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.

scholarly journals Biotin uptake by human colonic epithelial NCM460 cells: a carrier-mediated process shared with pantothenic acid

1998 ◽  
Vol 275 (5) ◽  
pp. C1365-C1371 ◽  
Author(s):  
Hamid M. Said ◽  
Alvaro Ortiz ◽  
Eric McCloud ◽  
David Dyer ◽  
Mary Pat Moyer ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Large Intestine ◽  
Pantothenic Acid ◽  
Epithelial Cell Line ◽  
Normal Microflora ◽  
Kinase C ◽  
Colonic Epithelial Cell Line ◽  
Uptake Velocity ◽  
Energy Dependent ◽  
First Time

Previous studies showed that the normal microflora of the large intestine synthesizes biotin and that the colon is capable of absorbing intraluminally introduced free biotin. Nothing, however, is known about the mechanism of biotin absorption in the large intestine and its regulation. To address these issues, we used the human-derived, nontransformed colonic epithelial cell line NCM460. The initial rate of biotin uptake was found to be 1) temperature and energy dependent, 2) Na+ dependent (coupling ratio of 1:1), 3) saturable as a function of concentration [apparent Michaelis constant ( K m) of 19.7 μM], 4) inhibited by structural analogs with a free carboxyl group at the valeric acid moiety, and 5) competitively inhibited by the vitamin pantothenic acid (inhibition constant of 14.4 μM). Pretreatment with the protein kinase C (PKC) activators phorbol 12-myristate 13-acetate (PMA) and 1,2-dioctanoyl- sn-glycerol significantly inhibited biotin uptake. In contrast, pretreatment with the PKC inhibitors staurosporine and chelerythrine led to a slight, but significant, increase in biotin uptake. The effect of PMA was mediated via a marked decrease in maximal uptake velocity and a slight increase in apparent K m. Pretreatment of cells with modulators of the protein kinase A-mediated pathway, on the other hand, showed no significant effect on biotin uptake. These results demonstrate, for the first time, the functional existence of a Na+-dependent, specialized carrier-mediated system for biotin uptake in colonic epithelial cells. This system is shared with pantothenic acid and appears to be under the regulation of an intracellular PKC-mediated pathway.

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.

scholarly journals Regulation of Histone Deacetylase 4 Expression by the SP Family of Transcription Factors

2006 ◽  
Vol 17 (2) ◽  
pp. 585-597 ◽  
Author(s):  
Fang Liu ◽  
Nabendu Pore ◽  
Mijin Kim ◽  
K. Ranh Voong ◽  
Melissa Dowling ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Dna Sequences ◽  
Chromatin Immunoprecipitation ◽  
Histone Deacetylases ◽  
Targeted Mutagenesis ◽  
Cellular Functions ◽  
Specific Expression ◽  
Protein Levels ◽  
Histone Deacetylase 4 ◽  
First Time

Histone deacetylases mediate critical cellular functions but relatively little is known about mechanisms controlling their expression, including expression of HDAC4, a class II HDAC implicated in the modulation of cellular differentiation and viability. Endogenous HDAC4 mRNA, protein levels and promoter activity were all readily repressed by mithramycin, suggesting regulation by GC-rich DNA sequences. We validated consensus binding sites for Sp1/Sp3 transcription factors in the HDAC4 promoter through truncation studies and targeted mutagenesis. Specific and functional binding by Sp1/Sp3 at these sites was confirmed with chromatin immunoprecipitation (ChIP) and electromobility shift assays (EMSA). Cotransfection of either Sp1 or Sp3 with a reporter driven by the HDAC4 promoter led to high activities in SL2 insect cells (which lack endogenous Sp1/Sp3). In human cells, restored expression of Sp1 and Sp3 up-regulated HDAC4 protein levels, whereas levels were decreased by RNA-interference-mediated knockdown of either protein. Finally, variable levels of Sp1 were in concordance with that of HDAC4 in a number of human tissues and cancer cell lines. These studies together characterize for the first time the activity of the HDAC4 promoter, through which Sp1 and Sp3 modulates expression of HDAC4 and which may contribute to tissue or cell-line-specific expression of HDAC4.

Pyridoxine uptake by colonocytes: a specific and regulated carrier-mediated process

2008 ◽  
Vol 294 (5) ◽  
pp. C1192-C1197 ◽  
Author(s):  
Zainab M. Said ◽  
Veedamali S. Subramanian ◽  
Nosratola D. Vaziri ◽  
Hamid M. Said
Keyword(s):  
Adult Mouse ◽  
Initial Rate ◽  
Apical Membrane ◽  
Membrane Vesicles ◽  
Water Soluble ◽  
Vitamin B ◽  
Cellular Functions ◽  
Normal Microflora ◽  
Water Soluble Vitamin ◽  
First Time

The water-soluble vitamin B6 (pyridoxine) is important for normal cellular functions, growth, and development. The vitamin is obtained from two exogenous sources: a dietary source, which is absorbed in the small intestine, and a bacterial source, where the vitamin is synthesized in significant quantities by the normal microflora of the large intestine. Evidence exists to suggest the bioavailability of the latter source of the vitamin, but nothing is known about the mechanism involved and its regulation. In this study, we addressed these issues using young adult mouse colonic epithelial (YAMC) cells and human colonic apical membrane vesicles (AMV) as models and using [3H]pyridoxine as the uptake substrate. The results showed the initial rate of [3H]pyridoxine uptake by YAMC cells to be 1) energy- and temperature- (but not Na-) dependent and to occur without metabolic alteration in the transported substrate; 2) saturable as a function of concentration with an apparent Km and Vmax of 2.1 ± 0.5 μM and 53.4 ± 4.3 pmol·mg protein−1·3 min−1, respectively; 3) cis-inhibited by unlabeled pyridoxine and its structural analogs, but not by the unrelated compounds theophylline, penicillamine, and isoniazid; 4) trans-stimulated by unlabeled pyridoxine; 5) amiloride sensitive; and 6) regulated by extracellular and intracellular factors. Uptake of pyridoxine by native human colonic AMV was also found to involve a carrier-mediated process. These studies demonstrate, for the first time, the functional existence of a specific and regulatable carrier-mediated process for pyridoxine uptake by mammalian colonocytes.

scholarly journals K2P2.1 (TREK-1) potassium channel activation protects against hyperoxia-induced lung injury

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Tatiana Zyrianova ◽  
Benjamin Lopez ◽  
Riccardo Olcese ◽  
John Belperio ◽  
Christopher M. Waters ◽  
...  
Keyword(s):  
Lung Injury ◽  
Lung Compliance ◽  
Alveolar Epithelial Cells ◽  
Pharmacological Intervention ◽  
Alveolar Epithelial ◽  
Protein Levels ◽  
Cell Counts ◽  
Novel Approach ◽  
Intracellular Ca ◽  
Cell Depolarization

AbstractNo targeted therapies exist to counteract Hyperoxia (HO)-induced Acute Lung Injury (HALI). We previously found that HO downregulates alveolar K2P2.1 (TREK-1) K+ channels, which results in worsening lung injury. This decrease in TREK-1 levels leaves a subset of channels amendable to pharmacological intervention. Therefore, we hypothesized that TREK-1 activation protects against HALI. We treated HO-exposed mice and primary alveolar epithelial cells (AECs) with the novel TREK-1 activators ML335 and BL1249, and quantified physiological, histological, and biochemical lung injury markers. We determined the effects of these drugs on epithelial TREK-1 currents, plasma membrane potential (Em), and intracellular Ca2+ (iCa) concentrations using fluorometric assays, and blocked voltage-gated Ca2+ channels (CaV) as a downstream mechanism of cytokine secretion. Once-daily, intra-tracheal injections of HO-exposed mice with ML335 or BL1249 improved lung compliance, histological lung injury scores, broncho-alveolar lavage protein levels and cell counts, and IL-6 and IP-10 concentrations. TREK-1 activation also decreased IL-6, IP-10, and CCL-2 secretion from primary AECs. Mechanistically, ML335 and BL1249 induced TREK-1 currents in AECs, counteracted HO-induced cell depolarization, and lowered iCa2+ concentrations. In addition, CCL-2 secretion was decreased after L-type CaV inhibition. Therefore, Em stabilization with TREK-1 activators may represent a novel approach to counteract HALI.

scholarly journals A novel role for WAVE1 in controlling actin network growth rate and architecture

2015 ◽  
Vol 26 (3) ◽  
pp. 495-505 ◽  
Author(s):  
Meredith O. Sweeney ◽  
Agnieszka Collins ◽  
Shae B. Padrick ◽  
Bruce L. Goode
Keyword(s):  
Actin Filament ◽  
Specific Activity ◽  
Inhibitory Effect ◽  
Actin Network ◽  
Inhibitory Effects ◽  
Cellular Functions ◽  
Network Growth ◽  
Assembly Kinetics ◽  
Filament Networks ◽  
First Time

Branched actin filament networks in cells are assembled through the combined activities of Arp2/3 complex and different WASP/WAVE proteins. Here we used TIRF and electron microscopy to directly compare for the first time the assembly kinetics and architectures of actin filament networks produced by Arp2/3 complex and dimerized VCA regions of WAVE1, WAVE2, or N-WASP. WAVE1 produced strikingly different networks from WAVE2 or N-WASP, which comprised unexpectedly short filaments. Further analysis showed that the WAVE1-specific activity stemmed from an inhibitory effect on filament elongation both in the presence and absence of Arp2/3 complex, which was observed even at low stoichiometries of WAVE1 to actin monomers, precluding an effect from monomer sequestration. Using a series of VCA chimeras, we mapped the elongation inhibitory effects of WAVE1 to its WH2 (“V”) domain. Further, mutating a single conserved lysine residue potently disrupted WAVE1's inhibitory effects. Taken together, our results show that WAVE1 has unique activities independent of Arp2/3 complex that can govern both the growth rates and architectures of actin filament networks. Such activities may underlie previously observed differences between the cellular functions of WAVE1 and WAVE2.

scholarly journals Specificity of Human Sulfiredoxin for Reductant and Peroxiredoxin Oligomeric State

Antioxidants ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 946
Author(s):  
Tom E. Forshaw ◽  
Julie A. Reisz ◽  
Kimberly J. Nelson ◽  
Rajesh Gumpena ◽  
J. Reed Lawson ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Antioxidant Enzymes ◽  
Hydrogen Sulfide ◽  
Active Site ◽  
Relative Efficacy ◽  
Oligomeric Structure ◽  
Cellular Functions ◽  
Oligomeric State ◽  
Structural Requirements ◽  
First Time

Human peroxiredoxins (Prx) are a family of antioxidant enzymes involved in a myriad of cellular functions and diseases. During the reaction with peroxides (e.g., H2O2), the typical 2-Cys Prxs change oligomeric structure between higher order (do)decamers and disulfide-linked dimers, with the hyperoxidized inactive state (-SO2H) favoring the multimeric structure of the reduced enzyme. Here, we present a study on the structural requirements for the repair of hyperoxidized 2-Cys Prxs by human sulfiredoxin (Srx) and the relative efficacy of physiological reductants hydrogen sulfide (H2S) and glutathione (GSH) in this reaction. The crystal structure of the toroidal Prx1-Srx complex shows an extended active site interface. The loss of this interface within engineered Prx2 and Prx3 dimers yielded variants more resistant to hyperoxidation and repair by Srx. Finally, we reveal for the first time Prx isoform-dependent use of and potential cooperation between GSH and H2S in supporting Srx activity.

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