Brain Research 881 (2000) 144–151 www.elsevier.com / locate / bres Research report Sodium-ascorbate cotransport controls intracellular ascorbate concentration in primary astrocyte cultures expressing the SVCT2 transporter * Jasminka Korcok, Raphael Yan, Ramin Siushansian, S. Jeffrey Dixon, John X. Wilson Department of Physiology, Faculty of Medicine and Dentistry, The University of Western Ontario, London, Ontario, Canada Accepted 8 August 2000 Abstract 1 Expression of the Na -ascorbate cotransporter, SVCT2, was detected in rat brain and in primary cultures of cerebral astrocytes by Northern blot analysis. SVCT2 expression in cultured astrocytes increased in response to the cyclic AMP analog, dibutyryl cyclic AMP. A 1 mathematical model of ascorbic acid transport was developed to evaluate the hypothesis that Na -ascorbate cotransport across the plasma membrane regulates the steady state intracellular concentration of ascorbic acid in these cells. The outcomes predicted by this model were compared to experimental observations obtained with primary cultures of rat cerebral astrocytes exposed to normal and pathologic conditions. Both cotransport activity and intracellular ascorbic acid concentration increased in astrocytes activated by dibutyryl cyclic 1 AMP. Conversely transport activity and ascorbic acid concentration were decreased by hyposmotic cell swelling, low extracellular Na 1 concentration, and depolarizing levels of extracellular K . In cells incubated for up to 3 h in medium having an ascorbic acid concentration typical of brain extracellular fluid, the changes in intracellular ascorbic acid concentration actually measured were not 1 significantly different from those predicted by modeling changes in Na -ascorbate cotransport activity. Thus, it was not necessary to specify alterations in vitamin C metabolism or efflux pathways in order to predict the steady state intracellular ascorbic acid concentration. These results establish that SVCT2 regulates intracellular ascorbic acid concentration in primary astrocyte cultures. They further indicate 1 that the intracellular-to-extracellular ratio of ascorbic acid concentration at steady state depends on the electrochemical gradients of Na and ascorbate across the plasma membrane.  2000 Elsevier Science B.V. All rights reserved. Theme: Neurotransmitters modulators transporters and receptors Topic: Uptake and transporters Keywords: Ascorbic acid; Transport; Mathematical model; Brain cell 1. Introduction vitamin through plasma membrane transporters. Intracellu- lar AA functions as an enzyme cofactor and may contrib- Vitamin C occurs almost entirely in its reduced form, ute to antioxidant defense in brain cells. The concentration ascorbic acid (AA), in normal brain [4,10,16,18,19]. While and redox state of vitamin C are crucial for these functions. the steady state concentration of AA is 200–400 mM in On the one hand, high concentrations of AA protect brain brain extracellular fluid, it is approximately 10-fold higher cells from ischemic, excitotoxic and oxidative injury in the cellular compartment. Brain cells cannot synthesize [9,15,16,23,35]. On the other hand, high concentrations of vitamin C from glucose de novo. Instead, they obtain the oxidized vitamin C (dehydroascorbic acid, DHAA) are cytotoxic [20] and especially neurotoxic [12]. Vitamin C transport systems have been characterized in Abbreviations: AA, ascorbic acid; DHAA, dehydroascorbic acid; an experimental model of brain cells, namely, primary dBcAMP, dibutyryl cyclic AMP; HPLC, high-performance liquid chroma- cultures of rat cerebral astrocytes. Cultured astrocytes tography achieve intracellular AA concentrations ([AA] ) as high as i *Corresponding author. Tel.: 11-519-661-3475; fax: 11-519-661- 8 mM when incubated with physiologic levels of extracel- 3827. E-mail address: john.wilson@med.uwo.ca (J.X. Wilson). lular AA for 3 h [24]. Longer incubation periods to do not 0006-8993 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0006-8993(00)02829-8