Journal
BIOCHEMICAL JOURNAL
Volume 428, Issue -, Pages 191-200Publisher
PORTLAND PRESS LTD
DOI: 10.1042/BJ20100064
Keywords
ascorbate (vitamin C); astrocyte; ferricyanide; intracellular pH (pH(i)); K562 cell; Na+/H+-exchanger (NHE)
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Funding
- Department of Biochemistry and Molecular Biology
- Monash University
- Nursing and Health Sciences (Monash University, Victoria, Australia)
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Ascorbate (vitamin C) is the major electron donor to a tPMET (transplasma membrane electron transport) system that was originally identified in human erythrocytes. This plasma membrane redox system appears to transfer electrons from intracellular ascorbate to extracellular oxidants (e.g. non-transferrin-bound iron). Although this phenomenon has been observed in nucleated cells, its mechanism and regulation are not well understood. In the present study we have examined both facets of this phenomenon in K562 cells and primary astrocyte cultures. Using ferricyanide as the analytical oxidant we demonstrate that tPMET is enhanced by dehydroascorbate uptake via facilitative glucose transporters, and subsequent accumulation of intracellular ascorbate. Additionally, we demonstrate that this stimulation is not due to ascorbate that is released from the cells, but is dependent only on a restricted intracellular pool of the vitamin. Substrate-saturation kinetics suggest an enzyme-catalysed reaction across the plasma membrane by an as-yet-unidentified reductase that relies on extensive recycling of intracellular ascorbate. Inhibition of ascorbate-sti mutated tPMET by the NHE (Na+/H+-exchanger) inhibitors amiloride and 5-(Nethyl-N-isopropyl)amiloride, which is diminished by bicarbonate. suggests that tPMET activity may be regulated by intracellular pH. In support of this hypothesis, tPMET in astrocytes was significantly inhibited by ammonium chloride-pulse-induced intracellular acidification, whereas it was signilicantly stimulated by bicarbonate-induced intracellular alkalinization. These results suggest that ascorbate-dependent tPMET is enzyme-catalysed and is modulated by NHE activity and intracellular pH.
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