Molecular mechanism of regulation of villus cell Na-K-ATPase in the chronically inflamed mammalian small intestine.

Na-K-ATPase located on the basolateral membrane (BLM) of intestinal epithelial cells provides a favorable intracellular Na+ gradient to promote all Na dependent co-transport processes across the brush border membrane (BBM). Down-regulation of Na-K-ATPase activity has been postulated to alter the absorption via Na-solute co-transporters in human inflammatory bowel disease (IBD). Further, the altered activity of a variety of Na-solute co-transporters in intact villus cells has been reported in animal models of chronic enteritis. But the molecular mechanism of down-regulation of Na-K-ATPase is not known. In the present study, using a rabbit model of chronic intestinal inflammation, which resembles human IBD, Na-K-ATPase in villus cells was shown to decrease. The relative mRNA abundance of α-1 and ß-1 subunits was not altered in villus cells during chronic intestinal inflammation. Similarly, the protein levels of these subunits were also not altered in villus cells during chronic enteritis. However, the BLM concentration of α-1 and ß-1 subunits was diminished in the chronically inflamed intestinal villus cells. An ankyrin-spectrin skeleton is necessary for the proper trafficking of Na-K-ATPase to the BLM of the cell. In the present study, ankyrin expression was markedly diminished in villus cells from the chronically inflamed intestine resulting in depolarization of ankyrin-G protein. The decrease of Na-K-ATPase activity was comparable to that seen in ankyrin knockdown IEC-18 cells. Therefore, altered localization of Na-K-ATPase as a result of transcriptional down-regulation of ankyrin-G mediates the down-regulation of Na-K-ATPase activity during chronic intestinal inflammation.

Regulation of sodium glucose co-transporter SGLT1 through altered glycosylation in the intestinal epithelial cells.

Inhibition of constitutive nitric oxide (cNO) production inhibits SGLT1 activity by a reduction in the affinity for glucose without a change in Vmax in intestinal epithelial cells (IEC-18). Thus, we studied the intracellular pathway responsible for the posttranslational modification/s of SGLT1. NO is known to mediate its effects via cGMP which is diminished tenfold in L-NAME treated cells. Inhibition of cGMP production at the level of guanylyl cyclase or inhibition of protein kinase G also showed reduced SGLT1 activity demonstrating the involvement of PKG pathway in the regulation of SGLT1 activity. Metabolic labeling and immunoprecipitation with anti-SGLT1 specific antibodies did not show any significant changes in phosphorylation of SGLT1 protein. Tunicamycin to inhibit glycosylation reduced SGLT1 activity comparable to that seen with L-NAME treatment. The mechanism of inhibition was secondary to decreased affinity without a change in Vmax. Immunoblots of luminal membranes from tunicamycin treated or L-NAME treated IEC-18 cells showed a decrease in the apparent molecular size of SGLT1 protein to 62 and 67 kD, respectively suggesting an alteration in protein glycosylation. The deglycosylation assay with PNGase-F treatment reduced the apparent molecular size of the specific immunoreactive band of SGLT1 from control and L-NAME treated IEC-18 cells to approximately 62 kD from their original molecular size of 75 kD and 67 kD, respectively. Thus, the posttranslational mechanism responsible for the altered affinity of SGLT1 when cNO is diminished is secondary to altered glycosylation of SGLT1 protein. The intracellular pathway responsible for this alteration is cGMP and its dependent kinase.

Prostaglandins, not the leukotrienes, regulate Cl(-)/HCO(3)(-) exchange (DRA, SLC26A3) in villus cells in the chronically inflamed rabbit ileum.

Previously studies have demonstrated that Cl(-)/HCO(3)(-) exchange was inhibited during chronic intestinal inflammation secondary to decrease in the affinity of the exchanger for Cl(-) rather than the number of transporters. Arachidonic acid metabolites (AAM) are elevated in the mucosa of the chronically inflamed small intestine. However, their role in the alteration of Cl(-)/HCO(3)(-) during chronic enteritis was unknown. Inhibition of AAM formation with arachidonyl trifluoro methylketone (ATMK) in chronically inflamed rabbit intestine reversed the diminished Cl(-)/HCO(3)(-) exchange activity. Kinetics studies showed that the reversal was secondary to restoration of the altered affinity of transporter. Downstream regulation of Cl(-)/HCO(3)(-) inhibition by AAM was determined to be by the cyclooxygenase pathway since only inhibition of cyclooxygenase with piroxicam treatment reversed the inhibited Cl(-)/HCO(3)(-) exchange. Further, DRA was shown to be the primary Cl(-)/HCO(3)(-) exchanger in villus cells. Kinetics and molecular studies indicated that the mechanism of inhibition of Cl(-)/HCO(3)(-) exchange by cyclooxygenase pathway metabolites was secondary to diminished affinity of the transporter for Cl(-) without a change in DRA BBM expression. Thus our data indicated that cyclooxygenase pathway metabolites mediate the inhibition of DRA during chronic intestinal inflammation.

Na-glutamine co-transporters B(0)AT1 in villus and SN2 in crypts are differentially altered in chronically inflamed rabbit intestine.

Glutamine is a major nutrient utilized by the intestinal epithelium and is primarily assimilated via Na-glutamine co-transport (NGcT) on the brush border membrane (BBM) of enterocytes. Recently we reported that B(0)AT1 (SLC6A19) mediates glutamine absorption in villus while SN2 (SLC38A5) does the same in crypt cells. However, how B(0)AT1 and SN2 are affected during intestinal inflammation is unknown. In the present study it was shown that during chronic enteritis NGcT was inhibited in villus cells, however, it was stimulated in crypt cells. Our studies also demonstrated that the mechanism of inhibition of NGcT during chronic enteritis was secondary to a reduction in the number of B(0)AT1 co-transporters in the villus cell BBM without a change in the affinity of the co-transporter. In contrast, stimulation of NGcT in crypt cells was secondary to an increase in the affinity of SN2 for glutamine without an alteration in the number of co-transporters. Thus, glutamine assimilation which occurs via distinct transporters in crypt and villus cells is altered in the chronically inflamed intestine.

Monocarboxylate 4 mediated butyrate transport in a rat intestinal epithelial cell line.

BACKGROUND: Short chain fatty acids (SCFA) are absorbed by carrier mediated uptake in the small intestine by pH-dependent SCFA/HCO3 (-) exchangers on the apical membrane of epithelial cells. Conventional assumption is that MCT1 mediates SCFA/HCO3 (-) exchange in the intestine. Further, due to the presence of multiple such anion exchangers, the identity of the intestinal SCFA/HCO3 (-) has been controversial. AIMS: The aim of this study was to determine the identities of the butyrate transporter in the intestinal epithelial cells (IEC-18). METHODS: IEC-18 cells were treated with specific siRNAs for MCT1 and MCT4, and butyrate and lactate uptake studies were performed. RESULTS: Alpha-cyano-4-hydroxycinnamic acid inhibited lactate uptake but not butyrate uptake in IEC-18 cells, indicating that these two substrates are transported via two different transporter systems. MCT1 siRNA treatment abolished both MCT1 mRNA by more than 95 % and protein expression by 83 % as evidenced by RTQ-PCR and western blotting experiments. However, MCT1 siRNA treatment inhibited butyrate uptake upto 24 %, whereas it inhibited lactate uptake significantly by 70 %. Treatment with MCT4 siRNA inhibited MCT4 mRNA expression by 75 % and protein expression by 85 % in these cells. MCT4 siRNA inhibited butyrate uptake by 40 %. Further, several non-steroidal anti-inflammatory drugs (NSAIDs) are transported by the butyrate transporter. Finally, MCT4 siRNA inhibited salicylate uptake by 27 % indicating direct evidence for the transport of salicylate by MCT4. CONCLUSIONS: These data indicate that MCT1 is the high affinity lactate transporter and MCT4 is the high affinity butyrate transporter in the intestinal epithelial cell line IEC-18.

Reciprocal regulation of the primary sodium absorptive pathways in rat intestinal epithelial cells.

Sodium absorption in the mammalian small intestine occurs predominantly by two primary pathways that include Na/H exchange (NHE3) and Na-glucose cotransport (SGLT1) on the brush border membrane (BBM) of villus cells. However, whether NHE3 and SGLT1 function together to regulate intestinal sodium absorption is unknown. Nontransformed small intestinal epithelial cells (IEC-18) were transfected with either NHE3 or SGLT1 small interfering RNAs (siRNAs) and were grown in confluent monolayers on transwell plates to measure the effects on Na absorption. Uptake studies were performed as well as molecular studies to determine the effects on NHE3 and SGLT1 activity. When IEC-18 monolayers were transfected with silencing NHE3 RNA, the cells demonstrated decreased NHE3 activity as well as decreased NHE3 mRNA and protein. However, in NHE3 siRNA-transected cells, SGLT1 activity, mRNA, and protein in the BBM were significantly increased. Thus, inhibition of NHE3 expression regulates the expression and function of SGLT1 in the BBM of intestinal epithelial cells. In addition, IEC-18 cells transected with silencing SGLT1 RNA demonstrated an inhibition of Na-dependent glucose uptake and a decrease in SGLT1 activity, mRNA, and protein levels. However, in these cells, Na/H exchange activity was significantly increased. Furthermore, NHE3 mRNA and protein levels were also increased. Therefore, the inhibition of SGLT1 expression stimulates the transcription and function of NHE3 and vice versa in the BBM of intestinal epithelial cells. Thus this study demonstrates that the major sodium absorptive pathways together function to regulate sodium absorption in epithelial cells.

Glucocorticoids differentially regulate Na-bile acid cotransport in normal and chronically inflamed rabbit ileal villus cells.

Previous studies have demonstrated that apical Na-bile acid cotransport (ASBT) is inhibited during chronic ileitis by both a decrease in the affinity as well as a decrease in the number of cotransporters. Methylprednisolone (MP), a commonly used treatment for inflammatory bowel disease (IBD, e.g., Crohn's disease), has been shown to reverse the inhibition of several other Na-solute cotransporters during chronic enteritis. However, the effect of MP on ASBT in the chronically inflamed ileum is not known. MP stimulated ASBT in villus cells from the normal rabbit ileum by increasing the cotransporter expression without a change in the affinity of the cotransporter for bile acid. Western blot studies demonstrated an increase in cotransporter expression. MP reversed the inhibition of ASBT in villus cells from the chronically inflamed ileum. Kinetic studies demonstrated that the mechanism of MP-mediated reversal of ASBT inhibition was secondary to a restoration of both affinity as well as cotransporter numbers. Western blot analysis demonstrated restoration of cotransporter numbers after MP treatment of rabbits with chronic ileitis. Thus MP stimulates ASBT in the normal ileum by increasing cotransporter numbers. MP reverses the inhibition of ASBT during chronic ileitis. However, MP restores the diminished affinity as well as cotransporter expression levels during chronic ileitis. Thus MP differentially regulates ASBT in the normal and in the chronically inflamed ileum.

Regulation of sodium-glutamine cotransport in villus and crypt cells by glucocorticoids during chronic enteritis.

BACKGROUND: Assimilation of the preferred nutrient of enterocytes is mediated primarily by sodium (Na)-dependent cotransport (NGct) in the intestine. The predominant NGcT in villus cells, B0AT1, is inhibited secondary to a decrease in cotransporter numbers during chronic intestinal inflammation. In contrast, NGcT mediated by SN2 in crypt cells is stimulated secondary to increased affinity of the cotransporter for glutamine during chronic ileitis. Glucocorticoid is a mainstay of treatment for inflammatory bowel disease. However, its effect on NGcT is not known. METHODS: The inhibition of B0AT1 in villus cells during chronic intestinal inflammation was reversed back to normal by methylprednisolone (MP). This was secondary to the restoration of the cotransporter numbers in the brush border membrane rather than an alteration in the affinity. The stimulation of NGcT in crypt cells during chronic ileitis was also restored back to its normal levels by MP treatment. This reversal was secondary to the restoration of the altered affinity of the cotransporter SN2 for glutamine. RESULTS: Kinetic studies and western blot analysis were consistent with functional studies for both cotransporters. Thus, glucocorticoids restore two uniquely altered Na-glutamine cotransporters, B0AT1 in villus and SN2 in crypt cells during chronic enteritis. CONCLUSIONS: These data indicate that glucocorticoids function as an upstream broad spectrum immune modulator in the chronically inflamed intestine.

Role of Sp1 and HNF1 transcription factors in SGLT1 regulation during chronic intestinal inflammation.

In a rabbit model of chronic intestinal inflammation, we previously demonstrated that the activity of Na-glucose cotransporter (SGLT1), SLC5A1, is inhibited. This inhibition is secondary to a decrease in the number of cotransporters, indicating that the regulation of SGLT1 during chronic inflammation is at the level of transcription. However, the regulation of SGLT1 expression and the transcription factors involved in the regulation are not yet known. In this report, we describe the cloning and characterization of rabbit SGLT1 promoter and the identification of transcription factors affected in villus cells during chronic intestinal inflammation. The promoter sequence for SGLT1 gene was identified by using the publicly available rabbit genomic sequence. Even though rabbit SGLT1 promoter did not have considerable overall homology with other mammalian SGLT1 promoters, two specificity protein 1 (Sp1) and a hepatocyte nuclear factor 1 (HNF1) binding sites were highly conserved among the species. Rabbit SGLT1 cDNA was encoded by 15 exons. Minimal promoter region determination showed that 196 nucleotides upstream of the transcription start site were sufficient for optimal promoter activity. This region encompassed two transcription factor binding sites, Sp1 and HNF1. For maximal SGLT1 promoter activity, these two transcription factor binding sites were essential, and their effect was synergistic, indicating that two separate regulatory pathways might be involved in their regulation. Using mobility shift assays, we further demonstrated that the binding of both Sp1 and HNF1 transcription factors to SGLT1 promoter regions were affected during chronic intestinal inflammation. Thus this report demonstrates that Sp1 and HNF1 transcription factors act in concert to regulate SGLT1 transcription in the chronically inflamed intestine.

Constitutive nitric oxide differentially regulates Na-H and Na-glucose cotransport in intestinal epithelial cells.

Previous in vivo studies suggest that constitutive nitric oxide (cNO) can regulate Na- glucose cotransport (SGLT1) and Na-H exchange (NHE3) in rabbit intestinal villus cells. Whether these two primary Na absorbing pathways are directly regulated by cNO and the mechanisms of this regulation in the enterocyte is not known. Thus nontransformed rat small intestinal epithelial cells (IEC-18) were treated with N(G)-nitro-l-arginine methyl ester (l-NAME), which directly decreased cNO in these cells. l-NAME treatment decreased SGLT1 in IEC-18 cells. Kinetic studies demonstrated that the mechanism of inhibition was secondary to a decrease in the affinity of the cotransporter for glucose without a change in the number of cotransporters. In contrast, l-NAME treatment increased NHE3 in IEC-18 cells. Kinetic studies demonstrated that the mechanism of stimulation was by increasing the number of the exchangers without a change in the affinity for Na. Quantitative RT-PCR (RTQ-PCR) and Western blot analysis of SGLT1 demonstrated no change in mRNA and protein, respectively. RTQ-PCR and Western blot analysis of NHE3 indicated that NHE3 was increased by l-NAME treatment by an increase in mRNA and protein, respectively. These results indicate that decreased cNO levels directly mediate the inhibition of SGLT1 and stimulation of NHE3 in intestinal epithelial cells. Thus cNO directly but uniquely regulates the two primary Na-absorptive pathways in the mammalian small intestine.

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

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

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

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.

Functional characterization, localization, and molecular identification of rabbit intestinal N-amino acid transporter.

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 473-amino-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 virus-mediated 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.