On the mechanism of folate transport in isolated intestinal epithelial cells

The mechanism of folic acid (FA) uptake was studied in isolated intestinal epithelial cells prepared from 2- to 6-wk-old chicks. The cells accumulated FA, reaching a level of three- to fivefold that at equilibrium. In the presence of the metabolic inhibitors, NaN3 or KCN, FA was taken up only until equilibration while accumulation of FA was inhibited. Addition of these inhibitors at a steady state of FA accumulation caused a release of intracellular FA. The kinetics of FA uptake were found to be saturable (Km = 3.5 x 10(-5) M), indicating a carrier-mediated mechanism. The steady-state level of FA accumulation was higher as the concentration of NA+ in the medium increased from 0 to 120 mM. This stimulation of FA uptake by Na+ was not due to the stimulation of glucose uptake, because in experiments carried out in the presence of phlorizin, a glucose-transport inhibitor, FA accumulation was not diminished. It is suggested that FA is taken up by a Na+-coupled transport system.

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Comparison of intestinal folate carrier clone expressed in IEC-6 cells and in Xenopusoocytes

AJP Cell Physiology ◽

10.1152/ajpcell.1998.274.1.c289 ◽

1998 ◽

Vol 274 (1) ◽

pp. C289-C294 ◽

Cited By ~ 59

Author(s):

Chandira K. Kumar ◽

Toai T. Nguyen ◽

Francis B. Gonzales ◽

Hamid M. Said

Keyword(s):

Folic Acid ◽

Epithelial Cells ◽

Cdna Clone ◽

Xenopus Oocytes ◽

Intestinal Epithelial Cells ◽

Maximal Velocity ◽

Mrna Levels ◽

Acidic Ph ◽

Intestinal Epithelial ◽

Folate Transport

We recently identified a cDNA clone from mouse small intestine, which appears to be involved in folate transport when expressed in Xenopus oocytes. The open reading frame of this clone is identical to that of the reduced folate carrier (RFC) (K. H. Dixon, B. C. Lanpher, J. Chiu, K. Kelley, and K. H. Cowan. J. Biol. Chem. 269: 17–20, 1994). The characteristics of this cDNA clone [previously referred to as intestinal folate carrier 1 (IFC-1)] expressed in Xenopus oocytes, however, were found to be different from the characteristics of folate transport in native small intestinal epithelial cells. To further study these differences, we determined the characteristics of RFC when expressed in an intestinal epithelial cell line, IEC-6, and compared the findings to its characteristics when expressed in Xenopus oocytes. RFC was stably transfected into IEC-6 cells by electroporation; its cRNA was microinjected into Xenopus oocytes. Northern blot analysis of poly(A)+RNA from IEC-6 cells stably transfected with RFC cDNA (IEC-6/RFC) showed a twofold increase in RFC mRNA levels over controls. Similarly, uptake of folic acid and 5-methyltetrahydrofolate (5-MTHF) by IEC-6/RFC was found to be fourfold higher than uptake in control sublines. This increase in folic acid and 5-MTHF uptake was inhibited by treating IEC-6/RFC cells with cholesterol-modified antisense DNA oligonucleotides. The increase in uptake was found to be mainly mediated through an increase in the maximal velocity ( V max) of the uptake process [the apparent Michaelis-Menten constant ( K m) also changed (range was 0.31 to 1.56 μM), but no specific trend was seen]. In both IEC-6/RFC and control sublines, the uptake of both folic acid and 5-MTHF displayed 1) pH dependency, with a higher uptake at acidic pH 5.5 compared with pH 7.5, and 2) inhibition to the same extent by both reduced and oxidized folate derivatives. These characteristics are very similar to those seen in native intestinal epithelial cells. In contrast, RFC expressed in Xenopus oocytes showed 1) higher uptake at neutral and alkaline pH 7.5 compared with acidic pH 5.5 and 2) higher sensitivity to reduced compared with oxidized folate derivatives. Results of these studies demonstrate that the characteristics of RFC vary depending on the cell system in which it is expressed. Furthermore, the results may suggest the involvement of cell- or tissue-specific posttranslational modification(s) and/or the existence of an auxiliary protein that may account for the differences in the characteristics of the intestinal RFC when expressed in Xenopus oocytes compared with when expressed in intestinal epithelial cells.

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Energetics of sugar transport by isolated intestinal epithelial cells: effects of cytochalasin B

AJP Cell Physiology ◽

10.1152/ajpcell.1979.237.1.c56 ◽

1979 ◽

Vol 237 (1) ◽

pp. C56-C63 ◽

Cited By ~ 42

Author(s):

G. A. Kimmich ◽

J. Randles

Keyword(s):

Steady State ◽

Epithelial Cells ◽

Transport System ◽

Cytochalasin B ◽

Intestinal Epithelial Cells ◽

Sugar Accumulation ◽

Facilitated Diffusion ◽

Intestinal Epithelial ◽

Passive System ◽

Concentration Gradients

The capability of isolated intestinal epithelial cells to establish concentration gradients of 3-O-methylglucose (3-OMG) by a Na+-dependent transport system is limited by concomitant function of a Na+-independent, facilitated diffusion transport system. Monosaccharides accumulated by the active system are continuously lost via the passive system, which acts to lower steady-state sugar gradients maintained by the cell. Cytochalasin B is a potent inhibitor of the passive system and allows the cells to establish a sugar gradient that is much higher than normal. When extracellular [3-;OMG] is 1 mM, cytochalasin induces sugar accumulation ratios of 30-;fold (+/- phlorizin) in contrast to control ratios of approximately 10-;fold. When [3-;OMG] is 0.1 mM, cytochalasin (0.1 mM) induces 40-;fold accumulation ratios. When changes in extracellular sugar concentration are considered, steady-state concentration gradients observed are 70-;fold. For a Na:sugar coupling stoichiometry of 1:1, gradients of this magnitude represent the approximate theoretical maximum for a transport system driven exclusively by the transmembrane electrochemical potential for Na+.

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Monochloramine induces reorganization of nuclear speckles and phosphorylation of SRp30 in human colonic epithelial cells: role of protein kinase C

AJP Cell Physiology ◽

10.1152/ajpcell.00090.2003 ◽

2003 ◽

Vol 285 (5) ◽

pp. C1294-C1303 ◽

Cited By ~ 8

Author(s):

Ya-Qin Zhu ◽

Yu Lu ◽

Xiao-Di Tan

Keyword(s):

Gastrointestinal Tract ◽

Epithelial Cells ◽

Intestinal Epithelial Cells ◽

Morphological Changes ◽

Sr Proteins ◽

Mrna Splicing ◽

Intestinal Epithelial ◽

Nuclear Speckles ◽

Colonic Epithelial Cells ◽

Stimulation Of

Intestinal epithelial cells are constantly stimulated by reactive oxidant metabolites (ROMs) in inflamed mucosa. Monochloramine (NH2Cl), a cell-permeant ROM, is particularly relevant to the pathogenesis of inflammation in the gastrointestinal tract. Nuclear speckles, a unique nuclear subcompartment, accumulate a family of proteins, namely, serine- and arginine-rich (SR) proteins. They play important roles in regulation of pre-mRNA splicing. Currently, little is known about the link between inflammatory stimulation and the pre-mRNA splicing process, although gene expression is changed in inflamed tissues. The present study was designed to investigate whether stimulation of human colonic epithelial cells (HT-29 and Caco-2 cell lines) with NH2Cl affects nuclear speckles and their components. By indirect immunofluorescence, nuclear speckles have been shown to undergo rapid aggregation after NH2Cl stimulation. By utilizing Western blotting, SRp30 (a subset of SR proteins) in intestinal epithelial cells was found to be phosphorylated after NH2Cl treatment, whereas other SR proteins were not responsive to NH2Cl stimulation. The cytotoxic effect of NH2Cl was excluded by both negative lactate dehydrogenase assay and propidium iodide staining. Therefore, NH2Cl-induced morphological changes on nuclear speckles and phosphorylated SRp30 do not result from intestinal epithelial injury. Furthermore, the effect of NH2Cl on nuclear speckles and SRp30 was blocked by bisindolylmaleimide I, a selective PKC inhibitor. Together, the available data suggest that stimulation of intestinal epithelial cells with NH2Cl results in a consequent change on pre-mRNA splicing machinery via a distinctive signal pathway involving activation of PKC. This effect may contribute to oxidant-induced pathophysiological changes in the gastrointestinal tract.

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