Figure 2. Isoosmotic low [NaCl] medium does not induce taurine release from cultured rat astrocytes but modestly enhances taurine release from rat cortical synaptosomes. (a) Effect of hypoosmotic or isoosmotic reductions in [NaCl] on [3H]taurine release from astrocytes. The data represent mean values 6SEM of integral 10-min releases under isoosmotic (Basal), hypoosmotic (Hypo), or isoosmotic low [NaCl] solutions. n = 4 for each group. *p,0.05, ***p,0.001, vs. basal. (b) Integral 5-min releases of [3H]taurine from synaptosomes exposed to isoosmotic (Basal), hypoosmotic (Hypo) or isoosmotic media with lowered [NaCl]. Means 6SEM of 3 experiments. ** p,0.01, vs. basal. doi:10.1371/journal.pone.0003543.g002 To verify the efficacy of DNDS as a VRAC blocker we tested the effect of DNDS on swelling-activated D-[3H]aspartate release in cultured astrocytes, which is entirely mediated by VRACs [47]. Two mM DNDS was sufficient to suppress D-[3H]aspartate release by ,70% (Fig. 5a), was similarly effective against astrocytic [3H]taurine release (,75%, data not shown), and nearly completely suppressed swelling-activated [3H]taurine release from cortical synaptosomes (Fig. 5b). To further explore whether DNDS may inhibit taurine transporters and in this way affects extracellular taurine levels in vivo, we tested the effects of DNDS on taurine uptake in cultured astrocytes. DNDS did not affect astrocytic [3H]taurine uptake up to the concentration of 32 mM, suggesting that this compound does not alter taurine transporter function (data not shown). Cortical Amino Acid Release Figure 3. Dependence of taurine and glutamate uptake on extracellular [Na+] in cultured astrocytes. Taurine and glutamate transport rates were measured in primary astrocyte cultures using [3H]taurine and D-[3H]aspartate. Extracellular concentrations of amino acids were adjusted to 10 mM using unlabeled taurine or L-glutamate. To compare glutamate versus taurine uptake, the values were normalized to uptake levels under basal conditions ([Na+]o = 135 mM). Note that under basal conditions absolute D-[3H]aspartate uptake rate (nmols/mg protein) was ,5-fold higher compared to taurine. Data are the mean values 6SEM of three experiments from each group. doi:10.1371/journal.pone.0003543.g003 Effects of the Ca2+ channel blocker Cd2+ on hypoosmotic medium-stimulated amino acid and taurine release in vivo To additionally explore the mechanisms responsible for the excitatory amino acid release in vivo, we tested for the contribution of alternative release mechanisms. One such mechanism is synaptic Ca2+-dependent release from a vesicular pool. Several reports suggest that hypoosmotic medium induces membrane depolarization and promotes increases in [Ca2+]i in synaptosomes and brain slices, which may trigger exocytotic neurotransmitter release [48–50]. To exclude the possible involvement of exocytosis in mediating hypoosmotic-induced amino acid release we used a broad spectrum blocker of voltage-sensitive Ca2+ channels, cadmium (Cd2+). Ca2+-free artificial cerebral spinal fluid contain- ing 300 mM Cd2+, given 20 minutes prior to and during Ca2+-free hypoosmotic medium perfusion, did not alter glutamate, aspartate or taurine release (Fig. 6a–c). Previous studies have found that 300 mM Cd2+, administered through microdialysis probes, is effective in blocking electrically stimulated serotonin release and membrane depolarization-induced norepinephrine release [51,52], and 30 mM Cd2+ is sufficient to reduce tonic glutamate release in the amygdala [53]. Thus, our data showing that amino acid release in vivo is insensitive to 300 mM Cd2+ suggests that hypoosmotic-stimulated excitatory amino acid release is not due to the stimulation of exocytosis. Effects of the glutamate transporter blocker DL-TBOA on hypoosmotic medium-stimulated excitatory amino acid release in vivo Low [Na+]e may potentially elevate dialysate glutamate and aspartate levels by inducing the reversal of glutamate transporters, PLoS ONE | www.plosone.org 5 October 2008 | Volume 3 | Issue 10 | e3543