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Mechanisms and regulation of vitamin C uptake: studies of the hSVCT systems in human liver epithelial cells.

ABSTRACT: Humans use two sodium-ascorbate cotransporters (hSVCT1 and hSVCT2) for transporting the dietary essential micronutrient ascorbic acid, the reduced and active form of vitamin C. Although the human liver plays a pivotal role in regulating and maintaining vitamin C homeostasis, vitamin C transport physiology and regulation of the hSVCT systems in this organ have not been well defined. Thus, this research used a human hepatic cell line (HepG2), confirming certain results with primary human hepatocytes and determined the initial rate of ascorbic acid uptake to be Na(+) gradient, pH dependent, and saturable as a function of concentration over low and high micromolar ranges. Additionally, hSVCT2 protein and mRNA are expressed at higher levels in HepG2 cells and native human liver, and the cloned hSVCT2 promoter has more activity in HepG2 cells. Results using short interfering RNA suggest that in HepG2 cells, decreasing hSVCT2 message levels reduces the overall ascorbic acid uptake process more than decreasing hSVCT1 message levels. Activation of PKC intracellular regulatory pathways caused a downregulation in ascorbic acid uptake not mediated by a single predicted PKC-specific amino acid phosphorylation site in hSVCT1 or hSVCT2. However, PKC activation causes internalization of hSVCT1 but not hSVCT2. Examination of other intracellular regulatory pathways on ascorbic acid uptake determined that regulation also potentially occurs by PKA, PTK, and Ca(2+)/calmodulin, but not by nitric oxide-dependent pathways. These studies are the first to determine the overall ascorbic acid uptake process and relative expression, regulation, and contribution of the hSVCT systems in human liver epithelial cells.

SUBMITTER: Reidling JC

PROVIDER: S-EPMC2604802 | biostudies-literature | 2008 Dec

REPOSITORIES: biostudies-literature

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Mechanisms and regulation of vitamin C uptake: studies of the hSVCT systems in human liver epithelial cells.

Reidling Jack C JC Subramanian Veedamali S VS Dahhan Tamara T Sadat Mohammed M Said Hamid M HM

American journal of physiology. Gastrointestinal and liver physiology 20081009 6

Humans use two sodium-ascorbate cotransporters (hSVCT1 and hSVCT2) for transporting the dietary essential micronutrient ascorbic acid, the reduced and active form of vitamin C. Although the human liver plays a pivotal role in regulating and maintaining vitamin C homeostasis, vitamin C transport physiology and regulation of the hSVCT systems in this organ have not been well defined. Thus, this research used a human hepatic cell line (HepG2), confirming certain results with primary human hepatocyt ...[more]

PMID: 18845575

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Project description:BackgroundThe use of gold nanoparticles (AuNPs) for diagnostic applications and for drug and gene-delivery is currently under intensive investigation. For such applications, biocompatibility and the absence of cytotoxicity of AuNPs is essential. Although generally considered as highly biocompatible, previous in vitro studies have shown that cytotoxicity of AuNPs in certain human epithelial cells was observed. In particular, the degree of purification of AuNPs (presence of sodium citrate residues on the particles) was shown to affect the proliferation and induce cytotoxicity in these cells. To expand these studies, we have examined if the effects are related to nanoparticle size (10, 11?nm, 25?nm), to the presence of sodium citrate on the particles' surface or they are due to a varying degree of internalization of the AuNPs. Since two cell types are present in the major barriers to the outside in the human body, we have also included endothelial cells from the vasculature and blood brain barrier.ResultsTransmission electron microscopy demonstrates that the internalized gold nanoparticles are located within vesicles. Increased cytotoxicity was observed after exposure to AuNPs and was found to be concentration-dependent. In addition, cell viability and the proliferation of both endothelial cells decreased after exposure to gold nanoparticles, especially at high concentrations. Moreover, in contrast to the size of the particles (10?nm, 11?nm, 25?nm), the presence of sodium citrate on the nanoparticle surface appeared to enhance these effects. The effects on microvascular endothelial cells from blood vessels were slightly enhanced compared to the effects on brain-derived endothelial cells. A quantification of AuNPs within cells by ICP-AES showed that epithelial cells internalized a higher quantity of AuNPs compared to endothelial cells and that the quantity of uptake is not correlated with the amount of sodium citrate on the nanoparticles' surface.ConclusionsIn conclusion the higher amount of citrate on the particle surface resulted in a higher impairment of cell viability, but did not enhance or reduce the uptake behavior in endothelial or epithelial cells. In addition, epithelial and endothelial cells exhibited different uptake behaviors for citrate-stabilized gold nanoparticles, which might be related to different interactions occurring at the nanoparticle-cell-surface interface. The different uptake in epithelial cells might explain the higher reduction of proliferation of these cells after exposure to AuNPs treatment although more detailed investigations are necessary to determine subcellular events. Nevertheless an extrinsic effect of sodium-citrate stabilized particles could not be excluded. Thus, the amount of sodium citrate should be reduced to a level on which the stability of the particles and the safety for biomedical applications are guaranteed.

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Mechanisms and regulation of vitamin C uptake: studies of the hSVCT systems in human liver epithelial cells.

Publications

Mechanisms and regulation of vitamin C uptake: studies of the hSVCT systems in human liver epithelial cells.

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