Cerebral ischemia and trauma lead to rapid increases in cerebral concentrations of cyclic AMP and dehydroascorbic acid (DHAA; oxidized vitamin C), depletion of intracellular ascorbic acid (AA; reduced vitamin C), and formation of reactive astrocytes. We investigated astrocytic transport of AA and DHAA and the effects of cyclic AMP on these transport systems. Primary cultures of astrocytes accumulated millimolar concentrations of intracellular AA when incubated in medium containing either M or DHAA. AA uptake was Nat-dependent and inhibited by 4,4 '-diisothiocyanostilbene-2,2 '-disulfonic acid (DIDS), whereas DHAA uptake was Na~-independent and DIDS-insensitive. DHAA uptake was inhibited by cytochalasin B, D-glucose, and glucose analogues specific for facilitative hexose transporters. Once inside the cells, DHM was reduced to AA. DHAA reduction greatly decreased astrocytic glutathione concentration. However, experiments with astrocytes that had been previously depleted of glutathione showed that DHM reduction does not require physiological concentrations of glutathione. Astrocyte cultures were treated with a permeant analogue of cyclic AMP or forskolin, an activator of adenylyl cyclase, to induce cellular differentiation and thus provide in vitro models of reactive astrocytes. Cyclic AMP stimulated uptake of M, DHAA, and 2-deoxyglucose. The effects of cyclic AMP required at least 12 h and were inhibited by cycloheximide, consistent with a requirement for de novo protein synthesis. Uptake and reduction of DHM by astrocytes may be a recycling pathway that contributes to brain M homeostasis. These results also indicate a role for cyclic AMP in accelerating the clearance and detoxification of DHAA in the brain.

The uptake of L-ascorbic acid (vitamin C) by astrocytes was studied using primary cultures prepared from the neopallium of newborn Swiss CD-1 mice or Sprague-Dawley rats. Initial uptake rates were significantly greater in mouse than in rat astrocytes. Exposure of cultures to 0.25 mM dibutyryl cyclic AMP for 2 weeks changed cell morphology from polygonal to stellate and stimulated ascorbate uptake, with the greatest stimulation occurring in mouse astrocytes. Uptake was specific for the vitamin since it was not diminished by the presence of other organic anions including acetate, formate, lactate, malonate, oxalate, p-aminohippurate, pyruvate and succinate. Ascorbate uptake was Na(+)-dependent but did not have a specific requirement for external Cl- (Cl-0). Substitution of Cl-0 by Br- or NO3- decreased ascorbate uptake rates by 20-31%; whereas substitution by gluconate or isethionate increased uptake by 20-31%. Ascorbate transport by astroglial cultures from both animal species was rapidly (less than or equal to 1 min) and reversibly inhibited by the anion transport inhibitors furosemide, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). The rapid and reversible effects of the impermeant inhibitors (SITS and DIDS) are consistent with direct inhibition of ascorbate transporters located in the astroglial plasma membrane.

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