Jamilur R. Talukder

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.

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.

Leukotriene D4 Requires PKCñ- Akt Signaling Pathway to Inhibit Na+-Dependent Alanine Cotransporter (ASCT1) in Enterocytes

Background: Varieties of inflammatory cytokines are produced during chronic enteritis including inflammatory bowel disease (IBD), that leads to malabsorption of nutrients and diarrhea. The inflammatory mediators are produced from different tissues of the body. Arachidonic acid metabolite derived via Lipoxygenase pathway, leukotriene D4 (LT) inhibits Na+-dependent alanine (Ala) cotransport (ASCT1, solute carrier, slc1a4) in the apical membrane of enterocytes by decreasing the affinity of cotransporter. However, the intracellular mechanism of LT-mediated inhibition of ASCT1 activity is unknown. Objective:To investigate the intracellular mechanism of leukotriene D4 (LT) mediated inhibition of ASCT1 activity in enterocytes. Methods: Rat intestinal epithelial cells (IEC-6) were grown on transwell plates. [3H]-Ala uptake was measured in 10 days postconfluent cells using a scintillation counter. IEC-6 cells were treated with different inhibitors in 8 days postconfluent to intercept different checkpoints of pathways for LT-mediated inhibition of ASCT1 activity. Intracellular Ca2+ and cAMP levels were measured. Immunoblotting, qRT-PCR, and immunocytochemistry were performed following the standard protocols. Results: LT treatment increased more than 2.5-fold [(cAMP)i] and [(Ca2+ )i]. PKA, PKC-δ and -θ inhibitor did not reverse the LT-mediated inhibition of ASCT1 activity. However, PKC-α inhibitor antagonized LT effect on ASCT1 activity. Further downstream of PKC-α pathway, tyrosine kinase (Akt) inhibitor also reversed LT-mediated inhibition of ASCT1 activity. Immunoblotting, qRT-PCR, immunocytochemistry, and Kinetics studies demonstrated that the mechanism of decreased affinity of ASCT1 by LT was due to decrease in affinity of ASCT1 for Ala transport that was restored by Akt inhibitor. Conclusion: Therefore, we conclude that LT inhibits ASCT1 activity by decreasing the affinity of ASCT1 to Ala through Ca2+-dependent PKCα-Akt pathway in enterocytes.