Vh. Guaiquil et al., EFFICIENT TRANSPORT AND ACCUMULATION OF VITAMIN-C IN HL-60 CELLS DEPLETED OF GLUTATHIONE, The Journal of biological chemistry, 272(15), 1997, pp. 9915-9921
Human myeloid leukemia cells (HL-60) transport only the oxidized form
of vitamin C (dehydroascorbic acid) and accumulate the vitamin in the
reduced form, ascorbic acid. We performed a detailed study of the role
of glutathione in the intracellular trapping/accumulation of ascorbic
acid in HL-60 cells. Uptake studies using HL-60 cells depleted of glu
tathione by treatment with L-buthionine-(S,R) sulfoximine and diethyl
maleate, revealed no changes in the cells' ability to transport dehydr
oascorbic acid and accumulate ascorbic acid. Similar transport and acc
umulation rates were obtained using HL-60 cells containing intracellul
ar glutathione concentrations from 6 mM to 1 mu M. HL-60 cells, contai
ning as little as 5 mu M glutathione, were able to accumulate up to 15
0 mM ascorbic acid intracellularly when incubated with dehydroascorbic
acid. Glutathione was capable of reducing dehydroascorbic acid by a d
irect chemical reaction, but only when present in a greater than 10-fo
ld stoichiometric excess over dehydroascorbic acid. The accumulation o
f ascorbic acid by HL-60 cells was strongly temperature-dependent and
was very inefficient at 16 degrees C. On the other hand, the direct ch
emical reduction of dehydroascorbic acid by excess glutathione proceed
ed efficiently at temperatures of 16 degrees C. Our data indicate that
glutathione-dependent reductases in HL-60 cells are not responsible f
or the ability of these cells to accumulate millimolar concentrations
of ascorbic acid. These findings indicate that alternative enzymatic m
echanisms are involved in the cellular reduction of dehydroascorbic ac
id.