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Keywords:

  • Dehydroascorbic acid;
  • Ascorbate;
  • Cyclic AMP;
  • Transport;
  • Astrocytes

Abstract

  1. Top of page
  2. Abstract

Abstract: 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 AA or DHAA. AA uptake was Na+-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, DHAA was reduced to AA. DHAA reduction greatly decreased astrocytic glutathione concentration. However, experiments with astrocytes that had been previously depleted of glutathione showed that DHAA 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 AA, 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 DHAA by astrocytes may be a recycling pathway that contributes to brain AA homeostasis. These results also indicate a role for cyclic AMP in accelerating the clearance and detoxification of DHAA in the brain.

Abbreviations used: AA, ascorbic acid; BSO, l-buthionine-SR-sulfoximine; dBcAMP, dibutyryl cyclic AMP; DHAA, dehydroascorbic acid; DIDS, 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid; GLUT, facilitative hexose transporter; GSH, glutathione; MEM, minimum essential medium.