Pharmacological comparison of swelling-activated excitatory amino acid release and Cl- currents in cultured rat astrocytes

Ordway Research Institute, Albany, NY 12208, USA.
The Journal of Physiology (Impact Factor: 5.04). 05/2006; 572(Pt 3):677-89. DOI: 10.1113/jphysiol.2005.103820
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Ubiquitously expressed volume-regulated anion channels (VRACs) are chloride channels which are permeable to a variety of small organic anions, including the excitatory amino acids (EAAs) glutamate and aspartate. Broad spectrum anion channel blockers strongly reduce EAA release in cerebral ischaemia and other pathological states associated with prominent astrocytic swelling. However, it is uncertain whether VRAC serves as a major pathway for EAA release from swollen cells. In the present study, we measured swelling-activated release of EAAs as D-[3H]aspartate efflux, and VRAC-mediated Cl- currents by whole-cell patch clamp in cultured rat astrocytes. We compared the pharmacological profiles of the swelling-activated EAA release pathway and Cl- currents. The expression of candidate Cl- channels was confirmed by RT-PCR. The maxi Cl- channel (p-VDAC) blocker Gd3+, the ClC-2 inhibitor Cd2+, and the MDR-1 blocker verapamil did not affect EAA release or VRAC currents. An antagonist of calcium-sensitive Cl- channels (CaCC), niflumic acid, had little effect on EAA release and only partially inhibited swelling-activated Cl- currents. The phorbol ester PDBu, which blocks ClC-3-mediated Cl- currents, had no effect on VRAC currents and up-regulated EAA release. In contrast, DCPIB, which selectively inhibits VRACs, potently suppressed both EAA release and VRAC currents. Two other relatively selective VRAC inhibitors, tamoxifen and phloretin, also blocked the VRAC currents and strongly reduced EAA release. Taken together, our data suggest that (i) astrocytic volume-dependent EAA release is largely mediated by the VRAC, and (ii) the ClC-2, ClC-3, ClC-4, ClC-5, VDAC, CaCC, MDR-1 and CFTR gene products do not contribute to EAA permeability.

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    • "In the subsequent experiments , we focused on approximately fivefold difference between reductions in L-glutamate and taurine content in 185 mOsm medium. It is generally accepted that during cell volume regulation, amino acids are predominantly released via the swellingactivated anion channel VRAC (see Abdullaev et al. 2006 and Introduction for additional references). Therefore, the simplest explanation for significant differences between hypo-osmotic reductions in the cellular contents of taurine versus L-glutamate and other amino acids is a higher permeability of VRAC to taurine. "
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    ABSTRACT: Hyponatremia and several other CNS pathologies are associated with substantial astrocytic swelling. To counteract cell swelling, astrocytes lose intracellular osmolytes, including l-glutamate and taurine, through volume regulated anion channel (VRAC). In vitro, when swollen by exposure to hypo-osmotic medium, astrocytes lose endogenous taurine faster, paradoxically, than l-glutamate or l-aspartate. Here, we explored the mechanisms responsible for differences between the rates of osmolyte release in primary rat astrocyte cultures. In radiotracer assays, hypo-osmotic efflux of preloaded [14C]taurine was indistinguishable from d-[3H]aspartate and only 30-40% faster than l-[3H]glutamate. However, when we used HPLC to measure the endogenous intracellular amino acid content, hypo-osmotic loss of taurine was ~5-fold greater than l-glutamate, and no loss of l-aspartate was detected. The dramatic difference between loss of endogenous taurine and glutamate was eliminated after inhibition of both glutamate reuptake (with 300 μM TBOA) and glutamate synthesis by aminotransferases (with 1 mM aminooxyacetic acid, AOA). Treatment with TBOA+AOA made reductions in the intracellular taurine and l-glutamate levels approximately equal. Taken together, these data suggest that swollen astrocytes actively conserve intracellular glutamate via reuptake and de novo synthesis. Our findings likely also explain why in animal models of acute hyponatremia, extracellular levels of taurine are dramatically elevated with minimal impact on extracellular l-glutamate. This article is protected by copyright. All rights reserved.
    Journal of Neurochemistry 01/2015; 135(1):176-185. DOI:10.1111/jnc.13229 · 4.28 Impact Factor
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    • "In contrast, the potent VRAC blocker DCPIB completely abolished hypoosmotic D-[ 3 H]aspartate release (Fig. 4A). The effect of DCPIB was consistent with literature findings [66] and confirmed the specificity of this assay. "
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    ABSTRACT: The contribution of oxidative stress to ischemic brain damage is well established. Nevertheless, for unknown reasons, several clinically tested antioxidant therapies failed to show benefits in human stroke. Based on our previous in vitro work, we hypothesized that the neuroprotective potency of antioxidants is related to their ability to limit release of the excitotoxic amino acids, glutamate and aspartate. We explored the effects of two antioxidants, tempol and edaravone, on amino acid release in the brain cortex, in a rat model of transient occlusion of the middle cerebral artery (MCAo). Amino acid levels were quantified using a microdialysis approach, with the probe positioned in the ischemic penumbra as verified by a laser Doppler technique. Two-hour MCAo triggered a dramatic increase in the levels of glutamate, aspartate, taurine and alanine. Microdialysate delivery of 10mM tempol reduced the amino acid release by 60-80%, while matching levels of edaravone had no effect. In line with these latter data, an intracerebroventricular injection of tempol but not edaravone (500 nmols each, 15 minutes prior to MCAo) reduced infarction volumes by ~50% and improved neurobehavioral outcomes. In vitro assays showed that tempol was superior in removing superoxide anion, whereas edaravone was more potent in scavenging hydrogen peroxide, hydroxyl radical, and peroxynitrite. Overall, our data suggests that the neuroprotective properties of tempol are likely related to its ability to reduce tissue levels of the superoxide anion and pathological glutamate release, and, in such a way, limit progression of brain infarction within ischemic penumbra. These new findings may be instrumental in developing new antioxidant therapies for treatment of stroke.
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    • "There is strong evidence in cultured astrocytes in vitro that astrocyte swelling leads to opening of volume-regulated anion channels (VRAC) to produce a regulatory volume decrease. Release of water through astrocytic VRAC is accompanied by substantial amounts of glutamate (Abdullaev et al., 2006; Haskew-Layton et al., 2008; Kimelberg et al., 2006; Liu et al., 2006). Among the first targets encountered by astrocytically-released glutamate are extrasynaptic NMDARs, leading to SICs and potentially interictal and ictal (seizure-like ) discharges. "
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