Figure 9. Effect of H2O2 on swelling-activated taurine release from cultured astrocytes and cortical synaptosomes. (a) The effect of H2O2 on swelling-activated [14C]taurine release from cultured astrocytes. Astrocytes were preloaded overnight with [14C]taurine and D-[3H]aspartate. 300 mM H2O2 was added to the media 10 min before and during exposure to hypoosmotic medium. For clarity, only [14C]taurine release is shown. The data are the mean values 6SEM of three experiments. **p,0.01 vs. hypotonic control. (b) The effect of H2O2 on swelling activated [3H]taurine release from rat cortical synaptosomes. Integral [3H]taurine release was measured for 5 minutes as described in Material and methods. 300 mM H2O2 was present in media 10 min before and during measurements of taurine release. Data are the mean values 6SEM of three independent experiments performed in quadruplicate. ***p,0.001 vs. basal release. doi:10.1371/journal.pone.0003543.g009 [23,22,28,43,70]. Consistent with its osmoregulatory role, taurine release in cultured astrocytes, hippocampal slices, and in vivo microdialysis experiments has been found to be potently upregulated under hypoosmotic conditions (reductions in [Na- Cl]e), but is insensitive to isoosmotic changes in [NaCl]e [23,41,66]. Therefore, our observation, that cortical taurine levels are strongly elevated by either hypoosmotic or isoosmotic low [NaCl] media, was rather surprising. Nevertheless, such a finding is not unique. A similar sensitivity of taurine to changes in [NaCl]e independent of changes in osmolarity has been found in two microdialysis studies measuring taurine levels in the rat hippo- campus and in slices prepared from the mouse brain stem [71,72]. Using an alternative approach, in which extracellular amino acids were sampled via a cortical cup, Phillis et al. also observed that replacement of extracellular NaCl with choline-Cl or MMDG-Cl strongly elevated superfusate levels of taurine but not those of glutamate or aspartate [73]. Cortical Amino Acid Release Besides the differences in sensitivity to isoosmotic [NaCl]e decreases, taurine release was (i) consistently delayed by ,5 min- utes, compared to the swelling-activate release of the excitatory amino acids, (ii) showed much slower inactivation, and (iii) was completely insensitive to the application of H2O2. Since the amino acid measurements were performed in the same samples, these data unequivocally point to different release mechanisms or different cellular sources. The idea of diverse transport pathways for taurine and other osmolytes has been suggested in the past, based on dissimilar properties of taurine and excitatory amino acid release in cultured astrocytes, hippocampal brain slices, and in a mammary cell line [35,66,74]. However, because the molecular identity of the volume-sensitive organic osmolyte release pathway is unknown, this hypothesis is difficult to address. An alternative hypothesis that would explain the atypical behavior of taurine is that in the cortex taurine and excitatory amino acids are released from different cellular pools. Glutamate is uniformly distributed in the brain, however it is somewhat more concentrated in neurons since its concentration in astrocytes is lowered by the activity of glutamine synthase [75]. In contrast, the cellular localization of taurine is highly heterogeneous. Depending on the brain region, taurine is concentrated within either glial cells or neurons. For instance, in the cerebellum and the putamen taurine is primarily localized to neurons, while in the thalamus, hypothalamus and brain stem it is concentrated in glial cells [76– 78]. In the cortex there have been conflicting reports suggesting that taurine is preferentially localized to either neurons or glial cells [79,80]. In order to model properties of astroglial and neuronal taurine release we performed experiments in primary rat astrocytes and cortical synaptosomes. In cultured astrocytes, taurine release was completely dissimilar to the release in vivo: it was absolutely insensitive to isoosmotic decreases in [NaCl]e, and was strongly potentiated by H2O2 in swollen cells. Since we were unable to mimic the astrocytic profile of taurine release in vivo, it suggests that astrocytes may contribute to only a negligible portion of taurine release in the cortex. On the other hand, similar to our in vivo data, swelling-activated taurine release in synaptosomes mimicked the in vivo release in that it was completely insensitive to H2O2 and sensitive to isoosmotic reductions in [NaCl]e (albeit to a much weaker degree than the in vivo response). Although these synaptosomal data do not perfectly match the in vivo results, they suggest a possibility that taurine release in the cerebral cortex originates from a neuronal pool. Ineffective uptake of extracellular taurine in vivo under hypoosmotic conditions and in response to isoosmotic reductions in [NaCl] may be determined by a lower density of taurine transporters and their high dependence on extracellular [Na+] and [Cl2] (see Fig. 3). In contrast, glutamate transporters are expressed at a very high density in the brain and driven by the transmembrane gradients of K + , Na+, and Cl2, and are therefore less sensitive to changes in [NaCl]e [54,75]. An integrated model, providing an explanation for our in vivo and in vitro data, is presented in Fig. 10. Relevance to pathophysiological amino acid and taurine release in ischemia There is ample evidence that VRAC contributes to pathological excitatory amino acid release in a number of neurological conditions associated with cell swelling, including ischemia, hyponatremia, hepatic encephalopathy, and traumatic brain injury (reviewed in [7,9]). In animal ischemia models, VRAC blockers reduce pathological elevations in the extracellular levels of excitatory amino acids [10,11,14,81], and potently protect the animal brain against ischemic damage [12,82,83]. Most recently, PLoS ONE | www.plosone.org 9 October 2008 | Volume 3 | Issue 10 | e3543