Figure 5. Effect of DNDS on swelling-activated D-[3H]aspartate release from cultured astrocytes and swelling-activated [3H]taurine uptake in cortical synaptosomes. (a) Cultured astrocytes preloaded with D-[3H]aspartate were superfused with hypoosmotic medium in the presence or absence of 2 mM DNDS. The data are the mean values of five experiments for each group 6SEM. *** p,0.001 hypo, vs. DNDS. (b) Release of preloaded [3H]taurine from cortical synaptosomes was measured under isoosmotic (BASAL) and hypoosmotic (HYPO) conditions in the presence or absence of 2 mM DNDS. The data are the mean values of integral 10-min [3H]taurine release 6SEM of three experiments performed in quadruplicate. ***p,0.001 vs. isoosmotic control (BASAL), ###p,0.001 vs. hypoos- motic control (HYPO). doi:10.1371/journal.pone.0003543.g005 of numerous volume-insensitive glutamate release pathways, we utilized perfusion of hypoosmotic medium via a microdialysis probe, rather than performing the studies in the ischemic brain. The major finding of our work is that hypoosmotic-medium induced release of excitatory amino acids and taurine exhibit significant differences in their kinetic properties, sensitivity to isoosmotic changes in extracellular [NaCl] and response to perfusion of the reactive oxygen species H2O2. Such differences indicate that these organic osmolytes are released from different cellular pools and/or via different release pathways. Cortical Amino Acid Release Properties of hypoosmotic medium-induced excitatory amino acid release are consistent with the involvement of VRAC Previous findings from the literature suggest that there is substantial similarity between swelling-activated amino acid release in vitro and in situ. In cultured astrocytes and neuronal cells, hypoosmotic medium promotes cell swelling and triggers the release of several uncharged or negatively charged amino acids such as glycine, alanine, taurine, glutamate, and aspartate [23,24,26,47,58,59]. Several in situ studies performed in brain slices have found that the release properties of isotope-labeled and endogenous excitatory amino acids and taurine are similar to those observed in vitro [36,60–62]. Such organic osmolyte release occurs via a non-saturable pathway, which is inhibited by a variety of Cl2 channel blockers, and therefore likely mediated by an anion channel. In vitro electrophysiological studies found that volume- regulated anion channels (VRAC) are permeable to glutamate, aspartate, taurine, and glycine, but not to the majority of other amino acids [21,22,63–65]. However, it is currently debated whether one or more permeability pathways contribute to the release of organic osmolytes [29–31]. Furthermore, some reports additionally proposed that hypoosmotic swelling may promote the release of excitatory amino acids via a Ca2+-independent mode of exocytosis [66,67]. In vivo, our present work and several previous studies found that hypoosmotic medium stimulates the release of VRAC-permeable amino acids (glutamate, aspartate, and taurine), while the levels of VRAC-impermeable amino acids (e.g., asparagine and glutamine) remain unaffected or decreased [38,39,42]. The broad spectrum Cl2 channel blocker DNDS inhibited the release of excitatory amino acids and taurine, at concentrations that block VRAC activity in vitro. We have found that DNDS is well tolerated in vivo, unlike other commonly used and more potent VRAC blockers, such as NPPB and phloretin. Consistent with the involvement of VRAC, increases in the extracellular levels of the excitatory amino acids were seen upon application of hypoosmotic medium (low [NaCl]e) but not in response to isoosmotic changes in [NaCl]e (NaCl replaced with mannitol). Furthermore, swelling-induced excitatory amino acid release was not blocked by inhibitors of two alternative glutamate and aspartate release pathways, i.e. reversal of glutamate transporters and exocytotic release. Although cell swelling is thought to be the primary stimulus responsible for initiating VRAC opening in the pathological brain, little else is known about the activation or modulation of VRACs in the intact tissue. Our recent in vitro work demonstrated that swelling activated excitatory amino acid release via VRAC is potently modulated by reactive nitrogen species and reactive oxygen species [68,69]. In the present microdialysis experiments, the reactive oxygen species H2O2 strongly increased hypoosmotic levels of glutamate and aspartate, but had little effect when administered under isoosmotic conditions. Taken together with the pharmacological data, these findings are in line with the idea that VRAC is the primary source of the excitatory amino acid release in response to hypoosmotic medium-induced (and pathological) cell swelling. Hypoosmotic medium-induced release of taurine differs from the excitatory amino acid release Unexpectedly, we found marked differences in taurine and excitatory amino acid release. Taurine is widely regarded as an important osmoregulatory molecule in the brain and in other tissues because it is one of the most abundant organic osmolytes and effectively permeates a putative VRAC-like pathway PLoS ONE | www.plosone.org 7 October 2008 | Volume 3 | Issue 10 | e3543