CEREBRAL ASTROCYTES TRANSPORT ASCORBIC-ACID AND DEHYDROASCORBIC ACID THROUGH DISTINCT MECHANISMS REGULATED BY CYCLIC-AMP

Cerebral ischemia and trauma lead to rapid increases in cerebral conce ntrations of cyclic AMP and dehydroascorbic acid (DHAA; oxidized vitam in C), depletion of intracellular ascorbic acid (AA; reduced vitamin C ), and formation of reactive astrocytes. We investigated astrocytic tr ansport of AA and DHAA and the effects of cyclic AMP on these transpor t systems. Primary cultures of astrocytes accumulated millimolar conce ntrations of intracellular AA when incubated in medium containing eith er AA or DHAA. AA uptake was Na+-dependent and inhibited by 4,4'-diiso thiocyanostilbene-2,2'-disulfonic acid (DIDS), whereas DHAA uptake was Na+-independent and DIDS-insensitive. DHAA uptake was inhibited by cy tochalasin B, D-glucose, and glucose analogues specific for facilitati ve hexose transporters. Once inside the cells, DHAA was reduced to AA. DHAA reduction greatly decreased astrocytic glutathione concentration . However, experiments with astrocytes that had been previously deplet ed of glutathione showed that DHAA reduction does not require physiolo gical concentrations of glutathione. Astrocyte cultures were treated w ith a permeant analogue of cyclic AMP or forskolin, an activator of ad enylyl cyclase, to induce cellular differentiation and thus provide in vitro models of reactive astrocytes. Cyclic AMP stimulated uptake of AA, DHAA, and 2-deoxyglucose. The effects of cyclic AMP required at le ast 12 h and were inhibited by cycloheximide, consistent with a requir ement for de novo protein synthesis. Uptake and reduction of DHAA by a strocytes may be a recycling pathway that contributes to brain AA home ostasis. These results also indicate a role for cyclic AMP in accelera ting the clearance and detoxification of DHAA in the brain.