Riboflavin uptake by human-derived colonic epithelial NCM460 cells

2000 ◽  
Vol 278 (2) ◽  
pp. C270-C276 ◽  
Author(s):  
Hamid M. Said ◽  
Alvaro Ortiz ◽  
Mary Pat Moyer ◽  
Norimoto Yanagawa
Keyword(s):  
Epithelial Cells ◽  
Large Intestine ◽  
Water Soluble ◽  
Cellular Regulation ◽  
Colonic Epithelial Cells ◽  
Normal Microflora ◽  
Intracellular Ca ◽  
Energy Dependent ◽  
Colonic Cells

Normal microflora of the large intestine synthesize a number of water-soluble vitamins including riboflavin (RF). Recent studies have shown that colonic epithelial cells posses an efficient carrier-mediated mechanism for absorbing some of these micronutrients. The aim of the present study was to determine whether colonic cells also posses a carrier-mediated mechanism for RF uptake and, if so, to characterize this mechanism and study its cellular regulation. Confluent monolayers of the human-derived nontransformed colonic epithelial cells NCM460 and [3H]RF were used in the study. Uptake of RF was found to be 1) appreciable and temperature and energy dependent; 2) Na+ independent; 3) saturable as a function of concentration with an apparent K mof 0.14 μM and V max of 3.29 pmol ⋅ mg protein−1 ⋅ 3 min−1; 4) inhibited by the structural analogs lumiflavin and lumichrome ( K i of 1.8 and 14.1 μM, respectively) but not by the unrelated biotin; 5) inhibited in a competitive manner by the membrane transport inhibitor amiloride ( K i = 0.86 mM) but not by furosemide, DIDS, or probenecid; 6) adaptively regulated by extracellular RF levels with a significant and specific upregulation and downregulation in RF uptake in RF-deficient and oversupplemented conditions, respectively; and 7) modulated by an intracellular Ca2+/calmodulin-mediated pathway. These studies demonstrate for the first time the existence of a specialized carrier-mediated mechanism for RF uptake in an in vitro cellular model system of human colonocytes. This mechanism appears to be regulated by extracellular substrate level and by an intracellular Ca2+/calmodulin-mediated pathway. It is suggested that the identified transport system may be involved in the absorption of bacterially synthesized RF in the large intestine and that this source of RF may contribute toward RF homeostasis, especially that of colonocytes.

scholarly journals A high-affinity and specific carrier-mediated mechanism for uptake of thiamine pyrophosphate by human colonic epithelial cells

2012 ◽  
Vol 303 (3) ◽  
pp. G389-G395 ◽  
Author(s):  
Svetlana M. Nabokina ◽  
Hamid M. Said
Keyword(s):  
Large Intestine ◽  
Apical Membrane ◽  
Vitamin B1 ◽  
Membrane Vesicles ◽  
Thiamine Pyrophosphate ◽  
Human Organ ◽  
Normal Microflora ◽  
Intracellular Ca ◽  
Energy Dependent ◽  
Apical Membrane Vesicles

All mammals require exogenous sources of thiamine (vitamin B1), as they lack the ability to synthesize the vitamin. These sources are dietary and bacterial (the latter is in reference to the vitamin, which is synthesized by the normal microflora of the large intestine). Bacterially generated thiamine exists in the free, as well as the pyrophosphorylated [thiamine pyrophosphate (TPP)], form. With no (or very little) phosphatase activity in the colon, we hypothesized that the bacterially generated TPP can also be taken up by colonocytes. To test this hypothesis, we examined [3H]TPP uptake in the human-derived, nontransformed colonic epithelial NCM460 cells and purified apical membrane vesicles isolated from the colon of human organ donors. Uptake of TPP by NCM460 cells occurred without metabolic alterations in the transported substrate and 1) was pH- and Na+-independent, but energy-dependent, 2) was saturable as a function of concentration (apparent Km = 0.157 ± 0.028 μM), 3) was highly specific for TPP and not affected by free thiamine (or its analogs) or by thiamine monophosphate and unrelated folate derivatives, 4) was adaptively regulated by extracellular substrate (TPP) level via what appears to be a transcriptionally mediated mechanism(s), and 5) appeared to be influenced by an intracellular Ca2+/calmodulin-mediated regulatory pathway. These findings suggest the involvement of a carrier-mediated mechanism for TPP uptake by colonic NCM460 cells, which was further confirmed by results from studies of native human colonic apical membrane vesicles. The results also suggest that the bacterially synthesized TPP in the large intestine is bioavailable and may contribute to overall body homeostasis of vitamin B1 and, especially, to the cellular nutrition of the local colonocytes.

Mechanism of thiamine uptake by human colonocytes: studies with cultured colonic epithelial cell line NCM460

2001 ◽  
Vol 281 (1) ◽  
pp. G144-G150 ◽  
Author(s):  
Hamid M. Said ◽  
Alvaro Ortiz ◽  
Veedamali S. Subramanian ◽  
Ellis J. Neufeld ◽  
Mary Pat Moyer ◽  
...  
Keyword(s):  
Large Intestine ◽  
Epithelial Cell Line ◽  
Cellular Functions ◽  
Protein Levels ◽  
Normal Microflora ◽  
Colonic Epithelial Cell Line ◽  
Intracellular Ca ◽  
Energy Dependent ◽  
First Time ◽  
Slc19a2 Gene

Thiamine (vitamin B1) is essential for normal cellular functions and growth. Mammals cannot synthesize thiamine and thus must obtain the vitamin via intestinal absorption. The intestine is exposed to a dietary thiamine source and a bacterial source in which the vitamin is synthesized by the normal microflora of the large intestine. Very little is known about thiamine uptake in the large intestine. The aim of this study was, therefore, to address this issue. Our results with human-derived colonic epithelial NCM460 cells as a model system showed thiamine uptake to be 1) temperature- and energy dependent, 2) Na+ independent, 3) increased with increasing buffer pH from 5 to 8 and after cell acidification but inhibited by amiloride, 4) saturable as a function of concentration, 5) inhibited by thiamine structural analogs but not by unrelated organic cations, and 6) inhibited by modulators of a Ca2+/calmodulin-mediated pathway. NCM460 cells and native human colonic mucosa expressed the recently cloned human thiamine transporter THTR-1 (product of the SLC19A2 gene) at both mRNA and protein levels. These results demonstrate for the first time that human NCM460 colonocytes possess a specific carrier-mediated system for thiamine uptake that appears to be under the regulation of an intracellular Ca2+/calmodulin-mediated pathway. It is suggested that bacterially synthesized thiamine in the large intestine may contribute to thiamine nutrition of the host, especially toward cellular nutrition of the local colonocytes.

Effects of 1,25(OH)2D3 on enterocyte basolateral membrane Ca transport in rats

1989 ◽  
Vol 256 (3) ◽  
pp. G613-G617 ◽  
Author(s):  
M. J. Favus ◽  
V. Tembe ◽  
K. A. Ambrosic ◽  
H. N. Nellans
Keyword(s):  
Vitamin D ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Cellular Transport ◽  
Dihydroxyvitamin D3 ◽  
Ca Transport ◽  
Intracellular Ca ◽  
Net Flux ◽  
Energy Dependent

One, twenty-five dihydroxyvitamin D3 [1,25(OH)2D3], commonly known as calcitriol, stimulates intestinal Ca absorption through increased activity of a cellular transport process. To determine whether transcellular Ca transport involves energy-dependent Ca efflux across enterocyte plasma membrane in vitamin D-sufficient rats, in vitro bidirectional Ca fluxes were measured under short-circuited conditions across proximal duodenum from rats fed diets adequate in vitamin D and containing a normal Ca diet (NCD), a low Ca diet (LCD), or fed NCD and injected with 50 ng of 1,25(OH)2D3 daily for 4 days before study. LCD or 1,25(OH)2D3 increased Ca net flux [Jnet, mucosal-to-serosal flux minus the serosal-to-mucosal flux] by increasing Ca mucosal-to-serosal flux (Jm----s) (mean +/- SE, NCD vs. LCD vs. 1,25(OH)2D3, 16 +/- 4 vs. 179 +/- 18 vs. 82 +/- 21 nmol.cm-2. h-1, P less than 0.0001). Initial ATP-dependent Ca uptake rates by duodenal basolateral membrane vesicles (BLMV) was greater in vesicles from rats fed NCD compared with LCD and not different from NCD injected with 1,25(OH)2D3. These studies suggest that in vitamin D-replete animals, 1,25(OH)2D3 increases epithelial Ca Jm----s by mechanisms that do not involve ATP-dependent BLM Ca efflux. ATP-dependent Ca exit from the cell under these conditions may play a role in intracellular Ca homeostasis rather than Ca absorption.

p38 mitogen-activated protein kinase inhibits calcium-dependent chloride secretion in T84 colonic epithelial cells

2003 ◽  
Vol 284 (2) ◽  
pp. C339-C348 ◽  
Author(s):  
Stephen J. Keely ◽  
Kim E. Barrett
Keyword(s):  
Protein Kinase ◽  
Epithelial Cells ◽  
Signaling Pathway ◽  
P38 Mapk ◽  
Mitogen Activated Protein Kinase ◽  
Muscarinic Agonist ◽  
Colonic Epithelial Cells ◽  
Mitogen Activated Protein ◽  
Intracellular Ca ◽  
Sb 203580

We have previously shown that Ca2+-dependent Cl−secretion across intestinal epithelial cells is limited by a signaling pathway involving transactivation of the epidermal growth factor receptor (EGFR) and activation of ERK mitogen-activated protein kinase (MAPK). Here, we have investigated a possible role for p38 MAPK in regulation of Ca2+-dependent Cl− secretion. Western blot analysis of T84 colonic epithelial cells revealed that the muscarinic agonist carbachol (CCh; 100 μM) stimulated phosphorylation and activation of p38 MAPK. The p38 inhibitor SB-203580 (10 μM) potentiated and prolonged short-circuit current ( I sc) responses to CCh across voltage-clamped T84 cells to 157.4 ± 6.9% of those in control cells ( n = 21; P < 0.001). CCh-induced p38 phosphorylation was attenuated by the EGFR inhibitor tyrphostin AG-1478 (0.1 nM–10 μM) and by the Src family kinase inhibitor PP2 (20 nM–2 μM). The effects of CCh on p38 phosphorylation were mimicked by thapsigargin (TG; 2 μM), which specifically elevates intracellular Ca2+, and were abolished by the Ca2+ chelator BAPTA-AM (20 μM), implying a role for intracellular Ca2+ in mediating p38 activation. SB-203580 (10 μM) potentiated I sc responses to TG to 172.4 ± 18.1% of those in control cells ( n= 18; P < 0.001). When cells were pretreated with SB-203580 and PD-98059 to simultaneously inhibit p38 and ERK MAPKs, respectively, I sc responses to TG and CCh were significantly greater than those observed with either inhibitor alone. We conclude that Ca2+-dependent agonists stimulate p38 MAPK in T84 cells by a mechanism involving intracellular Ca2+, Src family kinases, and the EGFR. CCh-stimulated p38 activation constitutes a similar, but distinct and complementary, antisecretory signaling pathway to that of ERK MAPK.

Interleukin-8 stimulates the migration of human colonic epithelial cells in vitro

1999 ◽  
Vol 97 (3) ◽  
pp. 385-390 ◽  
Author(s):  
Andrew J. WILSON ◽  
Keith BYRON ◽  
Peter R. GIBSON
Keyword(s):  
Cell Migration ◽  
Epithelial Cells ◽  
In Vitro Model ◽  
Interleukin 8 ◽  
Colonic Epithelium ◽  
Dependent Manner ◽  
Colonic Epithelial Cells ◽  
Tnf Α ◽  
Vitro Model

The migration of colonic epithelial cells (restitution) is an important event in the repair of mucosal injuries. Interleukin-8 (IL-8) is a physiological initiator of the chemotactic migration of leucocytes. This study aimed to determine whether IL-8 had a similar effect on migration in an in vitro model of wounded colonic epithelium. Cell migration over 24 h was assessed in circular wounds made in confluent monolayers of the human colon cancer cell line LIM1215. This migration was stimulated in a concentration-dependent manner by IL-8, with maximal effects of approx. 1.75-fold above basal migration. The motogenic effect of IL-8 was mediated independently of effects on cell proliferation. In contrast, it was partially dependent upon gene transcription and protein synthesis and involved the activation of pertussis-toxin-sensitive G-proteins. The short-chain fatty acids, acetate, propionate, butyrate and valerate, the activator of protein kinase C (phorbol-12-myristate-13-acetate) and tumour necrosis factor-α (TNF-α) all stimulated the secretion of IL-8. However, only the motogenic effect of TNF-α was dependent upon IL-8. In conclusion, IL-8 stimulated cell migration in an in vitro model of colonic epithelium, whereas the motogenic effect of at least one physiologically relevant factor was dependent upon an increase in its endogenous levels. If IL-8 stimulates colonic epithelial restitution in vivo, this would have ramifications for the control of repair processes following wounding of the colonic mucosa.

The VSL#3 probiotic formula induces mucin gene expression and secretion in colonic epithelial cells

2007 ◽  
Vol 292 (1) ◽  
pp. G315-G322 ◽  
Author(s):  
C. Caballero-Franco ◽  
K. Keller ◽  
C. De Simone ◽  
K. Chadee
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Cultured Cells ◽  
In Vivo Studies ◽  
Lactobacillus Species ◽  
Mucin Secretion ◽  
Colonic Epithelial Cells ◽  
Dnase Treatment

Several studies have stressed the importance of the microbiota in the maintenance of the gastrointestinal epithelium. Administration of probiotic bacteria, supplements composed of microbiota constituents, was previously shown to diminish symptoms in patients suffering from inflammatory bowel diseases. This raises the possibility that probiotics may play an active role in enhancing the intestinal barrier at the mucosal surface. In this study, we investigated whether the clinically tested VSL#3 probiotic formula and/or its secreted components can augment the protective mucus layer in vivo and in vitro. For in vivo studies, Wistar rats were orally administered the probiotic mixture VSL#3 on a daily basis for seven days. After treatment, basal luminal mucin content increased by 60%. In addition, we exposed isolated rat colonic loops to the VSL#3 probiotic formula, which significantly stimulated colonic mucin (MUC) secretion and MUC2 gene expression; however, MUC1 and MUC3 gene expression were only slightly elevated. The effect of the VSL#3 mucin secretagogue was also tested in vitro by use of LS 174T colonic epithelial cells. In contrast to the animal studies, cultured cells incubated with VSL#3 bacteria did not exhibit increased mucin secretion. However, the bacterial secreted products contained in the conditioned media stimulated a remarkable mucin secretion effect. Among the three bacterial groups ( Lactobacilli, Bifidobacteria, and Streptococci) contained in VSL#3, the Lactobacillus species were the strongest potentiator of mucin secretion in vitro. A preliminary characterization of the putative mucin secretagogue suggested that it was a heat-resistant soluble compound, which is not sensitive to protease and DNase treatment. These findings contribute to a better understanding of the complex and beneficial interaction between colonic epithelial cells and intestinal bacteria.

scholarly journals Streptococcus Thermophilus UASt-09 Upregulates Goblet Cell Activity in Colonic Epithelial Cells to a Greater Degree than other Probiotic Strains

2020 ◽  
Vol 8 (11) ◽  
pp. 1758
Author(s):  
Madhur D. Shastri ◽  
Wai Chin Chong ◽  
Ravichandra Vemuri ◽  
Christopher J. Martoni ◽  
Santosh Adhikari ◽  
...  
Keyword(s):  
Immune Response ◽  
Epithelial Cells ◽  
Bacterial Species ◽  
Cell Activity ◽  
Streptococcus Thermophilus ◽  
Mucosal Barrier ◽  
Intestinal Barrier Function ◽  
Colonic Epithelial Cells ◽  
Gastrointestinal Health

Probiotics have been widely used in maintaining gastrointestinal health, despite their actual mechanism remaining obscure. There are several hypotheses behind the beneficial effects of probiotics including the regulation of intestinal barrier function and improvement in immune responses in the gastrointestinal system. Multiple probiotics have been introduced in the market as effective dietary supplements in improving gastrointestinal integrity, but there are no or few studies that demonstrate their underlying mechanism. In the current study, we investigated and compared the efficacy of four probiotics (based on different bacterial species) in refining gastrointestinal health by improving mucus biosynthesis and intestinal immune response under in-vitro conditions. By analyzing the gene expression of mucus biosynthesis and intestinal immune response markers, we found that probiotic Streptococcus thermophilus UASt-09 showed promising potential in refining mucosal barrier and gastrointestinal health in human colonic epithelial cells, as compared to other commercial probiotics.

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