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.

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.

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.

Distribution and regulation of apical Cl/anion exchanges in surface and crypt cells of rat distal colon

1999 ◽  
Vol 276 (1) ◽  
pp. G132-G137 ◽  
Author(s):  
Vazhaikkurichi M. Rajendran ◽  
Henry J. Binder
Keyword(s):  
Spatial Distribution ◽  
Concentration Gradient ◽  
Apical Membrane ◽  
Membrane Vesicles ◽  
Distal Colon ◽  
Ph Homeostasis ◽  
Rat Distal Colon ◽  
Na Depletion ◽  
Crypt Cells ◽  
Apical Membrane Vesicles

Na depletion inhibits electroneutral Na-Cl absorption in intact tissues and Na/H exchange in apical membrane vesicles (AMV) of rat distal colon. Two anion (Cl/HCO3 and Cl/OH) exchanges have been identified in AMV from surface cells of rat distal colon. To determine whether Cl/HCO3 and/or Cl/OH exchange is responsible for vectorial Cl movement, this study examined the spatial distribution and the effect of Na depletion on anion-dependent 36Cl uptake by AMV in rat distal colon. These studies demonstrate that HCO3 concentration gradient-driven36Cl uptake (i.e., Cl/HCO3 exchange) is 1) primarily present in AMV from surface cells and 2) markedly reduced by Na depletion. In contrast, OH concentration gradient-driven36Cl uptake (i.e., Cl/OH exchange) present in both surface and crypt cells is not affected by Na depletion. In Na-depleted animals HCO3 also stimulates36Cl via Cl/OH exchange with low affinity. These results suggest that Cl/HCO3 exchange is responsible for vectorial Cl absorption, whereas Cl/OH exchange is involved in cell volume and/or cell pH homeostasis.

Cl-HCO3 and Cl-OH exchanges mediate Cl uptake in apical membrane vesicles of rat distal colon

1993 ◽  
Vol 264 (5) ◽  
pp. G874-G879 ◽  
Author(s):  
V. M. Rajendran ◽  
H. J. Binder
Keyword(s):  
Apical Membrane ◽  
Kinetic Constant ◽  
Membrane Vesicles ◽  
Distal Colon ◽  
Ph Gradient ◽  
Disulfonic Acid ◽  
Ph Homeostasis ◽  
Rat Distal Colon ◽  
Hyperbolic Curve ◽  
Apical Membrane Vesicles

This study describes Cl-HCO3 and Cl-OH exchanges as the mechanism for Cl uptake by apical membrane vesicles (AMV) of rat distal colon. Although HCO3 gradient-stimulated 36Cl uptake was additionally stimulated by the additional presence of a pH gradient, pH gradient-stimulated 36Cl uptake was not further enhanced by a HCO3 gradient. HCO3 gradient-stimulated and OH gradient-stimulated 36Cl uptake was not inhibited by voltage clamping, with K and its ionophore valinomycin, but was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, an anion exchange inhibitor, with an apparent inhibitory constant of 7.8 and 106.0 microM, respectively. Increasing intravesicular OH concentration in the absence of HCO3 (with fixed extravesicular Cl concentration) yielded a sigmoidal curve for 36Cl uptake. In contrast, increasing intravesicular OH concentration in the presence of equimolar intra- and extravesicular HCO3 (25 mM) yielded a saturable hyperbolic curve. Increasing extravesicular Cl concentration saturated both HCO3 gradient-stimulated and OH gradient-stimulated 36Cl uptake, with a kinetic constant for Cl of approximately 11.9 and 22.6 mM, respectively. We conclude that Cl uptake in AMV of rat distal colon occurs via two separate anion (Cl-HCO3 and Cl-OH) exchange processes. We speculate that one of these two anion exchanges may be responsible for transcellular Cl movement, while the other may be important in the regulation of intracellular pH homeostasis.

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.

Na+ transport in human proximal colonic apical membrane vesicles

1994 ◽  
Vol 106 (1) ◽  
pp. 125-133 ◽  
Author(s):  
Edward J. Cragoe ◽  
Pradeep K. Dudeja ◽  
James M. Harig ◽  
Melissa L. Baldwin ◽  
Krishnamurthy Ramaswamy ◽  
...  
Keyword(s):  
Apical Membrane ◽  
Membrane Vesicles ◽  
Na Transport ◽  
Apical Membrane Vesicles
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