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
Source: PubMed


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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    • "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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    • "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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