Ascorbic acid transport in mouse and rat astrocytes is reversibly inhibited by furosemide, SITS, and DIDS

Abstract

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.