Two Distinct Modes of Hypoosmotic Medium-Induced Release of Excitatory Amino Acids and Taurine in the Rat Brain In Vivo Rene´ e E. Haskew-Layton1,2, Alena Rudkouskaya1, Yiqiang Jin3, Paul J. Feustel1, Harold K. Kimelberg3, Alexander A. Mongin1* 1 Center of Neuropharmacology and Neuroscience, Albany Medical College, Albany, New York, United States of America, 2 Burke Medical Research Institute of Cornell University, White Plains, New York, United States of America, 3 Ordway Research Institute, Albany, New York, United States of America Abstract 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. Citation: Haskew-Layton RE, Rudkouskaya A, Jin Y, Feustel PJ, Kimelberg HK, et al. (2008) Two Distinct Modes of Hypoosmotic Medium-Induced Release of Excitatory Amino Acids and Taurine in the Rat Brain In Vivo. PLoS ONE 3(10): e3543. doi:10.1371/journal.pone.0003543 Editor: Schahram Akbarian, University of Massachusetts Medical School, United States of America Received August 15, 2008; Accepted October 3, 2008; Published October 28, 2008 Copyright: 2008 Haskew-Layton et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported in part by NIH grants F31 NS046961 (to REH-L), R01 NS035205 (to HKK), R21 NS052516 (to AAM), and AMC grant 201-311053 (to AAM). The funding agencies had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: MonginA@mail.amc.edu Introduction The release of organic osmolytes in response to cellular swelling is mediated by one or more volume-sensitive permeability pathways [1– 3]. Although this phenomenon occurs in all tissues, it has special significance in the brain since two major swelling-sensitive organic osmolytes, glutamate and taurine, also mediate or modulate neuronal communication [4–6]. Several neural pathologies, most notably cerebral ischemia, hyponatremia, hepatic encephalopathy and traumatic brain injury, are associated with pronounced cell swelling, which is largely restricted to astrocytes [7–9]. Pathological cell swelling is likely related to tissue damage since pharmacological inhibitors that block volume-sensitive anion permeability pathway(s) suppress the pathological release of the excitatory amino acids, glutamate and aspartate, and reduce infarct size in animal models of stroke and ischemia [10–15]. These findings have led to the proposal that the swelling-activated release of excitatory amino acids may play a critical role in promoting ischemic tissue damage [7,9,16]. The swelling-induced release of the excitatory amino acids glutamate and aspartate and the sulfonic acid taurine is thought to be mediated by Volume Regulated Anion Channels (VRACs), which are also termed in the literature as Volume Sensitive Outward Rectifying (VSOR) Cl2 channels or Volume-Sensitive Organic osmolyte/Anion Channels (VSOAC) [17–19]. VRACs are traditionally identified as volume-sensitive Cl2/anion channels that are activated in response to cell swelling in nearly all cell types studied. However, in spite of extensive research efforts, the molecular identity of these channels remains unknown [19,20]. The major physiological role of VRAC is cell volume regulation. Upon activation in swollen cells, VRACs mediate the release of inorganic and organic anions and, in conjunction with swelling- activated K+ channels, facilitate reductions in intracellular osmo- larity and subsequent regulatory volume decrease. In addition to Cl2, VRACs are also permeable to bicarbonate (HCO32), several other inorganic anions, as well as small organic osmolytes such as amino acids, polyols, and methylamines [2,21,22]. The evidence that taurine release is mediated by VRAC, or a very similar permeability pathway, largely stems from studies in cultured neuronal and glial cells, which show that both swelling- activated [ 3 H]taurine and 125I2 (Cl2) fluxes are inhibited by a variety of VRAC blockers, including the selective VRAC inhibitor DCPIB [23–27]. Several electrophysiological studies have con- PLoS ONE | www.plosone.org 1 October 2008 | Volume 3 | Issue 10 | e3543