Two Distinct Modes of Hypoosmotic Medium-Induced Release of Excitatory Amino Acids and Taurine in the Rat Brain In Vivo

A variety of physiological and pathological factors induce cellular swelling in the brain. Changes in cell volume activate several types of ion channels, which mediate the release of inorganic and organic osmolytes and allow for compensatory cell volume decrease. Volume-regulated anion channels (VRAC) are thought to be responsible for the release of some of organic osmolytes, including the excitatory neurotransmitters glutamate and aspartate. In the present study, we compared the in vivo properties of the swelling-activated release of glutamate, aspartate, and another major brain osmolyte taurine. Cell swelling was induced by perfusion of hypoosmotic (low [NaCl]) medium via a microdialysis probe placed in the rat cortex. The hypoosmotic medium produced several-fold increases in the extracellular levels of glutamate, aspartate and taurine. However, the release of the excitatory amino acids differed from the release of taurine in several respects including: (i) kinetic properties, (ii) sensitivity to isoosmotic changes in [NaCl], and (iii) sensitivity to hydrogen peroxide, which is known to modulate VRAC. Consistent with the involvement of VRAC, hypoosmotic medium-induced release of the excitatory amino acids was inhibited by the anion channel blocker DNDS, but not by the glutamate transporter inhibitor TBOA or Cd2+, which inhibits exocytosis. In order to elucidate the mechanisms contributing to taurine release, we studied its release properties in cultured astrocytes and cortical synaptosomes. Similarities between the results obtained in vivo and in synaptosomes suggest that the swelling-activated release of taurine in vivo may be of neuronal origin. Taken together, our findings indicate that different transport mechanisms and/or distinct cellular sources mediate hypoosmotic medium-induced release of the excitatory amino acids and taurine in vivo

Effect of the glutamate transporter inhibitor dl-TBOA on hypoosmotic medium induced amino acid release in the cortex and glutamate transporter reversal in cultured astrocytes.

<p>(a–b) Microdialysis probes implanted on opposite sides of the cortex were perfused with hypoosmotic medium in the presence or absence of 500 µM dl-TBOA, given 20 minutes prior to and during one hour hypoosmotic medium perfusion. The data represent average dialysate levels of glutamate (a), aspartate (b) ±SEM from 4 rats. ** p<0.01 HYPO vs. HYPO+TBOA. (c) DL-TBOA effectively prevented reversal of glutamate transporter in cultured astrocytes. Cultured astrocytes were superfused for one hour with 1 mM ouabain and additionally for 20 min high [KCl] (100 mM) plus ouabain to induce glutamate transporter reversal. 300 µM dl-TBOA was given 10 minutes prior to and during the high [KCl] perfusion in the presence of ouabain. The data are the average values ±SEM for three experiments in each group. ** p<0.01 KCl vs. KCl+TBOA.</p

Effect of hypoosmotic or isoosmotic low [NaCl] medium on amino acid levels measured in the rat cortex <i>in vivo</i>.

<p>(a–c) Microdialysis probes, implanted in the rat frontoparietal cortex, were perfused with hypoosmotic medium (−95 mM NaCl, −65% osmolarity) or isoosmotic low NaCl medium (−95 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) ±SEM 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.</p

Effect of H₂O₂ on swelling-activated taurine release from cultured astrocytes and cortical synaptosomes.

<p>(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 µM 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 ±SEM 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003543#s2" target="_blank">Material and methods</a>. 300 µM H₂O₂ was present in media 10 min before and during measurements of taurine release. Data are the mean values ±SEM of three independent experiments performed in quadruplicate. ***p<0.001 vs. basal release.</p

Effect of the Ca²⁺ channel blocker Cd²⁺ on hypoosmotic medium-induced amino acid release in the cortex.

<p>(a–c) Microdialysis probes were perfused with hypoosmotic medium (HYPO) in the presence or absence of 300 µM Cd²⁺ given 20 minutes prior to and during one-hour hypoosmotic medium perfusion. Each rat had two microdialysis probes implanted on opposite sides of the cortex (one perfused with HYPO alone and the other with HYPO+Cd²⁺). The data represent the average dialysate levels ±SEM of glutamate (a), aspartate (b), and taurine (c) from 5 rats.</p

Effect of the anion channel blocker DNDS on hypoosmotic medium-induced amino acid release in the rat cortex.

<p>(a–c) Microdialysis probes were perfused on opposite sides of the cortex with hypoosmotic medium (HYPO, −65% osmolarity) in the presence or absence of 10 mM DNDS, given one hour prior to and during one-hour hypoosmotic medium perfusion. The data represent average dialysate levels of glutamate (a), aspartate (b) and taurine (c) +/−SEM from 5 rats. * p<0.05, HYPO vs. HYPO+DNDS (glutamate); ** p<0.01, HYPO vs. HYPO+DNDS (aspartate); *** p<0.001 HYPO vs. HYPO+DNDS (taurine). Significance was determined by repeated measures ANOVA.</p

Hypothetical explanation of the experimental data showing differences in taurine and glutamate release <i>in vivo</i>.

<p>A reduction in medium osmolarity (↓[osm]_e) in the rat cortex causes an increase in the extracellular levels of the excitatory amino acids glutamate and aspartate and the sulfonic acid taurine via a mechanism sensitive to the anion channel blocker DNDS. Despite these similarities, the excitatory amino acid and taurine release demonstrate different kinetics and are likely mediated by different transport pathways (1 and 2) and/or originate from different cellular sources. The taurine pathway (1) but not the excitatory amino acid pathway (2) is activated by isoosmotic lowering of [NaCl]_e. Conversely, the swelling-activated excitatory amino acid release pathway (2) but not the taurine pathway (1) is potentiated by H₂O₂. Alternative transport pathways that were considered in this study include: [Na⁺]_e-dependent taurine transporters (3), [Na⁺]_e/[K⁺]_i-dependent glutamate transporters in neurons (4), and in astrocytes (5), which are sensitive to TBOA; and vesicular glutamate release (6), which is sensitive to the voltage-gated Ca²⁺ channel blocker Cd²⁺. Based on the similarities of excitatory amino acid release <i>in vivo</i> and in cultured astrocytes, we speculate that glutamate release <i>in vivo</i> largely originates from glial cells. Similarities between taurine release <i>in vivo</i> and in synaptosomes suggest that taurine release may be of a neuronal origin.</p

Effect of H₂O₂ on hypoosmotic medium induced amino acid release in the cortex.

<p>(a–c) Two microdialysis probes implanted on opposite sides of the cortex were perfused with hypoosmotic medium in the presence or absence of 1 mM H₂O₂ given 20 minutes prior to and during one-hour hypoosmotic medium perfusion. The data represent the average dialysate levels ±SEM of glutamate (a), aspartate (b) and taurine (c) from 9 rats. ** p<0.01 HYPO vs. HYPO+H₂O₂. In separate experiments, rats were perfused with 1 mM H₂O₂ alone (N = 5).</p

Dependence of taurine and glutamate uptake on extracellular [Na⁺] in cultured astrocytes.

<p>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 µM 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 ±SEM of three experiments from each group.</p