alexa The uptake of ascorbic acid into human umbilical vein endothelial cells and its effect on oxidant insult.


Journal of Nutrition & Food Sciences

Author(s): Ek A, Strm K, Cotgreave IA

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Abstract Intracellular reduced ascorbate (AA) levels in confluent cultures of human umbilical vein endothelial (HUVE) cells, grown under conventional conditions, were shown to be very low, ranging between undetectable, < 0.1 nmol/mg protein, and 0.3 nmol/mg protein. Reduced ascorbate was accumulated into the endothelial cells from M199 culture medium in time- and concentration-dependent manners, and was saturated at medium concentrations related to the normal plasma concentrations of the antioxidant (i.e. between 50 microM and 100 microM). Cells derived from different individuals demonstrated considerable inter-individual variation in these AA uptake parameters. The uptake of AA was sensitive to temperature and the presence of the structural analogue isoascorbate in the medium, indicating the involvement of an active transport mechanism. A role for the glucose transporter is, however, not indicated, as AA uptake was not sensitive to phloretin, an inhibitor of the cellular glucose transporter, nor greatly enhanced by depletion of glucose from the medium. Incubation of HUVE cells with dehydroascorbate (DHAA) caused a dose-dependent, but transient increase in intracellular AA. This indicates that HUVE cells are both competent in the uptake and intracellular reduction of oxidised ascorbate, and may resecrete AA into the medium. Indeed, reduced ascorbate in the medium was shown to be preferentially maintained in the presence of cells. The uptake of AA was not sensitive to the presence of DHAA in the medium, perhaps indicating different transporters for reduced and oxidised forms of ascorbate in these human cells. Pre-loading HUVE cells with AA was shown to protect control cells only weakly from the acute, sub-lethal toxicity of H2O2 generated by xanthine oxidase (1 U/mL or 10 U/mL). Protection was optimal at intracellular levels of 3-4 nmol AA/mg protein, with higher concentrations lacking a protective effect. Additionally, the presence of the iron chelator, desferoxamine, significantly protected GSH-depleted HUVE cells only in response to the peroxide, but did not potentiate the protective action of intracellular AA in either control or GSH-depleted cells. This indicates that ascorbate-driven redox-cycling of the Fe2+/Fe3+ does not hamper the intracellular protective function of ascorbate during hydrogen peroxide-derived oxidative stress. These results are discussed in terms of the central role of endothelial cells in the distribution of AA to the tissues of the body, the use of the HUVE cell system for model studies of the toxicity of oxidants in the human endothelium, and the balance between the antioxidant and pro-oxidant actions of AA.
This article was published in Biochem Pharmacol and referenced in Journal of Nutrition & Food Sciences

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