Cortical Amino Acid Release Figure 1. Effect of hypoosmotic or isoosmotic low [NaCl] medium on amino acid levels measured in the rat cortex in vivo. (a–c) Microdialysis probes, implanted in the rat frontoparietal cortex, were perfused with hypoosmotic medium (295 mM NaCl, 265% osmolarity) or isoosmotic low NaCl medium (295 mM NaCl +167 mM mannitol) for one hour. In these experiments, the rat brain was perfused with both the hypoosmotic and isoosmotic medium on opposite sides of the cortex. The data represent average dialysate levels of glutamate (a), aspartate (b), and taurine (c) 6SEM from 5 rats. ** p,0.01, hypoosmotic vs. isoosmotic low [NaCl], repeated measures ANOVA. (d–e) In several experiments dialysate levels of glutamine (d, N = 5), and asparagine (e, n = 3) were additionally measured on the ‘‘hypoosmotic’’ side of the brain. doi:10.1371/journal.pone.0003543.g001 when perfused with the same low [NaCl] isoosmotic medium (Fig 2a). In cortical synaptosomes, we found modest (,3-fold) 3 increases in [ H]taurine release under isoosmotic low [NaCI] conditions (2b). However, such increases were much smaller when compared to the releases induced by the hypoosmotic reduction in [NaCI] (,15 fold, Fig. 2b). This was in contrast to our in vivo data which showed very similar increases in taurine levels with both hypoosmotic and isoosmotic low [NaCl] medium (compare Figs 2b and 1c). Since taurine transporter function is dependent on the transmembrane Na+ gradient, we speculated that the increased levels of taurine seen in vivo upon application of low extracellular [NaCl] hypoosmotic or isoosmotic media may in part be due to inhibition of the taurine transporter. To address this issue, we evaluated how decreases in [Na+]e affect [3H]taurine uptake in cultured astrocytes. Given that glutamate and aspartate levels in vivo are insensitive to the isoosmotic decrease of [Na+]e (see Fig. 1a, b), we additionally compared the effect of low [Na+]e on [3H]taurine uptake to the uptake of D-[3H]aspartate. As seen in Fig. 3, decreases in [Na+]e to 50 mM (equivalent to the low [Na+]e used in the in vivo experiments) significantly inhibited both [3H]taurine and D-[3H]aspartate (L-glutamate) uptake, with taurine uptake inhibited to a greater extent. However, the observed in vitro difference in the transporters’ sensitivities to [Na+]e may not in itself be sufficient to explain the drastic sensitivity of taurine release to isoosmotic modulation of the [NaCl] observed in vivo. Effects of the anion channel blocker DNDS on hypoosmotic medium-stimulated amino acid and taurine release in vivo and in vitro Given that our data show that taurine and excitatory amino acid levels are differentially regulated in vivo by low [NaCl], we further investigated the potential mechanisms responsible for the elevated amino acid levels in response to hypoosmotic medium by using different amino acid transport inhibitors. Our first aim was to determine if the increases in extracellular excitatory amino acid levels is mediated by a VRAC-like pathway, as has been extensively shown in vitro. Therefore, we used the anion channel blocker DNDS, which has an IC50 for VRACs of ,1–2 mM [46]. Although DNDS has a low potency for inhibiting VRACs, it is one of the few anion channel inhibitors that can be used in microdialysis studies because it does not produce toxic effects or changes in the basal amino acid release levels. The more potent VRAC blockers NPPB or phloretin caused strong and progressive increases in microdialysate glutamate and aspartate levels, which likely reflect cytotoxicity (Y. Jin, R.E. Haskew-Layton, P.J. Feustel, H.K. Kimelberg, A.A. Mongin, unpublished observations). Ten mM DNDS, when added one hour prior to and during hypoosmotic medium exposure, significantly inhibited the hypo- osmotic-stimulated release of the excitatory amino acids glutamate and aspartate, but did not alter basal excitatory amino acid levels (Fig 4a, b). DNDS also potently inhibited hypoosmotic taurine release, and in addition, also reduced taurine levels under basal conditions (Fig. 4c). PLoS ONE | www.plosone.org 4 October 2008 | Volume 3 | Issue 10 | e3543