Lactoferrin ameliorates prostaglandin E2-mediated inhibition of Na+ -glucose cotransport in enterocytes.

Various immunoinflammatory cytokines are produced during chronic intestinal inflammation, which inhibits Na(+)-glucose cotransport (SGLT1) in villus cells. Lactoferrin (Lf), abundantly present in colostrum, is a multifunctional glycoprotein that is absorbed by receptor-mediated transcytosis in humans and animals and has been shown to exert anti-inflammatory effects. Therefore, this study aimed to examine whether Lf would prevent PGE2 effect on SGLT1 for glucose absorption in enterocytes. Intestinal epithelial cells (IEC-6) were grown on transwell plates, treated with phlorizin, PGE2, AH6809, and Lf, and 3-O-methyl d-glucopyranose (OMG) uptake was measured in 10 days postconfluent. Na(+)-dependent OMG uptake, phlorizin, and immunoblotting studies established the activity and apical membrane localization of SGLT1 in IEC-6 cells. PGE2 inhibited SGLT1 in a concentration- and time-dependent manner with an inhibitory constant (Ki) of 50.0 nmol/L and that was antagonized by prostanoid receptor inhibitor, AH6809. PGE2 did not alter Na(+)/K(+)-ATPase activity. In contrast, quantitative real-time polymerase chain reaction and Western blot analyses revealed that SGLT1-specific transcripts and protein expression level were decreased 3-fold by PGE2. Furthermore, PGE2 treatment increased intracellular cyclic adenosine monophosphate (cAMP) and Ca(2+) concentrations and decreased SGLT1 expression on the apical membrane, and these effects were ameliorated by Lf. Therefore, we conclude that Lf ameliorates the PGE2 inhibition of SGLT1 most likely via the Ca(2+)- and cAMP-signaling pathways.

Inflammatory cytokine TNF-α inhibits Na(+)-glutamine cotransport in intestinal epithelial cells.

Glutamine (Gln), a preferred fuel source for enterocytes, is critical for intestinal epithelial cell integrity and barrier function. Chronic enteritis inhibits apical Na(+)-Gln cotransport. It is not known whether inflammatory cytokines that are secreted during inflammation inhibit Na(+)-Gln cotransport. Thus, this study aimed to examine whether TNF-α would affect apical Na(+)-Gln cotransport in intestinal epithelial cells. In this study, the presence of Na(+)-Gln cotransport was established by measuring Gln uptake in 10 days postconfluent IEC-6 cells grown on transwell plates. Cation, amino acid specificity, and siRNA transfection studies established that Na(+)-Gln cotransport is mediated via B(0)AT1. Immunoblotting and immunofluorescence studies established the apical membrane localization of B(0)AT1 in IEC-6 cells. Tumour necrosis factor α (TNF-α) inhibited Na(+)-Gln cotransport in a concentration- and time-dependent manner with an inhibitory concentration of 1.53 nmol·L(-1). Quantitative real-time PCR and Western blot analyses indicated that TNF-α did not alter B(0)AT1-specific transcripts or protein expression level. Kinetic studies revealed that TNF-α inhibited Na(+)-Gln cotransport by reducing the affinity of the cotransporters for Gln, and this effect was antagonized by genistein. Thus, we conclude that the TNF-α inhibition of Na(+)-Gln cotransport occurs at the post-translational level, and that the IEC-6 cell line is an excellent system to study the role of cytokines in Na(+)-Gln cotransport.

Aldosterone induces active K⁺ secretion by enhancing mucosal expression of Kcnn4c and Kcnma1 channels in rat distal colon.

Although both Kcnn4c and Kcnma1 channels are present on colonic mucosal membranes, only Kcnma1 has been suggested to mediate K(+) secretion in the colon. Therefore, studies were initiated to investigate the relative roles of Kcnn4c and Kcnma1 in mediating aldosterone (Na-free diet)-induced K(+) secretion. Mucosal to serosal (m-s), serosal to mucosal (s-m), and net (86)Rb(+) (K(+) surrogate) fluxes as well as short circuit currents (I(sc); measure of net ion movement) were measured under voltage clamp condition in rat distal colon. Active K(+) absorption, but not K(+) secretion, is present in normal, while aldosterone induces active K(+) secretion (1.04 ± 0.26 vs. -1.21 ± 0.15 µeq·h(-1)·cm(-2); P < 0.001) in rat distal colon. Mucosal VO(4) (a P-type ATPase inhibitor) inhibited the net K(+) absorption in normal, while it significantly enhanced net K(+) secretion in aldosterone animals. The aldosterone-induced K(+) secretion was inhibited by the mucosal addition of 1) either Ba(2+) (a nonspecific K(+) channel blocker) or charybdotoxin (CTX; a common Kcnn4 and Kcnma1 channel blocker) by 89%; 2) tetraethyl ammonium (TEA) or iberiotoxin (IbTX; a Kcnma1 channel blocker) by 64%; and 3) TRAM-34 (a Kcnn4 channel blocker) by 29%. TRAM-34, but not TEA, in the presence of IbTX further significantly inhibited the aldosterone-induced K(+) secretion. Thus the aldosterone-induced Ba(2+)/CTX-sensitive K(+) secretion consists of IbTX/TEA-sensitive (Kcnma1) and IbTX/TEA-insensitive fractions. TRAM-34 inhibition of the IbTX-insensitive fraction is consistent with the aldosterone-induced K(+) secretion being mediated partially via Kcnn4c. Western and quantitative PCR analyses indicated that aldosterone enhanced both Kcnn4c and Kcnma1α protein expression and mRNA abundance. In vitro exposure of isolated normal colonic mucosa to aldosterone also enhanced Kcnn4c and Kcnma1α mRNA levels, and this was prevented by exposure to actinomycin D (an RNA synthesis inhibitor). These observations indicate that aldosterone induces active K(+) secretion by enhancing mucosal Kcnn4c and Kcnma1 expression at the transcriptional level.

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.

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.