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TRANSLATIONAL PHYSIOLOGY
1Ohio State University School of Medicine, Columbus, Ohio; and 2Section of Digestive Diseases, Department of Medicine, West Virginia University Medical Center, Morgantown, West Virginia
Submitted 24 October 2005 ; accepted in final form 14 March 2006
ABSTRACT
Neutral Na-amino acid cotransport by system ATB0 [e.g., Na-alanine cotransport (NAcT)] is an important means of assimilation of amino acids in the intestine. NAcT is inhibited during chronic intestinal inflammation by an alteration in the affinity for the amino acid. How glucocorticoids, a standard of treatment for diseases characterized by chronic intestinal inflammation, may affect NAcT during chronic enteritis is not known. Thus we first demonstrated that methylprednisolone (MP) stimulated NAcT in the normal intestine. The mechanism of stimulation was secondary to an increase in cotransporter numbers without an alteration in the affinity for the amino acid. Treatment with MP reversed the reduction in NAcT in villus cells from the chronically inflamed intestine. MP also alleviated the decrease in Na-K-ATPase activity in villus cells during chronic enteritis. However, MP treatment reversed the NAcT inhibition in villus cell brush border membrane vesicles from the inflamed intestine, which suggested an effect of MP at the level of the cotransporter itself. Kinetic studies demonstrated that the reversal of NAcT inhibition by MP was secondary to restoration in the affinity for the amino acid without a change in the Vmax. Unaltered steady-state mRNA and immunoreactive protein levels of NAcT also indicated that the number of cotransporters was unchanged after MP treatment in the chronically inflamed intestine. These results indicated that MP reversed NAcT inhibition in the chronically inflamed intestine by restoring the affinity of the transporter for the amino acid while it stimulated NAcT in the normal intestine by increasing the cotransporter numbers. Therefore, MP differentially regulates NAcT in the normal and chronically inflamed intestine.
glucocorticoids; inflammation; amino acid transport; sodium transport; intestinal absorption
One of the most common intestinal disorders is inflammatory bowel disease (IBD, e.g., Crohn’s disease). The most common morbidity of IBD is malabsorption of electrolytes, fluid, and nutrients, which leads to malabsorption, malnutrition, and weight loss in this condition (1, 4, 5). However, the mechanism of alteration of Na-dependent amino acid uptake in human inflammatory bowel diseases is unknown. In a mammalian model of chronic intestinal inflammation it was previously demonstrated that NAcT was inhibited (29). At the cellular level, NAcT was inhibited secondary to an effect at the level of the cotransporter in the BBM and as well as Na-K-ATPase on the basolateral membrane. However, inhibition of NAcT in villus cell BBM vesicles (BBMV) from the chronically inflamed intestine indicated that effect is indeed at the level of the cotransporter itself. Further kinetic and molecular studies demonstrated that the mechanism of inhibition was secondary to an alteration in the affinity for the amino acid without a change in the numbers of cotransporters (29). Given that Na-dependent amino acid uptake is present in the human small intestine and our demonstration that NAcT is reduced during chronic enteritis in rabbits (29) clearly raises the possibility that NAcT may be affected in IBD.
A wide variety of immune-inflammatory mediators are known to be endogenously produced in IBD, which may, at least in part, have an effect on electrolyte and nutrient transport pathways (5, 18, 20). However, it is not known whether a given immune-inflammatory mediator pathway is responsible for alterations seen with a specific transport pathway during chronic enteritis. Broad-spectrum immune suppression with glucocorticoids, a commonly used treatment of IBD, at least partially alleviates the malabsorption seen in these conditions (14). However, the cellular mechanism of action of glucocorticoids on NAcT in the chronically inflamed intestine, let alone in the normal intestine, is not known.
Given this background (9, 15, 17, 20, 23, 24), we looked at the effect of the glucocorticoid methylprednisolone (MP) on NAcT in the normal and chronically inflamed rabbit intestine. Glucocorticoids have been demonstrated to stimulate NAcT in neonatal pig intestine (13) while having no effect on this cotransport process in the rat small intestinal cell line IEC-6 (12). However, the mechanism of NAcT stimulation by glucocorticoids was not addressed in the former study. Thus, the effect of MP on NAcT is unclear in the normal mammalian small intestine. Furthermore, whether it may alleviate the inhibition of NAcT during chronic intestinal inflammation is unknown. Therefore, goals of this study were to 1) determine whether MP regulates NAcT in the normal mammalian small intestine and delineate this mechanism, 2) determine whether MP can reverse the inhibition of NAcT in the chronically inflamed intestine and delineate this mechanism, and 3) test the hypothesis that MP may regulate NAcT differentially in the normal and chronically inflamed intestine.
METHODS
Induction of chronic inflammation and drug treatment.
New Zealand White male rabbits (2.0–2.2 kg) were utilized. Chronic intestinal inflammation was produced in rabbits as previously reported (25). Pathogen-free rabbits were intragastrically inoculated with Eimeria magna oocytes or sham inoculated with 0.9% NaCl (control animals). None of the sham inoculations and 
80% of inoculations with coccidia resulted in chronic intestinal inflammation during days 13–15. Only enterocytes from those animals that had histologically confirmed chronic ileal inflammation were utilized for experiments. Control or inoculated rabbits were treated intramuscularly with saline (days 12 and 13 postinoculation) or with water-soluble 20 mg of MP sodium succinate per kilogram body weight per day (days 12 and 13 postinoculation) and killed on day 14 postinoculation. Animals were killed with an overdose of pentobarbital sodium through the ear vein according to protocols of the West Virginia University and the Ohio State University departments of Laboratory Animal Medicine.
Cell isolation. Villus cells were isolated from the intestine by a calcium chelation technique as previously described (25, 27). Previously established criteria were utilized to validate good separation of villus and crypt cells. Some of these criteria included 1) marker enzymes (e.g., thymidine kinase, alkaline phosphatase), 2) transporter specificity (e.g., Na:H on the BBM of villus, but not crypt cells), 3) differences in intracellular pH (e.g., intracellular pH is higher in crypt cells compared with villus), 4) morphological differences (e.g., villus cells are larger and with better developed BBM compared with crypt cells), and 5) differing rates of protein synthesis (e.g., higher synthesis rate in crypt cells compared with villus).
Previously established criteria were also utilized to study cells with good viability and to exclude cells that showed evidence of poor viability: 1) Trypan blue exclusion, 2) the demonstration of Na:H and Cl:HCO3 exchange activities, and 3) the ability of the cells to maintain a baseline pH or imposed acid or alkaline gradient and return to baseline pH after perturbations. At least one, frequently two, or all three criteria are used to assess viability of all cell preps. The cells were maintained in short-term culture for up to 6–8 h (25, 27).
Na-K-ATPase measurement. Na-K-ATPase was measured as Pi liberated (8) in cellular homogenates from the same amount of cells from normal or inflamed intestine as previously described (28). Enzyme specific activity was expressed as nanomoles of Pi released per milligram protein per minute.
BBMV preparation. BBMV from rabbit ileal villus cells were prepared by CaCl2 precipitation and differential centrifugation as previously reported (28). 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 villus cells. Intact cell uptakes were done as previously described (28–30). In brief, villus cells (100 mg wet wt) were washed and resuspended in HEPES buffer containing (in mM) 0.1 alanine, 4.5 KCl, 1.2 KH2PO4, 1.0 MgSO4, 1.25 CaCl2, 20 HEPES, and either 130 mM sodium chloride or choline chloride and was gassed with 100% O2 (pH 7.4 at 37°C). Ten µCi of 3H-alanine were added to 1 ml cell suspension in the HEPES buffer and 100 µl aliquots were removed at desired time intervals. The uptake was arrested by mixing with 10 ml ice-cold stop solution (choline-HEPES buffer). The mixture was filtered on 0.65 µm Millipore (HAWP) filters. Following two washes with ice cold-stop solution, the filter was dissolved in 5 ml Ecoscint and the radioactivity was determined.
BBMV uptake studies. Uptake studies were performed by rapid filtration technique as previously described (28–30). In brief, 5 µl of BBMV resuspended in 100 mM choline chloride, 0.10 mM MgSO4, 50 mM HEPES-Tris (pH 7.5), 50 mM mannitol, and 50 mM KCl were incubated in 95 µl of reaction medium that contained 50 mM HEPES-Tris buffer (pH 7.5), 0.1 mM alanine, 20 µCi 3H-alanine, 0.10 mM MgSO4, 50 mM KCl, 50 mM mannitol, 100 mM of either NaCl or choline chloride. The vesicles were voltage clamped with 10 µM valinomycin and 100 µM carbonyl cyanide p-(tri-fluoromethoxy) phenyl-hydrozone (FCCP). At desired times uptake was arrested by mixing with ice-cold stop solution [50 mM HEPES-Tris buffer (pH 7.5), 0.10 mM MgSO4, 75 mM KCl, and 100 mM choline chloride]. The mixture was filtered on 0.45 µm Millipore (HAWP) filter and washed with 10 ml ice-cold stop solution. Filters with BBMV were dissolved in Ecoscint and radioactivity was determined.
Northern blot studies.
Total RNA was extracted from rabbit ileal villus cells by the quanidinium isothiocynate-cesium chloride method as previously reported (7, 29). Messenger RNA was isolated from total RNA by oligo(dT)-cellulose chromatography (3). After denaturation messenger RNA was electrophoresed on 1.8% agarose-formaldehyde gel, transferred to nylon membrane (Schleicher and Schuell, Keene, NH), and incubated with prehybridization solution. Membranes were hybridized with 32P-labeled cDNA. Hybridized membrane was exposed to autoradiography film (NEN Research Products, Boston, MA). Human 
-actin DNA was utilized to ensure equal loading of messenger RNA onto the electrophoresis gels. -Actin DNA was random labeled with [32P]CTP with Klenow polymerase.
cDNA probe synthesis for Northern blot studies.
On the basis of rabbit Na-dependent neutral amino acid cotransporter sequence a 292-base pair fragment was selected for amplification (14, 29). Sense (5'-GTCTGCALLATCATCAACGT-3') and antisense (5'-CCGGTCTGTCCGTTTACCCAT-3') primers were designed and synthesized (GIBCO-BRL). Using rabbit enterocyte cDNA as template the 292 base pair probe was amplified by polymerase chain reaction (PCR). PCR was performed in a thermal cycler (Perkin-Elmer 2400) in a reaction volume of 100 µl consisting of 10 mM Tris·HCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl2, 0.2 mM each of dATP, dCTP, dGTP, and dTTP, 0.5% cDNA synthesis product, 0.2 mM oligonucleotide primers, and 2–5 units of Ampli-Taq gold polymerase. PCR profile consisted of 35 cycles each at 94, 52, and 72°C. Hot PCR with 
-dCTP-32P was simultaneously performed, and the PCR product was used as probes for Northern hybridization (29).
Western blot studies. BBMV (4 µg) were diluted in SDS buffer, boiled, and electrophoresed on a 12% SDS-PAGE gel. The gel was electroblotted onto a polyvinylidene difluoride membrane and blocked for 2 h in 5% BSA at room temperature. The membrane was incubated at room temperature with 1:3,000 anti-rabbit ATB0 antiserum (Alpha Diagnostics) followed by goat anti-rabbit IgG coupled to horseradish peroxidase (1:10,000, Pierce, Rockford, IL). After each incubation, the membrane was washed extensively with PBS-0.2% Tween 20. The signal was developed with the chemiluminescence Western blot kit (NEN Research Products).
Data presentation. When data are averaged, means ± SE are shown in the figures except when error bars are inclusive within the symbol. All uptakes were done in triplicate. The n number for any set of experiments refers to vesicle or isolated cell preparations from different animals. Preparations in which cell viability was <85% were excluded from analysis. Student’s t-test was used for statistical analysis.
RESULTS
Na-Amino acid cotransport in intact cells. We had previously demonstrated that Na-dependent amino acid uptake was present in villus, but not crypt cells from the normal and the chronically inflamed intestine. Furthermore, Na-dependent amino acid uptake was significantly diminished in villus cells from the chronically inflamed intestine (29). In this study we looked at Na-dependent amino acid uptake in villus cells from sham inoculated rabbits (control) and rabbits with chronic enteritis treated with MP. Na-stimulated alanine uptake was significantly enhanced in villus cells from control rabbits treated with MP (Fig. 1A). Also, Na-stimulated alanine uptake was significantly diminished in villus cells from the chronically inflamed intestine (Fig. 1). Furthermore, the inhibition of Na-stimulated alanine uptake seen in villus cells from the chronically inflamed intestine was almost completely reversed by MP treatment (Fig. 1B). These data indicated that MP stimulates NAcT in normal villus cells and reverses the inhibition of NAcT in villus cells from the chronically inflamed intestine.
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This study demonstrates that Na-amino acid cotransport is differentially regulated by glucocorticoids in the normal and chronically inflamed intestine. Treatment with glucocorticoids stimulates NAcT in the normal intestine. The mechanism stimulation by MP in the normal intestine is not secondary to an alteration in the Na-extrusion capacity of the villus cells. At the level of the cotransporter, the MP-mediated stimulation in the normal intestine is secondary to an increase in cotransporter numbers without an alteration in the affinity. The increase in cotransport numbers may be secondary to increased synthesis of the protein and/or membrane trafficking from cytosol to BBM.
As previously noted, glucocorticoids’ effect on Na-amino acid cotransport in the mammalian small intestine is poorly understood. It was shown to stimulate NAcT in neonatal pig intestine (13), but the mechanism of this stimulation was not deciphered. In another study it was suggested that glucocorticoids acutely accelerated intestinal nutrient transport possibly via diffusion (11). Yet in another study it was shown to have no effect on this cotransport process in the rat intestinal enterocytes (12). Thus the effect of an all too common medication, glucocorticoids, and the exact mechanism of regulation by this drug of an important pathway of amino acid assimilation in the mammalian small intestine were not well understood before this study.
In the chronically inflamed intestine, treatment with MP reverses the inhibition of NAcT. The mechanism of reversal by MP during chronic enteritis is not entirely secondary to an alteration in Na-K-ATPase activity. The MP-mediated reversal of inhibition in the chronically inflamed intestine is secondary to a restoration of the affinity of the cotransporter for the amino acid without an alteration in the cotransporter numbers.
Whereas malabsorption of electrolytes and nutrients have been well documented in IBD (1, 4, 5, 18, 20, 22), until recently the alterations that occur in Na-nutrient cotransport processes during chronic intestinal inflammation had not been investigated. In a rabbit model of chronic intestinal inflammation, unique alteration in Na-nutrient cotransport processes were observed (28–30). At the cellular level, Na-glucose, Na-amino acid, and Na-bile acid cotransport were inhibited secondary to an effect at the level of the cotransporter as well as a reduction in Na-K-ATPase in the chronically inflamed intestine. However, at the cotransporter level each pathway was uniquely altered during chronic enteritis. Na-glucose cotransport (SGLT-1) was inhibited by a decrease in the number of cotransporters without a change in the affinity for glucose in the chronically inflamed intestine (28). In contrast, NAcT was inhibited by a reduction in the affinity for the amino acid without a change in the number of cotransporters during chronic enteritis (29). Unlike these two Na-solute cotransport processes, Na-bile acid cotransport was inhibited by both a decrease in the affinity for the bile acid as well as in cotransporter numbers in the chronically inflamed ileum (30). Therefore, these three types of Na-dependent solute cotransport processes are inhibited by different mechanisms during chronic enteritis. Since a variety of immune-inflammatory mediators are known to be released in the chronically inflamed intestine, it is hypothesized that different immune-inflammatory mediators may regulate these three cotransport pathways in the chronically inflamed intestine.
This hypothesis may be further supported if a broad-spectrum immune modulator such as glucocorticoid were to reverse the malabsorption during chronic enteritis by reversing the same unique mechanism that resulted in the inhibition of each of the cotransporters during chronic enteritis. Indeed, we have previously demonstrated that MP reversed SGLT-1, which was inhibited by a decrease in cotransporter numbers, by restoring the cotransporter numbers without affecting the affinity for glucose (22). However, as this study demonstrates, NAcT, which was inhibited during chronic enteritis by a decrease in the affinity for the amino acid, was reversed by glucocorticoids by restoring the affinity for the amino acid without altering the cotransporter numbers. Thus it is likely that MP acts as an immune modulator to reverse the inhibition of SGLT-1 and NAcT in the chronically inflamed intestine rather than exerting a direct effect on the cotransporter itself.
Our studies to date demonstrate that, during chronic enteritis while both SGLT1 and NAcT are inhibited, their mechanisms of inhibition suggest that different molecular alterations are resulting in our observations. For example, inhibition of SGLT1 is secondary to a reduction in SGLT1 mRNA as well as a reduction in the protein on the BBM. Thus it is likely that SGLT1 is regulated at the transcriptional and posttranslational levels. The posttranslational alterations could be at the level of synthesis of the protein and/or secondary to altered trafficking of protein to the BBM. In contrast, NAcT is inhibited secondary to a reduction in the affinity of the cotransporter for the amino acid. Thus, unlike SGLT-1, NAcT is likely not regulated at the transcriptional level. Furthermore, at the posttranslational level it is also probably not regulated like SGLT-1. Most likely at this level it is regulated secondary to altered glycosylation and/or phosphorylation. Future studies should yield important insight into the regulation of these two Na-dependent cotransport processes during chronic intestinal inflammation.
It is also noteworthy that MP selectively regulates Na-solute cotransport processes in the normal intestine. For example, our previous studies demonstrated that MP did not affect SGLT-1 in the normal rabbit intestine. As previously noted, whether MP affected NAcT in prior studies was unclear. In any event, no mechanistic determinations were performed in these studies. This study demonstrated that, unlike SGLT-1, MP stimulates NAcT in villus cells from the normal rabbit intestine and the mechanism is by increasing BBM cotransporter numbers. The likely mechanisms of this increase in cotransporter numbers are at the level of transcription of mRNA and/or at the posttranslational level. With respect to the latter, the possible mechanisms include enhanced synthesis of the protein and/or altered trafficking to the BBM.
The hypothesis that MP acts as an immune modulator to reverse the inhibition of NAcT in the chronically inflamed intestine is also supported by the fact that MP had a different effect on NAcT in the normal intestine (MP increased cotransporter numbers) than it did in the chronically inflamed intestine. Similarly MP also had different effects on SGLT-1 (26) in the normal (no effect) and chronically inflamed intestine (MP reversed SGLT-1 inhibition by restoring cotransporter numbers). Therefore glucocorticoids most likely act as a broad-spectrum immune modulator to reverse specific alterations in cotransport processes seen during chronic enteritis. In contrast, glucocorticoids have different effects on at least some of these cotransporters in the normal intestine.
MP as an immune modulator may exert its effect at many levels. One possibility is that it normalizes the histological alterations seen in the chronically inflamed intestine (e.g., villus blunting, crypt hypertrophy). However, we have previously demonstrated that this brief duration of treatment with MP is not sufficient to restore the morphological alterations seen during chronic enteritis (26). Another more likely possibility is that formation of an immune-inflammatory mediator, which was responsible for the specific cotransport alteration during chronic enteritis, is inhibited by MP, allowing for the restoration of cotransporter activity. Specifically which immune-inflammatory mediators are responsible for the inhibition of Na-amino acid and Na-glucose cotransport in the chronically inflamed intestine will need to be delineated in future studies using specific pathway inhibitors, agonists, and antagonists.
This hypothesis of glucocorticoids having an effect on circulating immune-inflammatory mediators, which can inhibit electrolyte transport in the intestine, is supported by two previous studies. In one study the findings were consistent with circulating immune-inflammatory mediators altering electrolyte and fluid transport in nonhistologically involved areas of the intestine (4). In another study a single dose of methylprednisolone partially reversed the inhibition in Na and fluid absorption in patients with ulcerative colitis (21).
Methylprednisolone has also been demonstrated to partially reverse the inhibition of glucose stimulated Na flux in acute enteritis without altering the severe histological distortions seen in this condition. In that study, MP had no effect on glucose-stimulated Na flux in the normal piglet jejunum or on Na-K-ATPase in the acutely inflamed intestine. The mechanism of glucocorticoid-mediated reversal of Na-glucose cotransport inhibition was not further deciphered in that model of acute intestinal inflammation (19).
In conclusion, this study demonstrates that in the normal intestine glucocorticoids stimulate Na-amino acid cotransport by increasing cotransporter numbers without altering the affinity for the amino acid. It also demonstrates that the inhibition of Na-amino acid cotransport in the chronically inflamed intestine can be reversed by treatment with glucocorticoids. However, at the cotransporter level the mechanism of glucocorticoid-mediated reversal is secondary to restoration of affinity for the amino acid rather than altered cotransporter numbers. Therefore, it is postulated that glucocorticoids may act directly on the cotransporter in the normal intestine whereas they act as an immune modulator to reverse the inhibition of Na-amino acid cotransport in the chronically inflamed intestine. Thus the effects of MP on Na-amino acid cotransport are profoundly different at the cotransporter level in the normal and chronically inflamed intestine.
GRANTS
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-45062 and DK-58034 to U. Sundaram.
ACKNOWLEDGMENTS
The authors thank Dr. V. Ganapathy for helpful suggestions with molecular studies and M. Hua, graduate student, for technical assistance with some of the uptake studies.
Address for reprint requests and other correspondence: U. Sundaram, Section of Digestive Diseases, Dept. of Medicine, West Virginia Univ. School of Medicine, One Medical Center Drive, 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
This article has been cited by other articles:
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S. Coon, R. Kekuda, P. Saha, J. R. Talukder, and U. Sundaram Constitutive nitric oxide differentially regulates Na-H and Na-glucose cotransport in intestinal epithelial cells Am J Physiol Gastrointest Liver Physiol, June 1, 2008; 294(6): G1369 - G1375. [Abstract] [Full Text] [PDF]
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