 2007 ; accepted in final form 17 April 2008

ABSTRACT

In a rabbit model of chronic intestinal inflammation, we previously demonstrated inhibition of neutral Na-amino acid cotransport. The mechanism of the inhibition was secondary to a decrease in the affinity for amino acid rather than the number of cotransporters. Since leukotriene (LT)D₄ is known to be elevated in enterocytes during chronic intestinal inflammation, we used rat intestinal epithelial cell (IEC-18) monolayers to determine the mechanism of regulation of Na-alanine cotransport (alanine, serine, cysteine transporter 1: ASCT1) by LTD₄. Na-alanine cotransport was inhibited by LTD₄ in IEC-18 cells. The mechanism of inhibition of ASCT1 (solute carrier, SLC1A4) by LTD₄ is secondary to a decrease in the affinity of the cotransporter for alanine without a significant change in cotransporter numbers and is not secondary to an alteration in the Na⁺ extruding capacity of the cells. Real-time quantitative PCR and Western blot analysis results indicate that ASCT1 message and protein levels are also unchanged in LTD₄-treated IEC-18 cells. These results indicate that LTD₄ inhibits Na-dependent neutral amino acid cotransport in IEC. The mechanism of inhibition is secondary to a decrease in the affinity for alanine, which is identical to that seen in villus cells from the chronically inflamed rabbit small intestine, where LTD₄ levels are significantly increased.

leukotrienes; Na-amino acid cotransport; epithelial transport; chronic intestinal inflammation

In addition to functioning as a barrier and absorptive organ, intestinal epithelial cells (IEC) play an active role in inflammatory diseases by releasing multiple cytokines and inflammatory mediators. The constellation of IEC-derived inflammatory molecules includes interleukins, chemokines, prostaglandins, and nitrogen radicals (18). Inhibition of NaCl and nutrient absorption during chronic inflammation of the intestine has been well described (2, 14, 19). We have previously demonstrated that Na-alanine cotransport (e.g., ASCT1) is present on the brush-border membrane (BBM) of the absorptive villus but not the secretory crypt cells. Na-alanine cotransport is inhibited in villus cells from chronically inflamed rabbit small intestine (19). In vivo, the wide variety of immune-inflammatory mediators known to be endogenously produced in the chronically inflamed intestine have an effect either individually or synergistically on electrolyte and nutrient transport pathways (4, 14, 15). Furthermore, a variety of cytokines such as leukotriene B₄ (eicosanoid pathway product) is markedly increased in enterocytes in the chronically inflamed rabbit intestine (20). At present, it is not known whether a given immune-inflammatory mediator pathway such as 5-lipooxygenase pathway, which produces leukotrienes (LT), is responsible for the inhibition in the Na-dependent alanine uptake (ASCT1) during chronic enteritis in rabbit (19). Given this background, we studied the effect of LTD₄ on Na-dependent alanine uptake as representative of Na-neutral amino acid cotransport in a rat IEC model (IEC-18) in vitro. The favorable Na⁺ gradient for this cotransport is provided by Na⁺/K⁺-ATPase (8, 9, 11, 16). Thus LTD₄ can bring about cellular alterations in Na-alanine cotransport at the level of the cotransporter and/or the secondary to an alteration in Na⁺/K⁺-ATPase activity. Therefore, the aims of this study were to test the hypothesis that LTD₄ specifically inhibits Na-alanine cotransport in IEC-18 cells and to determine the cellular mechanisms of this alteration.

MATERIALS AND METHODS

Chemicals. LTD₄ was purchased from Cayman Chemical (Ann Arbor, MI), REV5901 from Calbiochem (San Diego, CA), tetramethylammonium chloride (TMA-Cl), tetramethylammonium hydroxide (TMA-OH), HEPES buffer, L-alanine, L-glutamine, and β-hydroxy butyric acid from Sigma Chemical (St. Louis, MO), and insulin from Novo Nordisk (Clayman, NC). DMEM, Leibovitz's L-15 medium powder (L-15), bovine fetal serum, Dulbecco's phosphate-buffered saline (DPBS), and all supplements were supplied from Invitrogen (Grand Island, NY). [³H]-L-alanine was purchased from Amersham Biosciences (Buckinghamshire, UK).

Cell culture. The normal, diploid, rat small IEC (IEC-18) line (CRL-1589 American Type Culture Collection, Manassas, VA) was used between passages 5 and 20. Density of IEC-18 cells were 2 x 10⁶ cells/well, grown on six-well Transwell plates (polyester membrane thickness 10 µm, pore size 0.4 µm; transparent inserts provide better cell visibility and monolayer formation under phase contrast microscopy), or 5 x 10⁶ cells/150-mm Petri dish (Corning, Corning, NY) in DMEM (high glucose 4.5 g/l, sodium bicarbonate 3.7 g/l) containing 2 mM L-glutamine, 10% vol/vol bovine fetal serum, 0.02% insulin, and 0.25% β-hydroxybutyric acid without any antibiotics in a humidified atmosphere of 10% CO₂ at 37°C. The medium was changed every 2–3 days, cells were treated on the eighth day, and experiments were carried out on the tenth day of postconfluence. Preliminary dose- and time-response experiments were performed to ascertain appropriate stimulus and length of incubation.

Amino acid transport. Transport studies were performed in six-well Transwell plates using triplicate wells for each time point. Each experiment was performed at 10 days postconfluence. Cells were rinsed once with oxygenated Leibovitz's (L-15, 100% oxygen) media (pH 7.4; pH was adjusted with TMA-OH), supplemented with 10% bovine fetal serum, 20 mM HEPES, and 200 U/l insulin (0.02%) and were incubated at room temperature for 1 h. Cells were washed once with TMA-HEPES buffer (in mM) (47 KCl, 1 MgSO₄, 1.2 KH₂PO₄, 20 HEPES, 125 CaCl₂, and 130 TMA-Cl; pH 7.4, pH was adjusted with TMA-OH) and were incubated with TMA-HEPES (Na-free) buffer for 10 min in room temperature. Cells were incubated with reaction mixture containing 200 µM of cold [³H]-L-alanine 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 (800 µl, 1 M) was added on the membrane in each transwell 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. The vial was kept in the dark for 48 h and counted for measuring [³H]-L-alanine in a scintillation counter (LS 6500; Beckman Coulter, Fullerton, CA).

Protein determination. Total protein was measured by the Bradford method (3), using the Bio-Rad protein assay kit (Hercules, CA) with BSA as standard.

Enzyme measurement. IEC-18 cells were cultured on 150-mm Transwell Petri dishes, the and treated group received LTD₄ (1 µM, for 48 h) at 8 days postconfluence. Cells were washed twice with ice-cold DPBS and harvested following scraping of the Transwell membrane. The cells were homogenized and centrifuged to make the membrane. Na⁺/K⁺-ATPase was measured as inorganic phosphate (P_i) formation in cellular homogenates from the same amount of cells (7, 19). Enzyme-specific activity was expressed as nmol of P_i released per mg protein per minute.

Isolation of RNA and expression analysis. IEC-18 cells were grown on Transwell Petri dishes, and treated cells received LTD₄ (1 µM) for 48 h at 8 days postconfluence. Total RNA was isolated from control and LTD₄-treated IEC-18 cells of 10 days postconfluence using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA). First-strand cDNA synthesis was performed using SuperScript III from Invitrogen Life Technologies. The cDNAs generated were used as templates for real-time PCR. Real-time PCR for ASCT1 and β-actin were performed using TaqMan Gene Expression Assays, assay ID Rn01786205_m1 and 4352931E, respectively, obtained from Applied Biosystems (Foster City, CA). Final unlabeled primer concentrations were 900 nM each, and FAM dye-labeled TaqMan MGB probe was 250 nM. Real-time PCR reactions were performed using TaqMan Universal PCR Master Mix from Applied Biosystems on an ABI 7300 real-time PCR system according to the manufacturer's instructions. All experiments were performed in triplicate.

Western blot analysis. IEC-18 cells were washed three times with ice-cold PBS and were collected from 150-mm Transwell dishes. BBM from control and LTD₄-treated IEC-18 cells were prepared according to the previously published protocol with slight modifications in that the process was stopped at the plasma membrane isolation without BBM vesicle preparation (10). Western blotting for ASCT1 was performed essentially according to the standard protocol (1). Briefly, BBM was 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, and 1 mM NaF). Protease inhibitor cocktail was added to RIPA buffer according to the manufacturer's recommendation (SAFC Biosciences, Lenexa, KS). Equal volume of 2x SDS/sample buffer (100 mM Tris, 25% glycerol, 2% SDS, 0.01% bromphenol blue, and 10% mercaptoethanol, pH 6.8) was added, and the proteins were separated on a 4–20% Ready Gel (Bio-Rad). After being transferred onto the nylon membrane (Schleicher and Schuell BioScience, Keene, NH), ASCT1 was probed by the primary rabbit polyclonal antibody for ASCT1 (Biotrend Chemikalien, Cologne, Germany). Horseradish peroxidase-conjugated secondary antibody (Jackson Immunoresearch Laboratories, West Grove, PA) was used to monitor the binding of the primary antibody. Enhanced chemiluminescence Western blotting detection reagent (GE Healthcare Bio-Sciences, Piscataway, NJ) was used to detect the immobilized ASCT1. The resultant chemiluminescence was detected with the use of X-ray films. The intensity of the bands was quantitated by using a molecular dynamics densitometric scanner. All experiments were performed in triplicate.

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

RESULTS

Alanine uptake in IEC-18 cells. To determine the presence of Na-dependent alanine cotransport in IEC-18 cells, alanine uptake studies were performed at 2 and 4 min in the presence or absence of Na. It was found that Na-dependent alanine uptake was present in IEC-18 cells (alanine uptake was 8.89 ± 0.51 nmol/mg protein in presence of Na and 1.90 ± 0.12 nmol/mg protein in absence of Na at 2 min, n = 6, P < 0.01; and 12.66 ± 0.65 nmol/mg protein in presence of Na and 2.46 ± 0.21 nmol/mg protein in absence of Na at 4 min, n = 6, P < 0.01; Fig. 1A). The data indicate that Na-alanine cotransport is present in IEC-18 cells.

View larger version (23K): [in this window] [in a new window]   Fig. 1. A: effect of extracellular Na+ on [3H]-L-alanine ([3H]Ala) uptake in rat intestinal epithelial cells (IEC-18). IEC-18 cells were grown with DMEM on six-well Transwell plates. [3H]Ala uptake was performed at 10 days postconfluence at 2 min and 4 min in presence of Na (+Na) or in absence of Na (–Na). Extracellular Na+ significantly stimulated alanine uptake, indicating that Na-dependent alanine cotransport is present in IEC-18 cells. B: IEC-18 cells were treated with either leukotriene (LT) D4 (1 µM, LTD4 group) or same volume of ethanol (vehicle) for control group at 8 days for a 48-h period. [3H]Ala 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 alanine uptake. Na-dependent alanine cotransport is significantly inhibited by LTD4 in IEC-18 cells in each time point (*P < 0.01). Data represent means ± SE, n = 6.

Effect of LTD₄ on Na⁺/K⁺-ATPase. Since Na⁺/K⁺-ATPase on the basolateral membrane provides the favorable Na⁺ gradient for Na-alanine cotransport, the reduction of Na-dependent alanine uptake (Fig. 1B) may be due to inhibition of Na⁺/K⁺-ATPase activity. Therefore, Na⁺/K⁺-ATPase activity was determined. As shown in Fig. 2, LTD₄ treatment did not alter Na⁺/K⁺-ATPase levels in IEC-18 cells. Na⁺/K⁺-ATPase levels were 26.2 ± 2.3 nmol·mg protein^–1·min^–1 in control and 25.2 ± 2.2 nmol·mg protein^–1·min^–1 in LTD₄-treated cells (n = 8). These data indicate that the inhibition of Na-alanine cotransport by LTD₄ is not secondary to an alteration in the Na⁺ extruding capacity of these cells.

View larger version (32K): [in this window] [in a new window]   Fig. 2. Effect of LTD4 on Na+/K+-ATPase activity in rat IEC-18 cells. IEC-18 cells were grown with DMEM on Transwell Petri dishes. Cells were treated with either LTD4 (1 µM, treated group) or same volume of ethanol (vehicle) for control group at 8 days postconfluence for a 48-h period. Na+/K+-ATPase was measured as inorganic phosphate (Pi) formation in cellular homogenates. Na+/K+-ATPase activity was not affected by treatment with LTD4. Data represent means ± SE, n = 8.

View larger version (45K): [in this window] [in a new window]   Fig. 3. The effects of REV5901 (LTD4 receptor antagonist, dose 10 µM) on LTD4 and Na-dependent alanine uptake in rat IEC-18 cells. Cells were grown with DMEM on six-well Transwell plates. IEC-18 cells were pretreated with REV5901 for 1 h before of LTD4 (1 µM) treatment; control group received same volume of ethanol (vehicle) at 8 days postconfluence for a 48-h period. Uptake experiments were performed in presence or absence of Na at 10 days postconfluence. [3H]Ala 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 alanine uptake. REV5901 antagonized the effect of LTD4 on Na-dependent alanine cotransport in IEC-18 cells, whereas REV5901 itself had no effect. These data indicate that the effect of LTD4 was specific on Na-alanine cotransport in IEC-18 cells. Data represent means ± SE, n = 6 (*P < 001).

View larger version (25K): [in this window] [in a new window]   Fig. 4. Effect of glutamine (Gln) on Na-dependent alanine (Ala) uptake in rat IEC-18 cells. IEC-18 cells were grown with DMEM on six-well Transwell plates. Uptake experiments were performed in presence or absence of Na at 10 days postconfluence. Reaction mixture contained 10 mM Gln as substrate for Gln group and 10 mM Ala for Ala group. [3H]Ala 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 Ala uptake. The data are presented as percent of control, and 25% of Na-dependent Ala uptake was inhibited by Gln, in contrast, more than 90% by alanine. These data indicate that the Na-dependent alanine, serine, cysteine cotransporter 1 (ASCT1) is predominantly functioning in IEC-18 cells. Data represent means ± SE, n = 6 (*P < 001).

View larger version (21K): [in this window] [in a new window]   Fig. 5. Kinetics of Na-dependent alanine uptake in rat IEC-18 cells. A: data are representative of 3 such experiments. IEC-18 cells were grown with DMEM on six-well Transwell plates. IEC-18 cells were treated with LTD4 (1 µM); the control group received same volume of ethanol (vehicle) at 8 days postconfluence for a 48-h period. Uptake experiments were performed in presence or absence of Na at 10 days postconfluence. [3H]Ala 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 alanine uptake. Uptake of alanine as a function of varying concentrations of extracellular alanine. Uptake for all concentrations was measured at 30 s. Isosmolarity was maintained by adjusting the concentration of mannitol. As extracellular alanine concentration was increased, uptake of alanine was stimulated and subsequently became saturated in control and LTD4-treated IEC-18 cells. B: analyses of these data with GraphPad Prism 4 software provided the kinetic parameters. The maximal rate of uptake (Vmax) of alanine was not affected by LTD4 treatment. However, the affinity (Km) for alanine uptake was significantly reduced by LTD4 treatment (n = 3, *P < 0.01). These data demonstrate that the mechanism of inhibition of Na-alanine cotransport by LTD4 in IEC-18 cells is secondary to a reduction in the affinity of the cotransporters for alanine without a change in the number of cotransporters.

View larger version (33K): [in this window] [in a new window]   Fig. 6. The mRNA levels for ASCT1 in rat IEC-18 cells. IEC-18 cells were grown with DMEM on Transwell Petri dishes. Cells were treated with either LTD4 (1 µM, treated group) or same volume of ethanol (vehicle) for control group at 8 days postconfluence for 48 h, and RNA was extracted by using TRIzol reagent. The messages for ASCT1 were quantitated by real-time PCR using TaqMan Gene Expression Assay, see MATERIALS AND METHODS for details. Data represent means ± SE, n = 3 in triplicate. These data indicate that the effect of LTD4 on ASCT1 is not at the level of the number of cotransporters.

View larger version (36K): [in this window] [in a new window]   Fig. 7. Western blot analysis for Na-dependent alanine cotransport, ASCT1 in rat IEC-18 cells. A: data are representative of 3 such experiments. IEC-18 cells were grown with DMEM on Transwell Petri dishes. Cells were treated with either LTD4 (1 µM, treated group) or same volume of ethanol for control group at 8 days postconfluence for 48 h. ASCT1 was probed by the primary rabbit monoclonal antibody for ASCT1. B: intensity of the bands was quantitated by using a molecular dynamics densitometric scanner, see MATERIALS AND METHODS for details. Data represent means ± SE, n = 3. These results indicate that the mechanism of inhibition of ASCT1 by LTD4 in IEC-18 cells is not secondary to a decrease in the number of cotransporters for alanine.

This study, for the first time, demonstrates that ASCT1 is present in IEC-18 cells and LTD₄ inhibits Na-alanine cotransport in IEC-18 cells by modulating ASCT1. This inhibition is not secondary to a reduction in the capacity of IEC-18 cells to extrude Na⁺. At the level of the Na-alanine cotransporter, the mechanism of inhibition is due to a decrease in the affinity of cotransporters in the LTD₄-treated IEC-18 cells.

Uptake studies for alanine indicate the IEC-18 cells possess Na-alanine cotransporter (Fig. 1). In addition, substrate inhibition studies indicate that a single transport system is responsible for Na-dependent alanine uptake in IEC-18 cells. This uptake is not inhibited more than 25% by 10 mM glutamine (Fig. 4), even with 50 mM glutamine (data not shown). In contrast, more than 92% Na-dependent alanine uptake was inhibited by 10 mM alanine. In concert, these findings indicate that Na-alanine cotransporter in IEC-18 cells is ASCT1. Doyle and McGivan (6) demonstrated Na-dependent alanine uptake in bovine renal epithelial cell line NBL-1. Furthermore, Pan and Stevens (13) observed the presence of a single amino acid cotransporter, system B, as the Na-dependent alanine uptake pathway in Caco-2 cells. This difference may be attributable to the differences in cell lines; Caco-2 cells are colon cancer cells, whereas IEC-18 are nontransformed IEC.

Given the numerous immune-inflammatory mediators that are produced in chronically inflamed intestine and given that at least some of them are capable of altering transport pathways, it is reasonable to postulate that different immune-inflammatory mediators may regulate different transport pathways during chronic enteritis. LTD₄ is known to be elevated in mucosa from the chronically inflamed intestine (20). Although Smith et al. (17) examined the effects of LTD₄ on electrolyte transport in intact tissue preparations of rat and rabbit ileum, whether this leukotriene may be responsible for ASCT1 inhibition during chronic enteritis was not previously known. This study demonstrates that LTD₄ inhibits Na-alanine cotransport in IEC-18 cells. This inhibition is specific since it is known that IEC-18 cells have LTD₄ receptors (Cyst 1 and 2) and they are inhibitable by a specific LTD₄ receptor blocker REV5901. Na-dependent alanine uptake inhibition by LTD₄ was reversed by REV5901, whereas REV5901 itself does not perturb alanine uptake in IEC-18 cells (Fig. 3). These alterations indicate that LTD₄ acts via LTD₄ receptor (either Cyst 1 or Cyst 2) to inhibit ASCT1 in IEC-18 cells. Figure 2 shows that LTD₄ treatment did not alter Na⁺/K⁺-ATPase activity in IEC-18 cells. These data indicate that the inhibition of Na-alanine cotransport by LTD₄ is not secondary to an alteration in the Na⁺ extruding capacity of these cells. Thus the inhibition of Na-dependent alanine uptake is an effect at the level of the cotransporter itself.

At the level of the cotransporter, kinetic studies demonstrated that the mechanism of inhibition of ASCT1 by LTD₄ is not secondary to altered maximal rate of alanine uptake (V_max). In contrast, the K_m for alanine uptake is significantly increased by LTD₄. Thus the mechanism of inhibition of Na-alanine cotransport is secondary to a reduction in the affinity of the cotransporters for alanine rather than a decrease in the number of cotransporters. This mechanism of inhibition (K_m phenomenon) for alanine is consistent with the rabbit model of chronically inflamed ileum.

Quantitative real-time PCR (RTQ-PCR) data demonstrates the presence of the message for ASCT1 in IEC-18 cells (Fig. 6) as the cotransporter responsible for Na-alanine cotransport. Functional inhibition studies are in agreement with this alteration (Fig. 4). Furthermore, Western blot analyses as shown in Fig. 7 demonstrate the presence of functional ASCT1 protein in IEC-18 cell BBM. The expected molecular weight of ASCT1 is 56 kDa (based on the amino acid sequence). The molecular weight of immunoreactive ASCT1 is 70 kDa. This could be due to the posttranslational modification of the protein. Both ASCT1 mRNA and immunoreactive protein levels remained unchanged in LTD₄-treated IEC-18 cells. These findings, in concert with kinetic studies, indicate that the mechanism of inhibition of ASCT1 by LTD₄ in IEC-18 cells is secondary to altered affinity of the cotransporter for amino acid rather than an alteration in the number of cotransporters.

In conclusion, Na-amino acid cotransporter, ASCT1, is inhibited by LTD₄ in IEC-18 cells by a mechanism similar to that seen in rabbit chronic intestinal inflammation. Neither inhibition is a consequence of a reduction in the Na⁺ extrusion capacity of the cell. At the level of the cotransporter, the mechanism of inhibition of Na-amino acid cotransport is secondary to a decrease in the affinity for alanine in vivo in rabbit and in vitro in IEC-18 cells. Thus LTD₄, which is known to be elevated in the mucosa of the chronically inflamed intestine, inhibits Na-alanine cotransport in IEC-18 cells in vitro by a mechanism identical to that seen in villus cells during chronic enteritis. Understanding the mechanisms involved in the downregulation of ASCT1 for alanine absorption is critical during chronic intestinal inflammation such as inflammatory bowel diseases where nutrient cotransporter functions are compromised and lead to nutrient malabsorption. Future studies are required to delineate the second messenger pathways involved in LTD₄-mediated downregulation of ASCT1.

GRANTS

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

FOOTNOTES

Address for reprint requests and other correspondence: U. Sundaram, Sect. of Digestive Diseases, Dept. of Medicine, PO Box 9161, Health Sciences Ctr., 1 Medical Center Dr., West Virginia Univ., Morgantown, WV 26506 (e-mail: usundaram{at}hsc.wvu.edu)

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.

REFERENCES

1. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K. Current Protocols in Molecular Biology. New York: John Wiley & Sons, 1995, p. 25–26.
2. Binder HJ, Ptak T. Jejunal absorption of water and electrolytes in inflammatory bowel diseases. J Lab Clin Med 76: 915–924, 1970.[Web of Science][Medline]
3. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254, 1976.[CrossRef][Web of Science][Medline]
4. Castro GA. Immunological regulation of electrolyte transport. In: Textbook of Secretory Diarrhea, edited by Lebenthan E and Duffey ME. New York: Raven, 1991, p. 31–46.
5. Christensen HN, Liang M, Archer EG. ASCT-1 is a neutral amino acid exchanger with chloride channel activity. J Biol Chem 242: 5237–5246, 1967.[Abstract/Free Full Text]
6. Doyle FA, McGivan JD. The bovine renal epithelial cell line NBL-1 expresses a broad specificity Na(+)-dependent neutral amino acid transport system (System B^o) similar to that in bovine renal brush border membrane vesicles. Biochim Biophys Acta 1104: 55–62, 1992.[Medline]
7. Forbush B. Assay of Na⁺/K⁺-ATPase in plasma membrane preparations: increasing the permeability of membrane vesicles using sodium dodecyl sulphate buffered with bovine serum albumin. Anal Biochem 128: 159–163, 1983.[CrossRef][Web of Science][Medline]
8. Ganapathy V, Brandsch M, Leibach FH. Intestinal transport of amino acids and peptides. In: Physiology of the Gastrointestinal Tract (3rd ed.), edited by Johnson LR. New York: Raven, 1994, p. 1773–1794.
9. Hopfer U. Membrane transport mechanisms for hexoses and amino acids in the small intestine. In: Physiology of the Gastrointestinal Tract (2nd ed.), edited by Johnson LR. New York: Raven, 1987, p. 1499–1526.
10. Knickelbein R, Aronson PS, Schron CM, Seifter J, Dobbins JW. Sodium and chloride transport across rabbit ileal brush border. II. Evidence for Cl-HCO3 exchange and mechanism of coupling. Am J Physiol Gastrointest Liver Physiol 249: G236–G245, 1985.[Abstract/Free Full Text]
11. Munck LK, Munck BA. Amino acid transport in the small intestine. Physiol Res 43: 335–346, 1994.[Web of Science][Medline]
12. Palacin M, Estevez R, Bertran J, Zorzano A. Molecular biology of mammalian plasma membrane amino acid transporters. Physiol Rev 78: 969–1054, 1998.[Abstract/Free Full Text]
13. Pan M, Stevens BR. Differentiation- and protein kinase C-dependent regulation of alanine transport via system B. J Biol Chem 270: 3582–3587, 1995.[Abstract/Free Full Text]
14. Powell DW. Immunophysiology of intestinal electrolyte transport. In: Handbook of Physiology. The Gastrointestinal System. Intestinal Absorption and Secretion. Bethesda, MD: Am. Physiol. Soc., 1991, sect. 6, vol. IV, chapt. 25, p. 591–641.
15. Sartor RB, Powell DW. Mechanisms of diarrhea in inflammation and hypersensitivity: immune system modulation of intestinal transport. In: Current Topics in Gastroenterology: Diarrheal Diseases, edited by Field M. New York: Elsevier Science, 1991, p. 75–114.
16. Schultz SG, Curran PF. Coupled transport of sodium and organic solutes. Physiol Rev 50: 637–718, 1970.[Free Full Text]
17. Smith PL, Montzka DP, McCafferty GP, Wasserman MA, Fondacaro JD. Effect of sulfidopeptide leukotrienes D₄ and E₄ on ileal ion transport in vitro in the rat and rabbit. Am J Physiol Gastrointest Liver Physiol 255: G175–G183, 1988.[Abstract/Free Full Text]
18. Stadnyk AW. Intestinal epithelial cells as a source of inflammatory cytokines and chemokines. Can J Gastroenterol 16: 241–246, 2002.[Web of Science][Medline]
19. Sundaram U, Wisel S, Fromkes JJ. Unique mechanism of inhibition of Na-amino acid co-transport during chronic ileal inflammation. Am J Physiol Gastrointest Liver Physiol 275: G483–G489, 1998.[Abstract/Free Full Text]
20. Sundaram U, Hassanain H, Suntres Z, Yu JG, Cooke HJ, Guzman J, Christofi FL. Rabbit chronic ileitis leads to up-regulation of adenosine A1/A3 gene products, oxidative stress, and immune modulation. Biochem Pharmacol 65: 1529–1538, 2003.[CrossRef][Web of Science][Medline]

This Article Abstract Full Text (PDF) All Versions of this Article: 295/1/G1    most recent 00498.2007v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Talukder, J. R. Articles by Sundaram, U. Search for Related Content PubMed PubMed Citation Articles by Talukder, J. R. Articles by Sundaram, U.